ELISA Handbook

G r a s p t h e P r o t e o m e
ELISA AND ELISPOT PRODUCTS

Table of Contents
ELISA the Pierce Way ...........................................................2 Introduction to ELISA ............................................................4 Typical ELISA Protocol ........................................................................4 Types of ELISAs...................................................................................4 Direct vs. Indirect Detection Techniques for ELISA .............................5 Developing an ELISA ...........................................................................6 Selecting an ELISA Plate .......................................................8 Coated Microplates ............................................................10 Blocking and Washing .........................................................24 Primary and Secondary Antibodies..........................................32 Affinity-Purified Primary Antibodies ..................................................32 Antibody Conjugates and Enzyme Detection .....................................33 Affinity-Purified Secondary Antibodies..............................................35 Antibody Labeling ..............................................................40 Fluorescent Labeling..........................................................................41 Enzyme Labeling................................................................................42 Choosing a Substrate ..........................................................46 Horseradish Peroxidase Substrates...................................................47 Alkaline Phosphatase Substrates.......................................................54 ELIFA System ...................................................................56 Bulk and Custom Offerings....................................................58 Recommended Reading.......................................................60
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Enzyme-linked immunosorbent assays (ELISAs) are designed for detecting

and quantitating substances such as peptides, proteins, antibodies and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same process. In an ELISA, an antigen must be immobilized to a solid surface. The antigen is then complexed with an antibody that is linked to an enzyme. Detection is accomplished by incubating this enzyme-complex with a substrate that produces a detectable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
Most commonly, ELISAs are performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform, as first described by Eva Engvall, et al.1 Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from unbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation. A detection enzyme may be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It may also be linked to a protein such as streptavidin if the primary antibody is biotin-labeled. However the enzyme is incorporated, the most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include -galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the necessary sensitivity level of the detection and the instrumentation available for detection (e.g., spectrophotometer, fluorometer or luminometer). Enzyme-linked immunospot assays (ELISPOTs) are a subclass of ELISAs that are used to count the number of cytokine-producing cells within a cell suspension. The difference between the two is that in ELISA, the substance containing the unknown is stuck at the bottom of the well, whereas in ELISPOT the substance within the unknown is placed in the well after the bottom of the well has been coated with cytokine-specific antibody. In both cases, the wells are typically contained within a generic microplate.
ELISA AND ELISPOT PRODUCTS
ELISA THE PIERCE WAY

STEP 1. Selecting an ELISA plate
96-well Plates (Product #s 15041 and 15042) 8-well Strip Plates (Product # 15031) Strip Well Ejector (Product # 15033) Choose a plate. Sealing Tape for Plates (Product # 15036) Reagent Reservoirs (Product # 15075) DNA Coating Solution (Product # 17250)
Pre-coated plates
Protein A (Product #s 15130 and 15132) Protein G (Product #s 15131 and 15133) Protein A/G (Product # 15138) Protein L (Product # 15190) Secondary Antibodies (Product #s 15134 and 15135) Anti-GFP (Product #s 15182, 15186 and 15188) NeutrAvidin Biotin-Binding Protein (Many options, see page 15-16) Streptavidin (Many options, see page 17-18)
Use a pre-coated plate for efficiency and consistency.
Biotin (Product # 15151) Nickel Chelate (Product #s 15142, 15242 and 15342) Copper Chelate (Product #s 15143, 15146, 15147 and 15148) Glutathione (Product #s 15140, 15240 and 15340) Poly-D-Lysine (Product #s 15600 and 15608) Collagen I (Product #s 15610 and 15618) Maleic Anhydride (Product #s 15110, 15112, 15100 and 15102) Maleimide (Product # 15150)
STEP 2. Blocking
StartingBlock Blocking Buffer in PBS (Product # 37538) and in TBS (Product # 37542) StartingBlock T20 Blocking Buffer (Contains 0.05% Tween-20) in PBS (Product # 37539) or TBS (Product # 37543) SuperBlock Buffer in PBS (Product # 37515) and in TBS (Product # 37535) SuperBlock T20 Blocking Buffer (Contains 0.05% Tween-20) in PBS (Product # 37516) or TBS (Product # 37536)
Block unoccupied sites.
Casein in PBS (Product # 37528) and in TBS (Product # 37532) BSA in PBS (Product # 37525) and in TBS (Product # 37520) SEA BLOCK Buffer (Product # 37527) BLOTTO in TBS (Product # 37530) Metal Chelate Buffer (Product # 37547)
STEP 3. Add sample and incubate

2
STEP 4. Formulate wash buffers

Phosphate Buffered Saline (PBS, Product # 28372) Tris Buffered Saline (TBS, Product #s 28376 and 28379) Modified Dulbeccos PBS (Product # 28374) Carbonate-Bicarbonate Buffer Packs (Product # 28382)
Choose a buffer. MES Buffered Saline (Product # 28390) BupH Borate Buffer Packs (Product # 28384) BupH Citrate-Carbonate Buffer Pack (Product # 28388)
Add detergent to buffers

[Skip this step if you use StartingBlock T20 Blocking Buffer in PBS (Product # 37539) or TBS (Product # 37543) or SuperBlock T20 Blocking Buffer in PBS (Product # 37516) or TBS (Product # 37536). These buffers already contain Tween-20 Detergent at optimized concentrations.]
Reduce nonspecific binding. Triton X-100 (Product # 28314) and Triton X-114 (Product # 28332) Nonidet P-40 (Product # 28324) Brij-35 (Product # 28316) and Brij-58 (Product # 28336)
Surfact-Amps Brand Detergents containing: Tween-20 (Product # 28320) and Tween-80 (Product # 28328)
STEP 5. Primary and Secondary Antibodies

For a complete list, visit the antibody selection guide on our web site (www.piercenet.com) accessible under the Products tab. For direct detection methods we offer: Monoclonal Antibodies Fluorescent Probes and Labeling Kits Enzyme Labeling Kits For indirect detection methods we offer: Biotinylation Kits
Incubate the membrane with antibodies. Protein A, Protein G and Protein L labeled with fluorescein, rhodamine, HRP, AP or biotin
HRP
Avidin, Streptavidin and NeutrAvidin Biotin-Binding Protein labeled with fluorescein, rhodamine, HRP or AP Secondary antibodies labeled with fluorescein, rhodamine, HRP, AP or biotin
STEP 6. Enzyme Substrates for Detection

Chemiluminescent Substrates: SuperSignal ELISA Pico Chemiluminescent Substrate (Product # 37070) SuperSignal ELISA Femto Chemiluminescent Substrate (Product # 37075) Chemifluorescent Substrate: Fluorogenic Peroxidase Substrate (Product #s 15162 and 15169) QuantaBlu
Add the detection reagent. Colorimetric Substrates: TMB Substrates (Product #s 34021, 34022, 34024 and 34028) ABTS Substrates (Product #s 34026 and 37615) OPD Substrate (Product #s 34005 and 34006) PNPP Substrates (Product #s 34045, 34047, 37620 and 37621)
HRP
SuperSignal Substrate
INTRODUCTION TO ELISA
Types of ELISAs
The most commonly used ELISA assay format is the sandwich assay (Figure 1). This type of assay is called a sandwich assay because the analyte to be measured is bound between two antibodies the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust.2 Competitive assays are often used when the antigen is small and has only one epitope, or antibody-binding site.3 Often the antigen is labeled instead of the antibody. Unlabeled antigen and the labeled antigen compete for binding to the capture antibody and a decrease in signal indicates the presence of the antigen in the sample. ELISPOT assays are a form of sandwich ELISAs. Either monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonals have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity. An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are considered a matched pair and are suitable for developing a sandwich ELISA. Another design consideration in choosing antibodies is cost. A polyclonal antibody is generally less expensive (~five-fold) to produce than a monoclonal. The specificity gained by using monoclonals for both the capture and detecting antibody must be weighed against the cost and time required for producing two monoclonal antibodies. Preparing a self-sandwich ELISA assay, in which the same antibody is used for the capture and detection, can limit the dynamic range and sensitivity of the final ELISA.
Typical ELISA Protocol

Coating antibody or antigen onto the microplate 1. Dilute the protein to be coated to a concentration of 2-10 g/ml in a buffer such as PBS or CarbonateBicarbonate and add 100 l of this solution per well. 2. Incubate for 18-20 hours at room temperature or 4C. 3. Block unoccupied sites with a blocking agent (200-300 l/well) such as StartingBlock Blocking Buffer. 4. Store plate at 4C with a dessicant for future use. Perform assay 5. Add sample to be tested (50-100 l/well) and incubate for 1 or more hours. 6. Wash using PBS with 0.05% Tween-20 or TBS with 0.05% Tween-20. 7. Add enzyme-antibody conjugate (100-200 l/well) diluted in blocking buffer and incubate for 1 hour. 8. Wash again. 9. Add substrate. 10. Detect.
Substrate Substrate Substrate E Substrate Primary antibody conjugate Ag E Secondary antibody conjugate Primary antibody Ag E E Biotinylated primary antibody E Ag E Substrate E Primary antibody #1 Ag Primary antibody #2 E Substrate E Substrate
Direct Assay Figure 1. Immunoassay formats.
Indirect Assay
Avidin-Biotin Complex
Capture Assay Sandwich or ELISPOT
Direct vs. Indirect Detection Techniques for ELISA

The direct detection method originated in the 1940s when Coons and colleagues labeled antibodies with a fluorescent tag to mark tissue antigens.4 In this technique, a labeled primary antibody reacts directly with the antigen (Figure 1). Direct detection is not widely used in ELISAs, but is quite common for immunohistochemical staining of tissues and cells. Advantages of direct detection Quick methodology, because only one antibody is used. Cross-reactivity of secondary antibody is eliminated. Disadvantages of direct detection Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Little signal amplification. The indirect, two-step method uses a labeled secondary antibody for detection (Figure 1). This was first described by Weller and Coons in 1954 and is still a popular method.5 First, a primary antibody is incubated with the antigen. This is followed by
incubation with a labeled secondary antibody that recognizes the primary antibody. For ELISA it is important that the antibody enzyme conjugate is of high specific activity. This is achieved when the antibody is affinity-purified and the enzyme conjugation chemistry preserves antibody specificity as well as enzyme activity. All Pierce antibody-enzyme conjugates fulfill these requirements. Advantages of indirect detection A wide variety of labeled secondary antibodies are commercially available. Versatility, because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Different visualization markers can be used with the same primary antibody. Disadvantages of indirect detection Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure.
Developing an ELISA
Optimizing immunoreagent concentrations and dilutions The goals in developing an ELISA assay are 1) to achieve the best signal:noise ratio for the sensitivity level desired, 2) to have a robust, reproducible assay for the sample being tested and 3) to be able to measure the antigen over a biologically relevant assay range (dynamic range). Therefore, ideal concentrations of each assay reagent must be established empirically. The signal generated by a sample containing analyte, relative to the signal of the same sample without analyte, is the signal:noise ratio. As the signal:noise ratio increases, the assay becomes better at measuring small amounts of antigen. Dilution ranges for assay reagents can vary widely, depending upon the detection system used. For example, most ELISA protocols based on enzyme-antibody conjugates using a colorimetric substrate recommend a 1:5,000 dilution (from a 1 mg/ml stock concentration) of the conjugate. But this ELISA may work equally well at 1:2,000 or 1:20,000 dilution. To establish the optimal dilutions, a checkerboard titration, also called a two-dimensional serial dilution, is performed. A checkerboard titration is a single experiment in which the concentration of two components is varied in a way that will result in a pattern (Figure 2). This method is used to optimize reagent concentrations when performing an indirect ELISA that uses a labeled secondary antibody. For example, the primary antibody is serially diluted across the plate, and the enzyme-labeled secondary antibody is serially diluted down the plate. This design permits analysis of different concentrations of the two reagents in each well to obtain the best signal:noise ratio.
Labeled Secondary Antibody Dilution
1:1 1:2 1:4 1:8 1:16 1:32 1:64
Primary Antibody Dilution

1:1 1:2 1:4 1:8 1:16 1:32 1:64 A B C D E F G H 1 2 2 3 4 6 7 8 9 10 11 12
Figure 2. A checkerboard titration using a precipitating substrate.
A more complex checkerboard titration for developing an ELISA can be seen in Figure 3. Here the coating or capture antibody is titered first by using high concentrations of biotinylated detecting antibody and streptavidin-HRP conjugate. Once the signal falls within the desired detection range (dynamic range), the checkerboard matrix in Figure 3 can be tested to optimize the concentrations of the biotinylated detecting antibody and streptavidin-conjugate.
(Coating Antibody at 2 g/ml)

0 pg/ml 80 pg/ml 400 pg/ml 2,000 pg/ml 0 pg/ml 80 pg/ml 400 pg/ml 2,000 pg/ml 0.032 0.225 0.555 2.870 0.041 0.337 0.831 3.720 0.031 0.219 0.545 2.620 0.040 0.335 0.886 **** 0.028 0.208 0.605 2.494 0.032 0.293 0.838 3.263 0.030 0.207 0.561 2.476 0.031 0.296 0.829 3.454 0.029 0.167 0.438 2.037 0.031 0.229 0.604 2.373 0.028 0.175 0.431 1.770 0.030 0.232 0.613 2.468 0.047 0.402 1.603 **** 0.074 0.448 1.466 **** 0.047 0.402 1.108 **** 0.067 0.411 1.348 **** 0.042 0.363 1.034 **** 0.060 0.436 1.243 **** 0.039 0.369 1.032 **** 0.059 0.419 1.255 **** 0.038 0.287 0.742 2.709 0.052 0.357 0.891 3.187 0.037 0.294 0.768 2.866 0.048 0.347 0.884 3.141 Biotin-Labeled Detecting Antibody Concentration 60 ng/ml 125 ng/ml 250 ng/ml 500 ng/ml
Standards
1:8,000
1:16,000
1:32,000
1:8,000
1:16,000
1:32,000
HRP-Conjugated Streptavidin Dilution 3A. The plate is divided into four equal quarters for four different detecting antibody concentrations. For each detecting antibody concentration, three different enzyme concentrations are tested on three different levels of standards, plus a zero. Prior to this checkerboard titration, a coating titration was performed to determine the optimal concentration for the coating antibody. From this checkerboard matrix, the following curves are generated: Human TNF Detecting Antibody Titration Curves (HRP-Conjugated Streptavidin at 1:32,000)
Absorbance at 450 nm-550 nm 3.0 2.5 2.0 1.5 1.0 0.5 0 0 500 1,000 Human TNF (pg/ml) 1,500 2,000 60 ng/ml 125 ng/ml 250 ng/ml 500 ng/ml Absorbance at 450 nm-550 nm 3.5 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 0 500 1,000 Human TNF (pg/ml) 1,500 2,000 1:8,000 1:16,000 1:32,000
HRP-Conjugated Streptavidin Titration Curves (Biotin-Labeled Antibody at 60 ng/ml)
3B. When dividing the mean O.D. for the 80 pg/ml standard by the O.D. for the zero standard, 250 ng/ml produced the greatest signal:noise ratio, suggesting the antibody is on a plateau. Using this detecting antibody at 250 ng/ml would provide optimal sensitivity, assay reproducibility and sample recovery.
3C. The standard curve is within the desired O.D. range at the 1:32,000 dilution of HRP-conjugated streptavidin. For increased sensitivity with a smaller assay range, a higher concentration of HRP-conjugated streptavidin may be selected. Poly-HRP streptavidin may also be used.
Figure 3. Checkerboard matrix: example of human TNF detecting antibody and enzyme titration.
References 1. Engvall, E. and Perlmann, P.O. (1971). Immunochemistry 8, 871-875. 2. Palomki, P. (1991). J. Immunol. Method 145, 55-63. 3. Rao, P.N. and Taraporewala, I.B. (1992). Steroids 57, 154-161. 4. Coons, A.A., et al. (1942). J. Immunol. 45, 159-170. 5. Weller, T.H. and Coons, A.H. (1954). Proc. Soc. Exp. Biol. (New York) 86, 789-794.
SELECTING AN ELISA PLATE

When binding an antibody, select a microplate with high-binding capacity. Choose a microplate with a minimum protein-binding capacity of 400 ng/cm2. It is also important that the CV value (coefficient of variation) of the protein binding be below 5%. Pierce ELISA plates undergo extensive quality testing and controlled manufacture and are guaranteed to have CVs less than 5%. Use Reacti-Bind 8-Well Strip Plates (Product # 15031) when partial-plate assays are performed frequently. The choice of plate color depends upon the signal being detected. Clear polystyrene plates are used for colorimetric signals and black or white opaque plates are used for fluorescent and chemiluminescent signals. Characteristics: Antibody Binding: High, 400 ng IgG/cm2 (1 cm2 = 100 l vol.) Binding Interaction: Hydrophobic/Ionic (-) Performance Certified: Yes. Well-to-well, CV 3% Well Shape (bottom): Flat Maximum Well Volume: 360 l Packaging: 1 plate per sealed tray Units per package: 100 plates
ImmunoWare Reagent Reservoirs

Allows easy dispensing of reagents by multi-channel pipettors.
Highlights: Sterile, sturdy reservoirs are molded from highimpact polystyrene Reservoirs facilitate repeated pick up of reagents by multichannel pipettors for delivery to 96-well plates
Ordering Information
Product # Description 15075 ImmunoWare Reagent Reservoirs
Sterile, 50 ml capacity
U.S. Pkg. Size Price 200/pkg. $ 99
Sealing Tape for 96-Well Plates

Highlight: Seal plates quickly and easily to allow mixing or prevent evaporation
Product # Description 15036 Sealing Tape for 96-Well Plates
Pre-cut pressure-sensitive sealing tape
U.S. Pkg. Size Price 100/pkg. $ 48
Reacti-Bind 96-Well Plates

Enhance antibody-binding properties in your ELISA applications.
Highlights: Plates are made of specially formulated polystyrene and treated to produce high antibodybinding characteristics Each lot is certified to ensure adherence to strict performance criteria
Characteristics: Antibody Binding: High, 400 ng IgG/cm2 (1 cm2 = 100 l vol.) Binding Interaction: Hydrophobic/Ionic Performance Certification: Well-to-well, CV 3% Well Shape (bottom): Flat Maximum Well Volume: 360 l Packaging: 1 plate per sealed tray
Reference Foster, B.A., et al. (1999). Science 286, 2507-2510.
U.S. Product # Description Pkg. Size Price 15041 Reacti-Bind 96-Well Plates Corner Notch 100/pkg. $278 15042 Reacti-Bind White Opaque 96-Well Plates 25/pkg. $ 84
Reacti-Bind 8-Well Strip Plates

No more loose-fitting strips!
Highlights: Special optically clear polystyrene formulation High antibody-binding surface improves the overall performance in immunoassay applications Plates are certified for performance Consistent lower coefficients of variation Eight-well strips fit snugly in the plate frame, virtually eliminating strip loss while in use even if turned upside down Strip guides help to avoid misorientation of the strip when returned to the holder Strips are gently removed from the frame with a strip well ejector Wells can be easily separated from the strips with a gentle twist, allowing you to work with individual wells or strips of less than eight wells
Characteristics: Antibody Binding: High, 400 ng IgG/cm2 (1 cm2 = 100 l vol.) Binding Interaction: Hydrophobic/Ionic Performance Certification: Well-to-well, CV 3% Well Shape (bottom): Flat Maximum Well Volume: 360 l Packaging: 5 plates per sealed tray
Product # Description 15031 Reacti-Bind 8-Well Strip Plates Corner Notch
Includes one Strip Well Ejector per package.
U.S. Pkg. Size Price 100/pkg. $462
Accessory 15033 Reacti-Bind Strip Well Ejector 1/pkg. $ 29
Reacti-Bind DNA Coating Solution

Quickly deposit DNA onto a plastic surface for hybridization.
Reacti-Bind DNA Coating Solution is the latest answer for efficient DNA surface coating of microplates and microsample tubes. Highlights: No more overnight incubations or heating microplates and waiting DNA Coating Solution promotes efficient immobilization of DNA onto microplates hybridization efficiency is related to efficiency of DNA surface immobilization.1 This solution promotes high-efficiency coating of DNA onto polystryene or polypropylene surfaces Target DNA coated onto microplates permits speed and flexibility in probing DNA coated onto plastic surfaces is stabilized sufficiently to allow storage for months prior to use DNA Coating Solution autoclaved to inactivate DNase
References 1. Hirayama, H., et al. (1996). Nucleic Acids Res. 24, 4098-4099. van Gijssel, H.E., et al. (2002). Cancer Epidem. Biomarker Prev. 11, 1622-1629.
Product # Description 17250 Reacti-Bind DNA Coating Solution
Sufficient for coating 5 x 96-well microplates (at 200 l/well).
U.S. Pkg. Size Price 100 ml $ 63
SELECTING AN ELISA PLATE
COATED MICROPLATES
There are several important points to consider when coating an ELISA plate. It is important to ensure that the coating solution is free of detergents, because detergents often compete for binding and cause low and/or uneven binding. For competitive assays, a lower coating concentration usually is chosen to ensure that the antibody is the limiting factor. Oddly, excessive concentrations of coating protein occasionally lead to less binding, a phenomenon known as the hook effect. A typical protein-coating solution concentration of 2-10 g/ml can be used as a starting point for successful plate-coating. Smaller molecules such as peptides and many drugs require chemically activated microplates such as Reacti-Bind Maleic Anhydride Activated Plates (Product # 15100 or 15110) or Maleimide Activated Plates (Product # 15150) to achieve covalent attachment to the plate surface. The maleic anhydride group reacts with primary amines (lysine, N-terminus) and the maleimide reacts with sulfhydryl groups (SH, reduced cysteine residues), forming a covalent bond (Figure 4). To increase the dynamic range of an assay for small molecules, they may be biotinylated and attached to a Reacti-Bind NeutrAvidin HBC (High Binding Capacity) Plate or a Reacti-Bind Streptavidin HBC Plate (Figure 5). These plates allow binding of more biotinylated oligos, peptides, etc. to the surface of the well, and are provided as clear, black or white opaque plates and in 96- or 384-well plate formats.
Reaction Scheme for Coupling both Large and Small Amine-Containing Molecules O C C ( )n C O C O Maleic Anhydride shown anchored to polystyrene well of a microplate. Both the well wall and base are activated. R H2N R pH 7.2 = Protein, peptide or other ligand O C N R Stable amide bond formation with amines, with a resulting hydrophilic well surface.
C C C O ( )n O
Reaction Scheme for Coupling both Large and Small Sulfhydryl-Containing Molecules Maleimide shown anchored to polystyrene well of a microplate. Both the well wall and base are activated. SH R pH > 6.5-7.5 ( )n R = Protein, peptide or other ligand S R
O
N ( )n
O
Stable thioether bond formation with sulfhydryls, with a resulting hydrophilic well surface.
Figure 4. Coupling reactions of Reacti-Bind Activated Plates.
10
Phosphopeptide Detection Assay Comparison of Biotin-Binding Protein Coated Plates

10 HBC 8 RBC CHC S/N Ratio 6
capability. Fusion proteins can be attached to a microplate in the proper orientation using Glutathione, Metal Chelate or Anti-GFP Coated Plates. Because the polystyrene surface of Reacti-Bind Coated Plates is precoated, immobilized proteins are physically separated from the surface and protected from its denaturing effects. Peptides and other small molecules, which are typically difficult to immobilize, can be biotinylated and attached to a streptavidin or NeutrAvidin Coated Plate with high efficiency. Precoated plates bind selectively to the desired target proteins, minimizing any contamination from other molecules that are present in the preparation. Studies have shown that the binding of antibodies to a microplate surface causes some denaturation.1 This can be solved in several ways. Antibodies can be bound to a binding protein, such as Protein A, Protein G, Protein A/G or Protein L, that is coated on the plate. Alternatively, the antibody may be biotinylated and immobilized onto streptavidin or NeutrAvidin Coated Plates. Either method physically separates the antibody from the surface of the plate and can result in proper orientation of the antigen-binding sites for better assay performance. Histidine-, green fluorescent protein (GFP)- and glutathione S-transferase (GST)-tagged fusion proteins are popular for manipulating recombinantly expressed proteins and developing ELISAs to detect the expression levels of these tagged proteins is simplified with microplates that are precoated with the ligand for capturing the fusion protein to be measured. Reacti-Bind Precoated Plates save time in developing ELISAs by 1) capturing the fusion protein from a crude cell lysate, 2) separating the fusion protein from the surface of the microplate to prevent denaturation and 3) properly orienting the fusion protein for detection. Table 1 lists the variety of Reacti-Bind Coated Plates available and the ligands they bind.
0 0 5 10 15 Biotinylated Phosphopeptide (pM/well)
Figure 5. Comparison of NeutrAvidin High Binding Capacity (HBC) Coated Plate, NeutrAvidin Regular Binding Capacity (RBC) Coated Plates and a competitors Streptavidin Coated High Binding Capacity Plates (CHC). Plates were incubated with various dilutions of biotinylated, phosphorylated peptide. After washing, the plates were incubated with mouse anti-phosphotyrosine antibody (1:1,000) and then detected using an anti-mouse-FITC conjugate (1:667). The Y-axis is described as the signal:noise (S/N) ratio.
The initial step in many microplate-based assays involves passive adsorption of proteins to the plate wells. Several problems arise from passive adsorption, including improper orientation, denaturation, poor immobilization efficiency and binding of contaminants along with the target molecule. Reacti-Bind Coated Plates from Pierce are designed to circumvent these problems. Antibodies can be covalently attached to a microplate through the Fc region using Protein A, G, or A/G coated plates, which orients them properly and preserves their antigen-binding
COATED MICROPLATES
11
COATED MICROPLATES
Table 1. Reacti-Bind Coated Polystyrene Microplates Protein A, G or A/G Protein L Secondary Antibodies NeutrAvidin Protein or Streptavidin Biotin HisGrab (Ni2+) or Glutathione Maleic Anhydride Maleimide Activated Anti-GFP Anti-GST For binding antibodies via their Fc regions For binding Fab antibody fragments and single-chain variable fragments (ScFvs) through the kappa light chain For binding antibodies, as an alternative to Protein A, G or L For binding biotinylated proteins, peptides or nucleic acids; also available in black or white opaque microplates For binding avidin, streptavidin or NeutrAvidin Biotin-Binding Protein For binding recombinantly expressed proteins containing polyhistidine or glutathione S-transferase For binding both large and small amine-containing molecules For binding sulfhydryl-containing molecules For capturing proteins expressing green fluorescent protein (GFP) tag For capturing proteins expressing glutathione S-transferase (GST)
0.8 Pierce Absorbance at 490 nm 0.6 Competitor B
0.4
Cell-based assays have become popular for detecting the effects of various drugs, toxins or growth factors on cellular growth. To enhance cell attachment to the surface of a 96-well plate, CellScreen Plates are coated with collagen or poly-D-lysine (Figure 6) and are irradiated to prevent bacterial growth. CellScreen Plates are offered in a variety of formats (96- or 384-well) for flexible assay design. Pierce also offers a custom plate-coating service for large batches of coated plates. Our automated plate-coating technology produces consistently coated plates that will meet your quality specifications. Take advantage of our coating expertise to expedite the development of your plate-based assay. See pages 58-59 for more information on custom-coated plates.
Reference 1. Schwab, C. and Bosshard, H.R. (1992). J. Immunol. Method 147, 125-134.
0.2
0.0 0 10,000 20,000 Cells/Well 30,000 40,000
Figure 6. Poly-D-Lysine Coated 96-Well Plates comparison (Pierce vs. Competitor B). Poly-D-Lysine plates (96-well white with clear bottom) from Pierce and Competitor B were tested for their ability to support attachment and spreading of rat cerebellar granule (RCG) cells. The graph represents cell proliferation assay results following a 24-hour cell incubation.
12
Reacti-Bind Protein A, G and A/G Coated Plates

Bind antibodies through their Fc portion, correctly orienting them for maximum antigen capture.
Highlights of using Reacti-Bind Plates for antibody capture: Retain antibody activity, which can be lost when antibodies are immobilized by passive adsorption Orient antibodies for maximum antigen-binding capacity Immobilize antibodies without prior purification Ensure minimal variation (< 5% well-to-well) from consistent coating Reduce nonspecific binding because plates are pre-blocked with SuperBlock Blocking Buffer Perform multiple immunoprecipitations Binds up to 2.5 g antibody/well (100 l coating volume) Protein A/G Consists of a fusion protein containing four antibody-binding sites from Protein A and two from Protein G Binds to the broadest spectrum of antibodies because it combines the specificities of Protein A and Protein G Binds to Fc region of antibodies, ensuring optimal orientation Binds well using a wide range of pH Protein A Binds strongly to IgG from rabbit, guinea pig, pig, dog and rhesus monkey Binds strongly to mouse IgG2a, IgG2b and IgG3 Binds to Fc region of antibodies for optimal orientation Protein G Binds strongly to IgG from many species including human, mouse, rabbit, sheep and goat Binds only to IgG no cross-reactivity with other antibody classes Binds to Fc region of antibodies for optimal orientation
0.8 0.7 Absorbance at 450 nm 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 0.16 0.312 0.625 1.25 2.5 Antibody Added per Well (ng) 5 10 Protein G Passively Bound Antibody
Figure 7. Properly oriented antibodies retain higher activity.

Reference Desai, S. and Hermanson, G. (1997). Previews 1(3), 2-7. Protein A Coated Plate References Asthagiri, A.R., et al. (1999). J. Biol. Chem. 274, 27119-27127. Lawrenson, I.D., et al. (2002). J. Cell Sci. 115, 1059-1072. Protein G Coated Plate References Lai, Z., et al. (2002). Proc. Natl. Acad. Sci. 99, 14734-14739. Rauch, J., et al. (2003). J. Biol. Chem. 278, 47508-47515.
Product # Description 15138 Reacti-Bind Protein A/G Coated 8-Well Strip Plates 15130 Reacti-Bind Protein A Coated 96-Well Plates 15132 Reacti-Bind Protein A Coated 8-Well Strip Plates 15131 Reacti-Bind Protein G Coated 96-Well Plates 15133 Reacti-Bind Protein G Coated 8-Well Strip Plates U.S. Pkg. Size Price 5 plates $170 5 plates 5 plates 5 plates 5 plates $141 $163 $156 $168
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Reacti-Bind Protein L Coated Plates
Great for binding ScFv and Fab fragments.
Protein L is an immunoglobulin-binding protein that has the unique ability to bind through kappa light chain interactions without interfering with an antibodys antigen-binding site. This gives Protein L the ability to bind a wider range of Ig classes and subclasses than any other antibody-binding proteins such as Protein A or Protein G. This also gives Protein L the unique ability to bind single-chain variable fragments (ScFvs) and Fab fragments. Highlights: Binds to all classes of Ig (IgG, IgM, IgA, IgE and IgD) Binds to the VL region of kappa light chains (human I, III and IV and mouse I) without interfering with antigen-binding sites Binds ScFvs Does not bind Bovine, Goat or Sheep Igs Binds weakly to Rabbit Igs Pre-blocked with SuperBlock Buffer to reduce nonspecific binding
References kerstrm, B. and Bjrck, L. (1989). J. Biol. Chem. 264, 19740-19746. Bjrck, L. (1988). J. Immunol. 140, 1194-1197. Kastern, W., et al. (1992). J. Biol. Chem. 267, 12820-12825. Nilson, B.H.K., et al. (1993). J. Immunol. Method 164, 33-40. Rhee, J., et al. (2001). J. Biol. Chem. 276, 6640-6644.
Product # Description 15190 Reacti-Bind Protein L Coated 96-Well Plates U.S. Pkg. Size Price 5 plates $168
Reacti-Bind Secondary Antibody Coated Plates

For species-specific antibody capture.
Highlights: Prevents antibody denaturation as a result of direct binding to polystyrene Unlike Protein A or Protein G Plates, these plates bind only to target species Antibody-binding capacity is higher than direct adsorption onto polystyrene Pre-blocked with SuperBlock Buffer to reduce nonspecific binding
References Gartner, W., et al. (2001). Cereb Cortex. 11, 1161-1169. Wagner, L., et al. (2000). J. Biol. Chem. 275, 24740-24751.
Product # Description 15134 Reacti-Bind Goat Anti-Mouse Coated Clear 96-Well Plates 15135 Reacti-Bind Goat Anti-Rabbit Coated Clear 96-Well Plates U.S. Pkg. Size Price 5 plates $130 5 plates $130
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Reacti-Bind Anti-GFP Coated Plates

Create more assay possibilities by capturing GFP fusion proteins on coated plates.
Highlights: Unique anti-GFP antibody binds all variants of GFP fusion proteins Coating is compatible with Poppers Cell Lysis Reagents and B-PER, M-PER and Y-PER Extraction Reagents Directly detect GFP-fusion proteins on white plates without interference from cellular autofluorescence or quenching agents Pre-blocked to save assay development time GFP excitation: 395 nm; emission 509 nm
Product # 15182 15186 15188 Description Pkg. Size Black 96-Well Plates with Blocker BSA 5 plates Clear 96-Well Plates with Blocker BSA 5 plates Clear 8-Well Strip Plates with Blocker BSA 5 plates U.S. Price $135 $135 $141
Reacti-Bind NeutrAvidin Coated Plates

The high affinity of avidin for biotin, without the nonspecific binding problems.
Highlights: Easy and gentle immobilization of biotin-containing conjugates Lowest nonspecific binding properties of all biotin-binding proteins NeutrAvidin Biotin-Binding Protein has no carbohydrate and an isoelectric point of 6.3 No denaturing of the protein component of a conjugate upon binding to the plate Ideal for binding small hydrophilic molecules (e.g., peptides) that typically exhibit poor binding directly to polystyrene Pre-blocked with your choice of Blocker BSA or SuperBlock Blocking Buffer Available in 96-well and 384-well formats
Protein Avidin Streptavidin NeutrAvidin Protein Isoelectric Point 10-10.5 5 6.3 Contains Carbohydrate Yes No No Nonspecific Binding High Low Ultralow
NeutrAvidin Coated Plate Characteristics

Binding Capacity Coat Volume Blocking Volume 96-Well Plate 15 pmoles/well 100 l/well 200 l/well 384-Well Plate 10 pmoles/well 50 l/well 100 l/well
References Brett, P.J., et al. (2002). J. Biol. Chem. 277, 20468-20476. Denlinger, L.C., et al. (2001). J. Immunol. 167, 1871-1876. Guixiang Dai, G., et al. (2002). J. Biol. Chem. 277, 161-168. Patil, S., et al. (1999). J. Biol. Chem. 274, 28575-28583. Singh, Y., et al. (1999). Infect. Immun. 67, 1853-1859.
Product # Description 15129 Clear 96-Well Plates with SuperBlock Blocking Buffer 15123 Clear 96-Well Plates with Blocker BSA 15128 Clear 8-Well Strip Plates with Blocker BSA 15127 Clear 8-Well Strip Plates with SuperBlock Blocking Buffer 15116 White 96-Well Plates with SuperBlock Blocking Buffer 15117 Black 96-Well Plates with SuperBlock Blocking Buffer 15400 Clear 384-Well Plates with SuperBlock Blocking Buffer 15401 White 384-Well Plates with SuperBlock Blocking Buffer 15402 Black 384-Well Plates with SuperBlock Blocking Buffer 15115 Reacti-Bind Biotin Binding Plate Sample Pack
One each of the following plates: Product #s 15120, 15121, 15127 and 15128
U.S. Pkg. Size Price 5 plates $123 5 plates 5 plates 5 plates 5 plates 5 plates 5 plates 5 plates 5 plates 4 plates $123 $172 $172 $135 $135 $262 $262 $262 $112
1.5 Absorbance at 450 nm
1.0
0.5
0 0.00
0.05 0.10 Units src kinase
0.15
Figure 8. Purified p60c-sr activity detection with TK peptide 2. Biotinylated tyrosine kinase peptide 2 was added to Reacti-Bind NeutrAvidin Coated Plates and incubated for 30 minutes. Wells were washed; samples containing p60c-src tyrosine kinase were added to phosphorylate the tyrosine residue on the peptide. Anti-phosphotyrosine monoclonal antibody conjugated to HRP was added. Tyrosine kinase activity was detected by 1-Step Turbo TMB Substrate. Kinase activity was quantitated by comparison with a standard curve generated using the phosphorylated form of the same peptide substrate.
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Reacti-Bind NeutrAvidin High Binding Capacity (HBC) Coated Plates
Unique technology for improved assay precision.
Pierces new patent-pending plate-coating technology offers a NeutrAvidin HBC Plate with a wider detection limit than our regular binding capacity plates. The standard curve exhibits greater linearity for detecting small biotinylated molecules such as peptides (Figure 9) and oligonucleotides, resulting in greater assay precision. Switch your assay over to Reacti-Bind NeutrAvidin HBC Coated Plates for binding small biotinylated ligands and see the difference for yourself. Highlights: Unique plate-coating technology results in high loading of NeutrAvidin Biotin-Binding Protein/well Improved sensitivity less nonspecific binding for improved signal:noise ratios Broader dynamic range extends the quantitative range so there is no need for dilutions Save time pre-blocked plates to reduce the number of assay steps Flexible assay formats coated plates offered in 96- and 384-well formats and in different colors
10 8 S/N Ratio 6 4 2 0 0 5 10 Biotinylated Phosphopeptide (pM/well) 15 HBC RBC CHC
Reacti-Bind NeutrAvidin HBC Coated Plate Characteristics

Product # Description 15507 Clear 96-Well Plates with SuperBlock Blocking Buffer* 15508 Clear 8-Well Strip Plates with SuperBlock Blocking Buffer* 15509 White 96-Well Plates with SuperBlock Blocking Buffer* 15510 Black 96-Well Plates with SuperBlock Blocking Buffer* 15511 Clear 384-Well Plates with SuperBlock Blocking Buffer* 15512 White 384-Well Plates with SuperBlock Blocking Buffer* 15513 Black 384-Well Plates with SuperBlock Blocking Buffer* U.S. Pkg. Size Price 5 plates $135 5 plates 5 plates 5 plates 5 plates 5 plates 5 plates $182 $146 $146 $285 $285 $285
* U.S. patent pending on high-binding capacity plate-coating technology.
Figure 9. Phosphopeptide detection assay comparison of biotin-binding protein-coated plates. Comparison of NeutrAvidin High Binding Capacity (HBC) Coated Plate, NeutrAvidin Regular Binding Capacity (RBC) Coated Plates and a competitors Streptavidin Coated High Binding Capacity Plates (CHC). Plates were incubated with various dilutions of biotinylated, phosphorylated peptide. After washing, the plates were incubated with mouse anti-phosphotyrosine antibody (1:1,000) and then detected using an anti-mouse-FITC conjugate (1:666). The Y-axis is described as the signal:noise (S/N) ratio.
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Reacti-Bind Streptavidin Coated Plates

The specific binding affinity of streptavidin for biotin in a microplate.
Highlights: Easy and gentle immobilization of biotin-containing conjugates Low nonspecific binding No denaturing of the protein component of a conjugate upon binding Ideal for binding small biotinylated hydrophilic molecules (e.g., peptides) that typically exhibit poor binding to polystyrene Pre-blocked with your choice of Blocker BSA or SuperBlock Blocking Buffer Reacti-Bind Streptavidin Coated Plate Characteristics
Binding Capacity Coat Volume Blocking Volume 96-Well Plate 5 pmoles/well 100 l/well* 200 l/well 384-Well Plate 4 pmoles/well 50 l/well 100 l/well
Product # Description 15120 Clear 8-Well Strip Plates with SuperBlock Blocking Buffer 15121 Clear 8-Well Strip Plates with Blocker BSA 15122 Clear 8-Well Strip Plates with SuperBlock Blocking Buffer 15124 Clear 96-Well Plates with SuperBlock Blocking Buffer 15125 Clear 96-Well Plates with Blocker BSA 15126 Clear 96-Well Plates with SuperBlock Blocking Buffer 15118 White 96-Well Plates with SuperBlock Blocking Buffer 15119 Black 96-Well Plates with SuperBlock Blocking Buffer 15405 Clear 384-Well Plates with SuperBlock Blocking Buffer 15406 White 384-Well Plates with SuperBlock Blocking Buffer 15407 Black 384-Well Plates with SuperBlock Blocking Buffer 15115 Reacti-Bind Biotin Binding Plate Sample Pack
One each of the following plates: Product #s 15120, 15121, 15127 and 15128
U.S. Pkg. Size Price 5 plates $183 5 plates $183 25 plates $717 5 plates $135
5 plates $135 25 plates $559 5 plates 5 plates 5 plates 5 plates 5 plates 4 plates $146 $146 $281 $281 $281 $112
* Product # 15125 is coated with 200 l of streptavidin coating solution. References Estrada, G., et al. (1996). Mol. Cell Probes 10, 179-185. Grobler, J.A., et al. (2002). Proc. Natl. Acad. Sci. 99, 6661-6666. Hong, P. W.-P., et al. (2002). J. Virol. 76, 12855-12865. Sthlinger, M.C., et al. (2001). Circulation 104, 2569-2575. Su, S.V., et al. (2004). J. Biol. Chem. In press.
Reacti-Bind Biotin Coated Plates

A simple format for testing the efficiency of avidin, streptavidin or NeutrAvidin Protein conjugations.
Reacti-Bind Biotin Coated Plates can be used in any immunoassay with NeutrAvidin Biotin-Binding Protein, streptavidin, avidin or other biotin-binding proteins. Highlights: Biotin group accessible for binding avidin, streptavidin or NeutrAvidin Biotin-Binding Protein Pre-blocked to reduce nonspecific binding Strip well plate format for the ultimate in convenience Coat volume: 200 l Blocking volume: 300 l
Product # Description 15151 Reacti-Bind Biotin Coated Clear 8-Well Strip Plates U.S. Pkg. Size Price 5 plates $123
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Reacti-Bind Streptavidin HBC Coated Plates
Take advantage of a Pierce technology that provides a broader dynamic range.
Reacti-Bind Streptavidin High Binding Capacity (HBC) Coated Plates are designed for binding biotinylated oligonucleotides and peptides with higher binding efficiency than other commercially available plates. Pierces proprietary coating technology (patent pending) has created a streptavidin-coated plate with four- to five-times the binding capacity of competitors plates. Using a Reacti-Bind Streptavidin HBC Plate can result in an assay with a broader dynamic range and better linearity, leading to improved assay precision. The figure below demonstrates this effect when measuring fluorescent polymerase chain reaction (PCR) products hybridized to biotinylated oligonucleotides bound to a Reacti-Bind High Binding Capacity (HBC) Plate and a leading competitors high binding capacity plate (CHC). Try the Reacti-Bind Streptavidin HBC Coated Plate and see what has been going undetected in your research.
180 160 140 S/N Ratio 120 100 80 60 40 20 0 0 5 10 15 20 25 Fluoresceinated Oligonucleotide (pM/well) 30 HBC CHC
Highlights: Broader dynamic range extends the quantitative range so theres no need for dilutions Better sensitivity increased binding capacity allows direct detection of small ligands not observed with regular binding capacity plates Superior assay precision standard curve demonstrates greater linearity Save time pre-blocked to reduce number of assay steps Flexible assay formats offered in 96- and 384-well formats and in different colors Reacti-Bind Streptavidin HBC Coated Plate Characteristics
Reference Wilson, D.S., et al. (2001). Proc. Natl. Acad. Sci. 98, 3750-3755.
Product # Description 15500 Clear 96-Well Plates with SuperBlock Blocking Buffer* 15501 Clear 8-Well Strip Plates with SuperBlock Blocking Buffer* 15502 White 96-Well Plates with SuperBlock Blocking Buffer* 15503 Black 96-Well Plates with SuperBlock Blocking Buffer* 15504 Clear 384-Well Plates with SuperBlock Blocking Buffer* 15506 Black 384-Well Plates with SuperBlock Blocking Buffer* 15505 White 384-Well Plates with SuperBlock Blocking Buffer* U.S. Pkg. Size Price 5 plates $146 5 plates 5 plates 5 plates 5 plates 5 plates 5 plates $194 $157 $157 $298 $298 $298
Figure 10. Comparison of Streptavidin High Binding Capacity (HBC) and competitors HBC coated plates. Fluoresceinated oligonucleotide hybridization assay comparison. Comparison of Reacti-Bind Streptavidin High Binding Capacity (HBC) Coated Plate with competing high binding capacity plate (CHC). Plates were incubated with a biotinylated oligonucleotide, washed and probed with a complementary oligonucleotide labeled with fluorescein at various dilutions. The Y-axis is described as the signal:noise (S/N) ratio.
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HisGrab Nickel Coated Plates

Bind recombinantly expressed fusion proteins containing a histidine tag for easy ELISA analysis.
These Ni2+ chelate-coated plates provide a simple format for protein:protein interaction studies.
1.0 Absorbance at 450 nm 0.8 0.6 0.4 0.2 0 0 1.15 3.125 6.25 12.5 ng ATF 25 50 100 Pierce Brand Q
Highlights: Detergents used to lyse cells do not inhibit binding to Ni2+coated plates as they do with plain polystyrene The detection limit is 1 ng of histidine fusion protein Better binding for more sensitive assays compared to other commercially available nickel-activated plates1 Can be custom-made with other metals. Contact Pierce Bulk & Custom Sales or your local distributor Special blocking solution (Product # 37547) improves signal:noise ratio for enhanced assay sensitivity
Product # 15142 15242 15342 Description Clear 8-Well Strip Plates White 96-Well Plates Black 96-Well Plates Pkg. Size 5 plates 5 plates 5 plates U.S. Price $168 $168 $168
Figure 11. Binding comparison of histidine-tagged ATF fusion protein.

References 1. Desai, S., et al. (1997). Previews 1(4), 12-15. Pan, W., et al. (2003). J. Biol. Chem. 278, 27820-27827. Tu, Y., et al. (1999). Mol. Cell. Biol. 19, 2425-2434.
Related Pierce Products: Product # Description 37547 Blocker Metal Chelate Compatible Formulation U.S. Pkg. Size Price 20 ml $ 32
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HisGrab Copper Coated High Binding Capacity (HBC) Plates
Boost assay performance by binding more histidine-tagged protein.
HisGrab Copper Coated High Binding Capacity (HBC) Plates use an exclusive coating process to increase the amount of histidine-tagged protein that will bind to the plate surface. This is ideal for high-throughput screening applications that need improved sensitivity and greater dynamic range.
60,000 50,000 Bound RFU 40,000 30,000 20,000 8.9 pmoles 10,000 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 PHT Fusion Protein Applied, Units 1 HBC Copper Coated Nickel Coated 36.5 pmoles
Highlights: Quantitate previously undetectable histidine-tagged proteins Wider dynamic range with four-fold greater capacity than regular nickel chelate-coated plates Special blocking solution (Product # 37547) improves signal:noise ratio for enhanced assay sensitivity Copper chelate provides greater binding capacity
Product # 15143 15146 15147 15148 Description Clear 96-Well Plates* Clear 8-Well Strip Plates* White 96-Well Plates* Black 96-Well Plates* Pkg. Size 5 plates 5 plates 5 plates 5 plates U.S. Price $186 $197 $186 $186
Related Pierce Products: Product # Description 37547 Blocker Metal Chelate Compatible Formulation U.S. Pkg. Size Price 20 ml $ 32
Figure 12. Binding comparison of a histidine-tagged fluorescent fusion protein to standard HisGrab Nickel Coated and Copper Coated High Binding Capacity (HBC) Plates. HisGrab Copper Coated HBC Plates exhibit a four-fold greater capacity for binding purified polyhistidine-tagged protein when assayed using a 100 l volume. Incubation time was two hours for the binding of polyhistidine-tagged fusion protein.
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Reacti-Bind Glutathione Coated Plates

Bind fusion proteins containing GST.
These plates provide easy quantitation of antibodies raised against GST-fused proteins.
Absorbance at 450 nm
0.8 GST bound to Pierce Glutathione Coated Plate 0.6
Highlights: The lower detection limit is 1 ng of GST fusion protein Simple format for protein:protein interaction studies Detergents used to lyse cells dont inhibit binding to precoated plates as they do with plain polystyrene Pre-blocked with SuperBlock Buffer to reduce nonspecific binding
References McKevitt, M., et al. (2003). Genome Res. 13, 1665-1674. Pullen, S.S., et al. (1999). J. Biol. Chem. 274, 14246-14254. Yarwood, S.J., et al. (1999). J. Biol. Chem. 274, 14909-14917.
0.4 GST bound to non-coated, pre-blocked plate
0.2
0.0 0.0
5.0 10.0 15.0 20.0 25.0 30.0 35.0 Amount of GST applied to each well (ng)
40.0
Product # 15140 15240 15340 Description Clear 8-Well Strip Plates White 96-Well Plates Black 96-Well Plates Pkg. Size 5 plates 5 plates 5 plates U.S. Price $141 $141 $141
Reacti-Bind Anti-GST Coated Plates

A unique alternative to glutathione-coated plates for binding GST fusion proteins.
1.0 Anti-GST (Pierce) Absorbance at 450 nm 0.8 0.6 0.4 0.2 0 0 10 Purified GST Conc. (ng/ml) 1,000
Anti-GST antibodies often provide better binding of GST fusion proteins because protein expressing GST may not fold properly or it may become denatured during the extraction process. This could result in poor binding to the glutathione ligand. Highlights: Lower detection limit is 7.5 ng of GST Binds native or denatured forms of GST; an alternative to glutathione-coated plates Pre-blocked with SuperBlock Buffer Poppers Cell Lysis Reagents will not interfere with GST fusion protein binding
Figure 13. Reacti-Bind Anti-GST Coated Plates were tested for their ability to bind and detect GST. The plates were assayed with purified GST that was detected using a rabbit anti-GST antibody (1 mg/ml) followed by donkey anti-rabbit HRP conjugate. The signal was developed with 1-Step Turbo TMB Substrate (Product # 34022).
Product # Description 15145 Clear 8-Well Strip Plates U.S. Pkg. Size Price 5 plates $141
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CellScreen Coated Plates
Our coated plate product line is growing cells.
The plate-coating experts at Pierce have developed CellScreen Coated Plates, a line of plates for cell-based assays. These coated plates enable firm attachment and efficient growth of cells for cell-based applications such as reporter assays, receptor binding assays, cytotoxicity assays, apoptosis assays and cell proliferation assays. The CellScreen Product Line includes Poly-D-Lysine and Collagen I coatings in 96- and 384-well plate formats. CellScreen Plates are produced using high-quality raw materials and automation-compatible plates and lids for your high-throughput applications. Following Pierces proprietary coating procedure, all CellScreen Plates are treated with a predetermined radiation dose. This validated irradiation process provides a more consistently coated plate product for cell-based assays. Pierce has extensive microplate-coating capabilities using manufacturing and quality assurance equipment designed for efficient production and testing of coated microplates in large quantities. This results in economical pricing for customers. Highlights: High-quality base plate and lid designed for automated systems Post-treated using a validated irradiation process Produced using automated production and quality analysis equipment Reliable for use in high-throughput screening equipment and robotics Highly controlled procedures result in superior lot-to-lot consistency and assurance of quality Economical for high-volume use contact Pierce for a bulk quote Large lot size capability, consistency in production and quality assurance
CellScreen Coated Plate Characteristics

Plate Type Plate Bottom Well Volume Growth Surface Area Lid 96-Well Polystyrene Flat 300 l 3.3 mm2/well Included, contains condensation rings Plate Colors Available Clear and black with clear bottom 384-Well Polystyrene Flat 100 l 1.6 mm2/well Included Clear and black with clear bottom
CellScreen Poly-D-Lysine Coated 96- and 384-Well Plates*
Product # Description 15600 Clear 96-Well Plates 15608 Black with Clear Bottom 384-Well Plates U.S. Pkg. Size Price 5 plates $ 51 5 plates $122
CellScreen Collagen I Coated 96- and 384-Well Plates*
Product # Description 15610 Clear 96-Well Plates 15618 Black with Clear Bottom 384-Well Plates U.S. Pkg. Size Price 5 plates $ 51 5 plates $122
* All CellScreen Coated Plates are available at a special price in bulk quantities of 200 or more plates. Interested in another base-plate color or coating? In addition to our catalog offering, the Pierce ChoiceCoat Custom Plate-Coating Service can custom-coat Poly-D-Lysine, Poly-L-Lysine or Collagen on 96- or 384-well plates. Contact Bulk & Custom Sales for information regarding quantity, availability and pricing.
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Reacti-Bind Maleic Anhydride Plates

Proteins and other primary amine-containing compounds covalently attach to the microplate.
Great for immobilization of compounds that do not normally stick to plain polystyrene plates. Highlights: Spontaneously react with primary amines Maleic anhydride retains its integrity and coupling availability for months
References Batista, F.D. and Neuberger, M.S. (2000). EMBO J. 19, 513-520. Brett, P.J., et al. (2002). J. Biol. Chem. 277, 20468-20476. Dumoutier, L., et al. (2001). J. Immunol. 166, 7090-7095. Leu, S.-J., et al. (2003). J. Biol. Chem. 278, 33801-33808. Liao, Y.-F., et al. (2002). J. Biol. Chem. 277, 14467-14474. Marston, E.L., et al. (2002). Clin. Diagn. Lab. Immunol. 9, 446-452.
Product # 15110 15112 15100 15102 Description Clear 96-Well Plates Clear 96-Well Plates Clear 8-Well Strip Plates Clear 8-Well Strip Plates Pkg. Size 5 plates 25 plates 5 plates 25 plates U.S. Price $ 67 $309 $ 90 $359
Reaction Scheme for Coupling both Large and Small Amine-Containing Molecules O C C ( )n C O C O Maleic Anhydride shown anchored to polystyrene well of a microplate. Both the well wall and base are activated. R H2N R pH 7.2 = Protein, peptide or other ligand O C N R H H C C C O ( )n O Stable amide bond formation with amines, with a resulting hydrophilic well surface.
Reacti-Bind Maleimide Activated Plates

A convenient alternative to amine-reactive chemistries for attaching sulfhydryl-containing compounds.
Maleimide groups specifically and covalently conjugate sulfhydryl groups at neutral pH, creating a stable thioether bond. Highlights: Pre-blocked to reduce nonspecific binding Convenient 8-well strip format Easy (spontaneous) immobilization of peptides derivatized with a terminal cysteine and proteins with free sulfhydryl
References Ostrowski, M., et al. (2002). J. Virol. 76, 4241-4250. Reinhard, M., et al. (1999). J. Biol. Chem. 274, 13410-13418.
Product # Description 15150 Clear 8-Well Strip Plates U.S. Pkg. Size Price 5 plates $111
Reaction Scheme for Coupling both Large and Small Sulfhydryl-Containing Molecules Maleimide shown anchored to polystyrene well of a microplate. Both the well wall and base are activated. SH R pH 6.5-7.5 ( )n R = Protein, peptide or other ligand H Stable thioether bond formation with sulfhydryls, with a resulting hydrophilic well surface. R
O O
N ( )n
O
N S
O
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BLOCKING AND WASHING

Blocking Unoccupied Sites
In an ELISA, it is important to block the unoccupied sites on the surface of the well to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay. A variety of blocking buffers ranging from nonfat milk to highly purified proteins have been used to block unreacted sites. The blocking buffer should improve the sensitivity of the assay by reducing the background interference. An individual blocking buffer will not be compatible with every system; for this reason, a variety of blockers in both Tris buffered saline (TBS) and phosphate buffered saline (PBS) are available. The proper choice of blocker for a given assay depends on the antigen itself and on the type of enzyme conjugate to be used. For example, with applications using an alkaline phosphatase conjugate, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding. For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Many factors can influence nonspecific binding, including various protein:protein interactions unique to a given ELISA. The most important parameter when selecting a blocker is the signal:noise ratio, which is measured as the signal obtained with a sample containing the target analyte as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background and a reduced signal:noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the enzyme, again causing a reduction of the signal:noise ratio. When developing any new ELISA, it is important to test several different blockers for the highest signal:noise ratio in the assay. No single blocking agent is ideal for every occasion because each antibody-antigen pair has unique characteristics. Pierce offers a complete line of blocking buffers for ELISA including StartingBlock, SuperBlock, Casein, BSA, SEA BLOCK and BLOTTO Blocking Buffers. StartingBlock Buffer contains no biotin or serum protein and is compatible with a wide range of detection systems. StartingBlock Buffer works almost immediately on ELISA plates, requiring no incubation step. SuperBlock Blocking Buffer is a highly purified non-serum protein solution that is the ideal blocking agent in many assays. The blocking ability of SuperBlock Buffers and their ability to maintain a high signal:noise ratio is superior to most other formulations. SuperBlock Blocking Buffers also accomplish blocking very quickly, often in as little as 10 minutes. Blocker Casein is a 1% (w/v) ready-to-use solution of Hammersten Grade casein that can be used to block nonspecific sites. Since casein is a purified protein, it is less likely than BLOTTO or other complex protein mixtures to cross-react with the antibodies and cause high background. Blocker BSA is a 10% solution of high-quality bovine serum albumin (BSA). BSA is a commonly used blocking agent for all immunoassay applications. Blocker BSA is concentrated and must be diluted before use. One to three percent BSA solutions are commonly used for blocking nonspecific sites. SEA BLOCK Blocking Buffer is made from steelhead salmon serum. As a non-mammalian protein blocker, the risk of background caused by nonspecific interactions is minimized. Blocker BLOTTO is a ready-to-use blocking buffer made from nonfat dry milk. Nonfat dry milk contains endogenous biotin and should not be used with avidin-biotin systems.
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StartingBlock Blocking Buffer

StartingBlock Blocking Buffer simplifies the selection of a blocker for ELISA applications.
Although no blocking buffer is ideal for every system, you can improve the odds dramatically with StartingBlock Blocking Buffer because it is compatible with the widest variety of antibodies. For example: StartingBlock Blocking Buffers are compatible with biotin-containing systems, while milk-based protein blockers interfere. StartingBlock Buffers do not cross-react with rabbit antibodies, while many other blockers do. StartingBlock Blocking Buffers are also free of potentially interfering serum proteins. StartingBlock Blocking Buffers offer a high level of performance regardless of the system you choose for your ELISA or Western blotting application. In fact, they may be the only blockers you ever use. Highlights: Compatible with a wide range of detection systems Works in both ELISA and Western applications Does not cross-react with rabbit antibodies Serum protein-free Biotin-free Shorter blocking times No-wait blocking capability Superior signal:noise ratios in ELISA applications Signal:noise ratios in the range of 10:1-20:1 have been realized with StartingBlock Blocking Buffer
Product # Description 37538 StartingBlock (PBS) Blocking Buffer
A protein-based blocker formulation in phosphate buffered saline (pH 7.5) for use in ELISA and Western blotting applications.
U.S. Pkg. Size Price 1 liter $127
37542
StartingBlock (TBS) Blocking Buffer

A protein-based blocker formulation in Tris buffered saline (pH 7.5) for use in ELISA and Western blotting applications.
1 liter
$127
Starting Block Blocking Buffers are also available with an optimized amount of Tween-20 Detergent to provide the lowest background.
Product # Description 37539 StartingBlock T20 (PBS) Blocking Buffer
A protein-based blocker formulation in phosphate buffered saline at pH 7.5 with 0.05% Tween-20 and Kathon Antimicrobial Agent.
U.S. Pkg. Size Price 1 liter $139
37543
StartingBlock T20 (TBS) Blocking Buffer

A protein-based blocker formulation in Tris buffered saline at pH 7.5 with 0.05% Tween-20 and Kathon Antimicrobial Agent.
1 liter
$139
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SuperBlock Blocking Buffers
Guaranteed to be biotin-free.
Our most popular blocking buffer, SuperBlock Blocking Buffer, now comes in both dry and liquid formats! Many researchers have discovered that SuperBlock Blocking Buffer is the only blocking buffer needed for all of their applications. Highlights: Fast blocking blocks ELISA plates in two minutes or membranes in five to 10 minutes Non-serum protein solution yields a very high signal:noise ratio Plates blocked with SuperBlock Blocking Buffer can be stored dry for up to 12 months Liquid formulations available in PBS or TBS Biotin-free
Product # Description 37515 SuperBlock (PBS) Blocking Buffer 37535 SuperBlock (TBS) Blocking Buffer U.S. Pkg. Size Price 1 liter $118 1 liter $118
SuperBlock Blocking Buffers are also available with an optimized amount of Tween-20 Detergent to provide the lowest background.
Product # Description 37516 SuperBlock T20 (PBS) Blocking Buffer
(Contains 0.05% Tween-20)
U.S. Pkg. Size Price 1 liter $129 1 liter $129
37536
SuperBlock T20 (TBS) Blocking Buffer

(Contains 0.05% Tween-20)
SuperBlock Dry Blend (TBS) Blocking Buffer

Delivers the ultimate in space-saving convenience.
Highlights: Delivers even more economy and stability Each pouch reconstitutes to form 200 ml of SuperBlock Blocking Buffer in TBS Room-temperature storage; small packaging takes up minimal shelf space
References Ikeda, K., et al. (2003). J. Biol. Chem. 278, 7725-7734. Leclerc, G.J. and Barredo, J.C. (2001). Clin. Cancer Res. 7, 942-951. Subbarayan, V., et al. (2001). Cancer Res. 61, 2720-276. Walters, R.W., et al. (2002). Cell 100, 789-799.
Product # Description 37545 SuperBlock (TBS) Blocking Buffer Dry Blend Blocking Buffer
Each pouch yields 200 ml when reconstituted.
U.S. Pkg. Size Price 5 pouches $ 98
SEA BLOCK Blocking Buffer

No mammalian proteins, reducing the risk of nonspecific interaction.
Highlights: Made from steelhead salmon serum Functions as a universal blocker Offers reduced background Can be diluted up to 1:10 with buffer
References Hypolite, J.A., et al. (2001). Amer. J. Physiol. Cell. Physiol. 280, C254-264. Wang, L., et al. (2002). J. Clin. Invest. 110, 1175-1184.
Product # Description 37527 SEA BLOCK Blocking Buffer U.S. Pkg. Size Price 500 ml $121
26
Blocker Casein
Ready-to-use solution (1% w/v) of Hammersten Grade casein for blocking nonspecific sites.
Highlights: Preformulated for ease of use Use when skim milk demonstrates high background problems Thimerosal-free formulation
References Nemzek, J.A., et al. (2000). Amer. J. Physiol. Lung Cell. Mol. Physiol. 278, L512-520. Stuyver, L.J., et al. (2003). Antimicrob. Agents Chemother. 47, 244-254. Wolfman, J.C., et al. (2002). Mol. Cell. Biol. 22, 1589-1606.
Product # Description 37532 Blocker Casein in TBS
1% (w/v) Casein Hammersten Grade in TBS, Contains Kathon Antimicrobial Reagent as preservative, pH 7.4.
U.S. Pkg. Size Price 1 liter $ 83
37528
Blocker Casein in PBS

1% (w/v) Casein Hammersten Grade in PBS, Contains Kathon Antimicrobial Reagent as preservative, pH 7.4.
1 liter
$ 83
Blocker BLOTTO
Ready-to-use blocking buffers made of nonfat dry milk.
Highlights: Preformulated for ease of use Anti-foaming agent added Available in TBS Buffer Merthiolate-free formulation
References Goretzki, L., et al. (2000). J. Biol. Chem. 275, 28625-28633. Abrams, E.T., et al. (2003). J. Immunol. 170, 2759-2764.
Product # Description 37530 Blocker BLOTTO in TBS
5% (w/v) nonfat powdered milk in TBS, 0.01% Anti-foam A, contains Kathon Antimicrobial Reagent as preservative, pH 7.4.
U.S. Pkg. Size Price 1 liter $ 79
Blocker BSA
For all blocking applications.
Highlights: 10% solutions of high-quality bovine serum albumin Concentrated formulation saves storage space No waiting for powder to dissolve with this ready-to-dilute liquid concentrate
Product # Description 37525 Blocker BSA in PBS (10X) 37520 Blocker BSA in TBS (10X) U.S. Pkg. Size Price 200 ml $102 125 ml $ 98
27

Surfact-Amps 20 Purified Detergent Solution
Specially purified form of Tween-20 Detergent.
Highlights: Guaranteed < 1 milliequivalent of peroxides and carbonyl in a 10% solution Enhances signal:background ratio
Reference Ebong, S.J., et al. (2001). Infect. Immun. 69, 2099-2106.
Product # Description 28320 Surfact-Amps 20 Purified Detergent Solution U.S. Pkg. Size Price 6 x 10 ml $ 82
Blocker Metal Chelate Compatible Formulation

Designed to improve assay sensitivity for metal chelate-based assay systems.
Highlights: Improves assay sensitivity when using HisGrab Metal Chelate High Binding Capacity (HBC) and regular HisGrab Metal Chelate Coated plates Use at a 1:1,000 dilution to dilute assay reagents for best signal:noise ratio Thimerosal-free formulation
Product # Description 37547 Blocker Metal Chelate Compatible Formulation
Use concentrate at 1:1,000 dilution, pH 7.4.
U.S. Pkg. Size Price 20 ml $ 32
Blocking Buffers Application Chart Product # 37538 37542 37539 37543 37515 37535 37516 37536 37527 37520 37525 37532 37528 37530 37547 Blocking Buffer StartingBlock (PBS) Blocking Buffer StartingBlock (TBS) Blocking Buffer StartingBlock T20 (PBS) Blocking Buffer StartingBlock T20 (TBS) Blocking Buffer SuperBlock Blocking Buffer in PBS SuperBlock Blocking Buffer in TBS SuperBlock T-20 PBS Blocking Buffer SuperBlock T-20 TBS Blocking Buffer SEA BLOCK Blocking Buffer Blocker BSA in TBS Blocker BSA in PBS Blocker Casein in TBS Blocker Casein in PBS Blocker BLOTTO in TBS Blocker Metal Chelate ELISA Western blot Dot Blot Immunohistochemistry DNA/RNA Hybridizations U.S. Price $127 $127 $139 $139 $118 $118 $129 $129 $121 $ 98 $102 $ 83 $ 83 $ 79 $ 32
28
Surfact-Amps Purified Detergent Solutions

Nothing could be easier nothing protects better!
Highlights: Disrupts nonspecific protein interactions Ampules are packed under nitrogen for maximum shelf life and are pre-scored for easy opening Gentle non-denaturing at low concentrations Clear and pure 10% solutions less than 1.0 eq/ml peroxides and carbonyl content Unparalleled convenience solutions are purified, prediluted and ampuled
Product # Description Tween-20-Based Detergents 28320 28328 Surfact-Amps 20
(Active Ingredient: Tween-20)
U.S. Pkg. Size Price
6 x 10 ml $ 82 6 x 10 ml $ 82
Surfact-Amps 80
(Active Ingredient: Tween-80)
Triton-based Detergents 28314 28332 Surfact-Amps X-100

(Active Ingredient: Triton X-100)
6 x 10 ml $ 82 6 x 10 ml $192
Surfact-Amps X-114
(Active Ingredient: Triton X-114)
Nonidet-based Detergent 28324 Surfact-Amps NP-40

(Active Ingredient: Nonidet P-40)
6 x 10 ml $ 82
Brij-based Detergents 28316 28336 20150 Surfact-Amps 35

(Active Ingredient: Brij-35)
6 x 10 ml $ 82 6 x 10 ml $119 950 ml $ 75
Surfact-Amps 58
(Active Ingredient: Brij-58)
Brij -35, 30% Solution
Active ingredient is supplied as a purified 10% aqueous solution ampuled under nitrogen.
Surfact-Pak Detergent Sampler

Convenient 10-sample package of detergents allows for testing and experimentation.
Product # Description 28340 Surfact-Pak Detergent Sampler
Contains: Surfact-Amps Purified Detergents Surfact-Amps X-100 Surfact-Amps 35 Surfact-Amps 20 Surfact-Amps NP-40 Surfact-Amps 80 Surfact-Amps X-114 Surfact-Amps 58 Octyl -Glucoside Octyl -Thioglucopyranoside CHAPS
U.S. Pkg. Size Price Kit $239

10 ml 10 ml 10 ml 10 ml 10 ml 10 ml 10 ml 100 mg 100 mg 100 mg
29

CHAPS & CHAPSO
Zwitterionic detergents that are ideal for protecting the native state of proteins.
OH O N H N
+
Product # Description 28300 CHAPS
O HO S O O
U.S. Pkg. Size Price 5g $ 99 100 g 5g $829 $209
(3-[(3-Cholamidopropyl)dimethylammonio]-1propanesulfonate)
HO
OH
CHAPS
M.W. 614.88
CHAPSO
M.W. 630.88
28299 28304
CHAPS CHAPSO
(3-[(3-Cholamidopropyl)dimethylammonio]-2hydroxy-1-propanesulfonate)
Highlights: Non-denaturing Able to disrupt nonspecific protein interactions Less protein aggregation than nonionic detergents Electrically neutral
Washing the Microplate

Many immunoassay procedures include a series of incubations with different immunochemical reagents separated by wash steps. Washing steps are necessary to remove unbound reagents and reduce background, thereby increasing the signal:noise ratio. Insufficient washing will allow high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the well. A variety of buffers may be used. Occasionally, washing is performed in a physiologic buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS) without any additives. More commonly, a detergent such as 0.05% Tween-20 (Product # 28320) is added to the buffer to help remove nonspecifically bound material. Another common technique is to use a dilute solution of the blocking buffer along with some added detergent. Including the blocking agent and adding a detergent in wash buffers helps to minimize background in the assay. For best results, use high-purity detergents, such as Surfact-Amps Detergents.
BupH Dry Buffers The most advanced, versatile, time-saving buffer product line available. The ultimate in convenience 1. Reach for the sealed foil pack sitting conveniently on your bench top. 2. Open, pour into beaker and add water. 3. The fresh buffer is ready to use in practical aliquots so theres no waste. The ultimate in versatility 1. Routine buffers are designed for use in Western blotting, dialysis, cross-linking, ELISAs, immunohistochemistry, protein plate-coating, biotinylation and other applications. 2. Using one buffer source maintains consistency and eliminates variables within the lab. The ultimate in integrity 1. BupH Buffers are protected from contamination and are fresh every time. 2. Carry out applications with confidence in buffer quality. 3. Test-assured with the Pierce commitment to quality management standards. The ultimate in time savings 1. Making routine buffers is no longer time-consuming. 2. No component measurement, pH adjustment, quality validation, preparation tracking or refrigeration hassles. 3. Move forward with your work by eliminating re-tests due to buffer problems.
30
BupH Phosphate Buffered Saline Packs

Great wash buffer!
Each pack yields 500 ml of 0.1 M phosphate, 0.15 M NaCl, pH 7.0 when dissolved in 500 ml deionized water (20 liters total).
Product # Description 28372 BupH Phosphate Buffered Saline Packs U.S. Pkg. Size Price 40 pack $109
BupH Tris Buffered Saline

Great wash buffer!
Each pack yields 500 ml of 25 mM Tris, 0.15 M NaCl, pH 7.2 when dissolved in 500 ml deionized water (10 pack makes 5 liters total; 40 pack makes 20 liters total).
Product # 28380 28376 28379 Description BupH Tris-Glycine Buffer Packs BupH Tris Buffered Saline Packs BupH Tris Buffered Saline Packs Pkg. Size 40 pack 40 pack 10 pack U.S. Price $ 98 $109 $ 49
Surfact-Amps 20 Purified Detergent Solution

Specially purified form of Tween -20.
Highlights: Can be added to PBS or TBS wash buffers to improve performance Guaranteed < 1 milliequivalent of peroxides and carbonyl in a 10% solution Enhances signal:background ratio
Product # Description 28320 Surfact-Amps 20 U.S. Pkg. Size Price 6 x 10 ml $ 82
31
PRIMARY AND SECONDARY
ANTIBODIES
host species may be chosen. Another option to reduce background is to use a secondary antibody that has been pre-adsorbed to serum proteins from other species. This pre-adsorption process removes antibodies that have the potential to cross-react with serum proteins, including antibodies, from those species. To expedite the process of choosing the appropriate secondary antibody, visit the Secondary Antibody Selection Guide located under the Products tab on the Pierce web site. Antibodies for ELISA are typically used as dilute solutions, ranging from 1/100-1/500,000 dilutions beginning from a 1 mg/ml stock solution. The optimal dilution of a given antibody with a particular detection system must be determined experimentally. More sensitive detection systems require that less antibody be used, which can result in substantial savings on antibody costs and allow a limited supply of antibody to be stretched out over more experiments. It also produces a side benefit of reduced background because the limited amount of antibody shows increased specificity for the target with the highest affinity. Antibody dilutions are typically made in the wash buffer containing a blocking agent. The presence of a small amount of blocking agent and detergent in the antibody diluent often helps to minimize background. Pierce offers a wide variety of ImmunoPure Labeled Secondary Antibodies for use in ELISA. The labels include biotin, fluorescein, rhodamine, horseradish peroxidase and alkaline phosphatase. For the complete list of labeled secondary antibodies please refer to pages 36-38.
Affinity-Purified Primary Antibodies

The choice of a primary antibody will depend on the antigen to be detected and what antibodies are available to that antigen. A huge number of primary antibodies are available commercially and can be identified quickly by searching sites such as www.antibodyresource.com on the Internet. Alternatively, a primary antibody may be made to recognize the antigen of interest. For more information on producing a custom antibody, see the Antibody Production and Purification technical section of the Pierce Technical Handbook and Catalog. Both polyclonal and monoclonal antibodies work well for ELISA. Polyclonal antibodies are less expensive and less time-consuming to produce and they often have a high affinity for the antigen. Monoclonal antibodies are valued for their specificity, purity and consistency that result in lower background. Crude antibody preparations such as serum or ascites fluid are sometimes used for ELISA, but the impurities present may increase background. To obtain antibodies with the greatest specificity, they can be affinitypurified using the immobilized antigen. For more information on affinity purification, order your FREE Affinity Purification Handbook from a Pierce Customer Service representative at 800-874-3723 or 815-968-0747, or from your local Perbio Branch office or distributor. A wide variety of labeled secondary antibodies can be used for ELISA detection. The choice of secondary antibody depends upon the species of animal in which the primary antibody was raised (the host species). For example, if the primary antibody is a mouse monoclonal antibody, the secondary antibody must be an anti-mouse antibody obtained from a host other than the mouse. The host species of the secondary antibody often will not affect the experiment. However, secondary antibodies are available from several different host species and, if a secondary antibody causes high background in a particular assay, another
32
Antibody Labels The choice of secondary antibody also depends upon the type of label that is desired. Many different labels can be conjugated to antibodies. Radioisotopes were used extensively in the past, but they are expensive, have a short shelf life, offer no improvement in signal:noise ratio and require special handling. Alternative labels are biotin, fluorophores and enzymes. The use of fluorophores requires fewer steps; however, special equipment is needed to view the fluorescence. Also, a photograph must be taken if a permanent record of the results is desired. Enzymatic labels are used most commonly and, although they require extra steps, they can also be extremely sensitive. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes that are used extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme. For a detailed comparison of these two enzymes, see Table 3. AP, a 140 kDa protein that is generally isolated from calf intestine, catalyzes the hydrolysis of phosphate groups from a substrate molecule, resulting in a colored or fluorescent product or the release of light as a byproduct. AP has optimal enzymatic activity at a basic pH (pH 8-10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. As a label for ELISA, AP offers a distinct advantage over other enzymes. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period. HRP is a 40 kDa protein that catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product or the release of light as a byproduct. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnover rate, good stability, low cost and wide availability of substrates make HRP the enzyme of choice for most applications.
Table 3. Comparison of horseradish peroxidase and alkaline phosphatase enzymes. Size Price Stability (Storage) Number of Substrates Kinetics pH optimum Horseradish Peroxidase 40 kDa Relatively Inexpensive Stable at < 0C Many Rapid 5-7 Alkaline Phosphatase 140 kDa Relatively Expensive Unstable at < 0C Few Slower 8-10
Antibody Conjugates and Enzyme Detection

After blocking, the primary antibody is incubated in the microplate, allowing it to bind to the antigen. The time required for this step typically ranges from 1 hour at room temperature to overnight at 4C, depending upon the affinity of the antibody for its antigen. The primary antibody may have been conjugated (labeled) with an enzyme or biotin molecule to create a direct ELISA. Care must be taken that the antibody conjugate is stored properly and that it has been diluted in the blocking buffer with or without Tween-20 Detergent when performing the ELISA. Peroxidase-conjugated antibodies may be stored in Guardian Peroxidase Conjugate Stabilizer (Product # 37548) to a 1:1,000 dilution or to the desired assay dilution range. This stabilizer prevents the loss of enzyme activity over time, removes the need to aliquot and freeze conjugates, and allows the antibody conjugate to be stored at 4C. An alternative to the direct method is to use a labeled secondary antibody that recognizes the primary antibody species. For example, a goat anti-mouse secondary antibody will bind to a mouse monoclonal primary antibody. Many secondary antibody conjugates are commercially available with common enzyme labels such as AP or HRP. If the primary antibody has a biotin tag, then the secondary reagent would be a streptavidin or NeutrAvidin Enzyme Conjugate, with an alternative being the avidin-biotin complex (ABC) with a biotinylated enzyme (Figure 1, page 5). Selecting the appropriate protocol for an ELISA depends upon the availability of the primary antibody, the sensitivity level desired and the nature of the sample being tested. Pierce provides a variety of secondary antibodies, NeutrAvidin Protein and streptavidin conjugates to choose from in designing an ELISA with low background and high reproducibility.
PRIMARY & SECONDARY
ANTIBODIES
33
ANTIBODIES
The final stage in all ELISA systems is a detection step in which a substrate is introduced, and the bound enzyme (indicating the presence of analyte) converts that substrate to a detectable product. The intensity of the signal produced when the substrate is added should correlate to the concentration of the primary antibody and the respective antigen. Enzyme-labeled reagents may be detected using chromogenic, chemifluorescent or chemiluminescent substrates. When performing ELISAs, a soluble substrate is used to generate a signal in solution. Chemiluminescent signals emit light that is measured by a luminometer while fluorescent substrates require an excitation for light to be emitted and detected by a fluorometer (Table 4).
Enzyme-antibody conjugates often yield best results when diluted in the same material that was used for blocking with the addition of high-quality Tween-20 Detergent to a concentration of 0.05%. Figure 14 compares SuperBlock Blocking Buffer with Tween-20 Detergent to other proteins for use as conjugate diluents. It has been demonstrated that Tween-20 Detergent, as a component of the primary and secondary antibody diluents, often provides increased signal:noise ratios. Pierce Surfact-Amps 20 (Product # 28320) is a highly purified Tween-20 Detergent solution, free of peroxides and carbonyls that can cause ELISA artifacts.
1.25 Absorbance at 490 nm 1.00 0.75 0.50 0.25 0.0 SuperBlock Blocking Buffer with .05% Tween-20 Detergent 3% BSA 10% Skim Milk
Background Levels
Figure 14. SuperBlock Blocking Buffers vs. other proteins for use as conjugate diluents. SuperBlock Blocking Buffers with 0.05% Tween-20 Detergent is the superior diluent. Table 4. Choosing a detection method. ELISA Colorimetric Substrates Medium/low sensitivity Generally less expensive Many substrates available Slow signal generation Enzyme catalyzed quickly Small linear range/poor low-end linearity Flexible (stopped, nonstopped and kinetic assays) Spectrophotometer provides quantifiable results Chemifluorescent Substrates High sensitivity Generally more expensive Few substrates available Rapid signal generation Enzyme activity maintained Large linear range/enhanced low-end linearity Flexible (stopped, nonstopped and kinetic assays) Fluorometer provides quantifiable results Chemiluminescent Substrates High sensitivity Generally more expensive Few substrates available Rapid signal generation Enzyme catalyzed quickly Large linear range/enhanced low-end linearity Nonflexible Luminometer provides quantifiable results
Detection Method
34
Affinity-Purified Secondary Antibodies

ImmunoPure Affinity-Purified Antibodies are available unconjugated or conjugated with biotin, alkaline phosphatase, horseradish peroxidase, fluorescein or rhodamine. F(ab)2 fragments of antibodies to immunoglobulins are also available in unconjugated or conjugated forms. These F(ab)2 fragments of antibodies are especially useful in assays in which binding between the Fc portions of antibodies and Fc receptor-bearing cells must be eliminated. ImmunoPure Polyclonal Antibodies are purified by immunoaffinity chromatography using antigens coupled to agarose gels. Affinity purification helps to eliminate nonspecific antibodies, resulting in an increase in sensitivity and specificity, with a decrease in background. The purification process involves an elution procedure, yielding antibodies with high avidity. These antibodies exhibit both maximal binding to antigens and minimal cross-reactivity to other molecules. Conjugated antibodies are affinity-purified prior to the conjugation process. Selected ImmunoPure Antibodies have been further purified by passing them through immobilized serum proteins from other species. This additional processing minimizes cross-reactivities to other species serum proteins and is indicated by min x Species Sr Prot. The key to abbreviations for the individual species is shown in Table 5.
Table 5. Key to abbreviations for individual species. Bv = Bovine Ch = Chicken Gt = Goat Gu = Guinea Pig Ha = Hamster Hn = Human Hs = Horse Ms = Mouse Rb = Rabbit Rt = Rat Sh = Sheep Sw = Swine
ImmunoPure Polyclonal Conjugated Antibodies contain bovine serum albumin as a stabilizer. Table 6 lists the typical working dilutions for the conjugated antibodies when doing ELISAs.
Table 6. Typical dilution ranges recommended for Pierce ImmunoPure Polyclonal Conjugated Antibodies when doing ELISAs. Conjugate Alkaline Phosphatase Peroxidase ELISA 1:5,000-1:50,000 1:5,000-1:200,000 (for SuperSignal ELISA Products)
Storing Enzyme Conjugates Pierce provides a variety of reagents to help preserve enzyme conjugate activity. Typically, conjugates are aliquoted in 50-100 l increments using purified ethylene glycol (Product # 29810) as a preservative for -20C storage. Conjugates can maintain activity for up to two years. An alternative to aliquoting is to use SuperFreeze Peroxidase Conjugate Stabilizer (Product # 31503), diluting the conjugate 1:1 in the stabilizer and storing it at -20C for up to one year as a stock solution. Guardian Peroxidase Stabilizer/Diluents (Product #s 37548 and 37552) allow peroxidase conjugates to be reconstituted and stored at 4C as a 1:1,000 dilution or a 1:100,000 dilution stock solution. Conjugate Stabilizers
Product # Description 37548 Guardian Peroxidase Conjugate Stabilizer/Diluent 37552 Guardian Peroxidase Conjugate Stabilizer/Diluent 31503 SuperFreeze Peroxidase Conjugate Stabilizer 29810 Ethylene Glycol
(50% aqueous solution)
U.S. Pkg. Size Price 200 ml $ 73 1 liter 25 ml 200 ml $223 $ 49 $ 75
PRIMARY & SECONDARY
ANTIBODIES
35

ANTIBODIES
Host Rabbit Donkey Mouse Rabbit Rabbit Rabbit Rabbit Product # Pkg. Size/Price Unconj. Biotin-LC 31104 31720 2 mg/$113 1.5 ml/$123 31108 1.5 mg/$91 31107 31730 1.5 mg/$117 1 ml/$144 31105 31732 2 mg/$106 1.5 mg/$112 31153 31753 2 mg/$122 1.5 ml/$138 31133 31733 2 mg/$114 1.5 ml/$146 31109 0.5 mg/$113 31115 1.5 mg/$93 31120 2 mg/$77 31116 2 mg/$78 31130 2 mg/$81 31118 0.5 mg/$77 31119 1.5 mg/$84 31122 2 mg/$101 31132 1.5 mg/$93 31123 1.5 mg/$93 31136 2 mg/$90 31124 0.5 mg/$89 31140 2 mg/$95 31128 2 mg/$109 31129 0.5 mg/$84 31131 0.5 mg/$90 31135 2 mg/$110 31750 1.5 mg/$114 31587 1.5 mg/$93 31760 1.5 mg/$112 31770 31529 1.5 mg/$112 2 mg/$87 31652 1.5 mg/$90 Fluorescein Rhodamine 31501 1.5 mg/$106 Peroxidase 31401 1.5 ml/$135 Alk. Phos. 31512 1 mg/$117 31509 31650 1.5 mg/$98 1.5 mg/$99 31553 1.5 mg/$116 31533 1.5 mg/$116 31400 1 ml/$149 31402 1.5 ml/$125 31403 1.5 ml/$148 31433 1.5 ml/$144
Specificity Anti-CHICKEN Anti-GOAT
Description Chicken IgY (H+L) Goat IgG (H+L) Goat IgG (H+L) (min x HnMsRb Sr Prot)* Goat IgG (H+L) Goat IgG [F(ab)2] Goat IgG (Fc)
Anti-GOAT F(ab)2 Fragment of Host Antibody Anti-HAMSTER
Goat IgG (H+L) (min x Hn Sr Prot)* Hamster IgG (H+L) Hamster IgG (H+L)
31300 1 ml/$156 31405 1 ml/$138 31337 1 ml/$178 31302 0.5 ml/$162
Goat Rabbit Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Mouse
Anti-HORSE Anti-HUMAN
Horse IgG (H+L) Human IgG (H+L) Human IgG Gamma Chain Specific Human IgG (H+L) (min x BvHsMs Sr Prot)* Human IgG [F(ab)2] Human IgG [F(ab)2] (min x BvHsMs Sr Prot)* Human IgG (Fc) (min x BvHsMs Sr Prot)* Human IgM (Fc5) Human IgM () Human IgA () Human IgA + IgG + IgM (H+L) Human Kappa Chain Human Lambda Chain Human IgG (H+L) (min x Ms Sr Prot)*
31656 2 mg/$94
31410 2 ml/$116
31310 1 ml/$144
31774 1.5 ml/$114
31531 1.5 mg/$82
31412 1.5 ml/$120 31312 1 ml/$170 31414 1.5 ml/$152 31416 1.5 ml/$154 31415 2 ml/$151
31575 2 mg/$119 31778 0.5 mg/$120 31577 2 mg/$98 31782 2 ml/$144 31780 0.5 mg/$120
31417 2 ml/$139 31418 2 ml/$139
31314 1 ml/$159 31316 1 ml/$159
31420 1.5 ml/$121
*See Table 5 on page 35 for the Key to Abbreviations.
36
Product # Pkg. Size/Price Unconj. Biotin-LC 31137 31784 1.5 mg/$104 1 ml/$128 31143 31786 2 mg/$99 1.5 ml/$113 31142 31789 2 mg/$101 1.5 ml/$137 31163 1 mg/$117 31165 1 mg/$84 31539 1 mg/$111 31169 1 mg/$143 31171 2 mg/$142 31160 2 mg/$84 31164 1.5 mg/$79 31166 2 mg/$109 31168 2 mg/$99 31170 1.5 mg/$95 31172 2 mg/$114 31176 1.5 mg/$111 31182 2 mg/$90 31184 1.5 mg/$110 31181 1.5 mg/$89 31188 2 mg/$104 31190 1.5 mg/$99 31192 2 mg/$105 31194 2 mg/$106 31196 2 mg/$138 31198 2 mg/$124 31185 1 mg/$118
Specificity Anti-HUMAN continued
Description Human IgG (H+L) (min x BvHsMs Sr Prot)* Human IgG (H+L) Human IgG (Fc)
Host Mouse Rabbit Rabbit Goat
Fluorescein
Rhodamine
Peroxidase
Alk. Phos.
31535 1.5 mg/$104
31423 1.5 ml/$151
31318 1 ml/$158
Anti-HUMAN F(ab)2 Fragment of Host Antibody
Human IgG (Fc)
Human IgG (H+L) Human IgA + IgG + IgM (H+L) Mouse IgA () (min x Hn Sr Prot)* Mouse IgA + IgG + IgM (H+L) Mouse IgG (H+L) Mouse IgG (H+L) (min x BvHnHs Sr Prot)* Mouse IgG [F(ab)2] Mouse IgG (Fc) Mouse IgG (Fc) (min x BvHnHs Sr Prot)* Mouse IgM () Mouse IgM () (min x BvHnHs Sr Prot)* Mouse IgG + IgM (H+L) Mouse IgG + IgM (H+L) (min x BvHnHs Sr Prot)* Mouse IgG (H+L) Mouse IgG (H+L) Mouse IgG (H+L) (min x Hn Sr Prot)* Mouse IgG [F(ab)2] Mouse IgG (Fc) Mouse IgM () Mouse IgG + IgM (H+L) Mouse IgG (H+L) (min x BvHnHs Sr Prot)*
Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Goat Horse Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Goat
Anti-MOUSE
31800 2 ml/$109 31802 1.5 mg/$125 31803 2 ml/$144 31805 2 ml/$138
31569 2 mg/$95 31541 1.5 mg/$101 31543 2 mg/$113 31547 2 mg/$114
31660 2 mg/$99 31661 1.5 mg/$99
31663 2 mg/$111
31804 31992 0.5 mg/$114 2 mg/$139 31585 1.5 mg/$154 31807 31586 2 ml/$138 1.5 mg/$128
31662 2 mg/$138 31664 1.5 mg/$159
31430 2 ml/$131 31432 1.5 ml/$128 31436 2 ml/$139 31437 2 ml/$148 31439 1.5 ml/$167 31440 2 ml/$168
31320 1 ml/$156 31322 1 ml/$161 31324 1 ml/$167 31325 1 ml/$162 31327 1 ml/$185 31326 1 ml/$207
31444 2 ml/$135 31446 1.5 ml/$167
31328 1 ml/$167 31330 1 ml/$215
31806 1.5 mg/$109 31810 1.5 ml/$120 31812 1 ml/$125 31811 1.5 ml/$157 31813 1.5 ml/$151 31814 1.5 ml/$167 31815 1.5 ml/$146
31561 1.5 mg/$87
31665 1.5 mg/$82
31559 1.5 mg/$106 31555 1.5 mg/$100 31557 1.5 mg/$124 31558 1.5 mg/$117 31565 1 mg/$140
31450 1.5 ml/$132 31452 1 ml/$138 31666 31451 1.5 mg/$106 1.5 ml/$161 31455 1.5 ml/$156 31456 1.5 ml/$171 31457 1.5 ml/$143 31438 0.5 ml/$148
31329 1 ml/$139 31334 0.5 ml/$157 31331 1 ml/$158 31332 1 ml/$158 31333 1 ml/$212 31335 1 ml/$145
PRIMARY & SECONDARY
ANTIBODIES
37

ANTIBODIES
Host Goat Product # Pkg. Size/Price Unconj. Biotin-LC 31178 1 mg/$147 31186 1 mg/$149 Fluorescein Rhodamine Peroxidase Alk. Phos. 31442 0.5 ml/$166 31448 0.5 ml/$134 31458 0.5 ml/$114 31460 2 ml/$135 31462 1.5 ml/$161 31461 2 ml/$148 31463 2 ml/$149 31464 1 ml/$148
Specificity Anti-MOUSE F(ab)2 Fragment of Host Antibody
Description Mouse IgM ()
Anti-RABBIT
Mouse IgM () Goat (min x BvHnHs Sr Prot)* Mouse IgG + IgM Goat (H+L) (min x BvHnHs Sr Prot)* Rabbit IgG (H+L) Donkey (min x BvGtHnHsMsRtSh Sr Prot)* Rabbit IgG (H+L) Goat Rabbit IgG (H+L) (min x Hn Sr Prot)* Rabbit IgG [F(ab)2] Rabbit IgG (Fc) Rabbit IgG (H+L) (min x GtHnMsSh Sr Prot)* Rabbit IgG (H+L) Goat Goat Goat Mouse Goat
Anti-RABBIT F(ab)2 Fragment of Host Antibody
31210 2 mg/$82 31212 1.5 mg/$94 31234 2 mg/$82 31216 2 mg/$94 31213 31824 1.5 mg/$115 1 ml/$138 31214 1 mg/$88 31217 1 mg/$132 31220 2 mg/$94
31821 0.5 ml/$109 31820 1.5 mg/$114 31822 1.5 ml/$114 31823 2 ml/$121
31568 0.5 mg/$93 31670 2 mg/$112 31583 1.5 mg/$106 31573 2 mg/$114
31345 0.5 ml/$143 31340 1 ml/$157 31342 1 ml/$152 31343 1 ml/$159 31341 1 ml/$156
31584 1 mg/$117 31579 1 mg/$120 31581 1 mg/$143 31629 2 mg/$94
31674 1 mg/$114
Rabbit IgG (Fc) Anti-RAT Rat IgG (H+L) Rat IgG [F(ab)2] Rat IgG (Fc) Rat IgM () Rat IgG (H+L) Rat IgG (H+L) (min x Ms Sr Prot)* Rat IgG (H+L)
Goat Goat Goat Goat Goat Rabbit Rabbit Rabbit
31830 2 ml/$109
31680 2 mg/$94
Anti-RAT F(ab)2 Fragment of Host Antibody
31226 2 mg/$92 31228 2 mg/$128 31218 2 mg/$95 31219 0.5 mg/$123 31227 1 mg/$101
31833 2 ml/$125 31832 2 ml/$152 31834 1.5 mg/$100 31836 0.5 mg/$128
31621 2 mg/$102 31631 2 mg/$139
31470 2 ml/$112 31474 2 ml/$128 31475 2 ml/$137 31476 2 ml/$190
31350 1 ml/$137
31353 1 ml/$146 31354 1 ml/$207
Anti-SHEEP
Rat IgG + IgM (H+L) Rat IgG (H+L) (min x Ms Sr Prot)* Sheep IgG (H+L) Sheep IgG (Fc) Sheep IgG [F(ab)2]
Goat Mouse Rabbit Rabbit Rabbit 31225 1 mg/$118 31240 2 mg/$111 31241 2 mg/$112 31244 2 mg/$96
31625 1 mg/$143 31633 31682 0.5 mg/$121 0.5 mg/$121 31840 31627 1.5 mg/$115 1.5 mg/$88 31841 1.5 ml/$133 31844 1.5 ml/$136
31480 1.5 ml/$125 31441 1.5 ml/$141 31481 1.5 ml/$136
31360 1 ml/$143 31356 1 ml/$152 31344 1 ml/$152
38
DyLight Conjugates
Excellent alternatives to CyDye Fluors.
The new DyLight Conjugates are an excellent alternative to Cy3 and Cy5 Fluors, providing greater photostability and fluorescence over a broad range of pH values. The absorption spectra of the DyLight 547 Fluor and the DyLight 647 Fluor closely matches that of the Cy3 and Cy5 Fluors, respectively, enabling a seamless transition from CyDye to the DyLight Fluors without purchasing new filters. Highlights: Molar ratio (dye:protein) optimized to provide excellent fluorescent intensity Antibody conjugates are affinity-purified DyLight Flours have demonstrated improved performance in several applications: ELISA Immunohistochemical (IHC) staining Western blotting Flow cytometry Plate-based functional assays
DyLight Spectral Characteristics

Dye DyLight 547 DyLight 647 Excitation (nm) 557 652 Emission (nm) 574 673 Extinction Coefficient (min) 150,000 M-1 cm-1 250,000 M-1 cm-1
Product # Description 31010 DyLight 547 Fluor, Goat Anti-Mouse IgG (H + L) Conjugated 31015 DyLight 647 Fluor, Goat Anti-Mouse IgG (H + L) Conjugated 31020 DyLight 547 Fluor, Goat Anti-Rabbit IgG (H+L) Conjugated 31025 DyLight 647 Fluor, Goat Anti-Rabbit IgG (H+L) Conjugated 21424 DyLight 547 Fluor, Streptavidin Conjugated 21824 DyLight 647 Fluor, Streptavidin Conjugated Pkg. Size 1 ml (1 mg/ml) 1 ml (1 mg/ml) 1 ml (1 mg/ml) 1 ml (1 mg/ml) 1 ml (1 mg/ml) 1 ml (1 mg/ml) U.S. Price $155 $155 $155 $155 $145 $145
Biotin Detection Ordering Information

Avidin Unconjugated 21121, 10 mg, $67 21128, 20 mg, $113 31000, 10 mg, $112 21122, 1 mg, $90 21125, 5 mg, $272 Enzyme-labeled Peroxidase: Alk. Phos: Peroxidase: Alk. Phos: Peroxidase: Alk. Phos: 21123, 2 mg, $108 29994, 5 mg, $191 21321, 100 units, $102 31001, 2 mg, $185 31002, 2 mg, $229 21126, 1 mg, $125 21124, 2 mg, $225 21127, 5 mg, $389 21324, 1 mg, $152 21323, 3 mg, $349 Fluorescent-labeled Fluorescein: 21221, 5 mg, $104 Phycoerythrin: 21021, 1 mg, $119 Fluorescein: Fluorescein: Rhodamine: Texas Red Dye: R-Phycoerythrin: Allophycocyanin: 31006, 5 mg, $149 21224, 1 mg, $154 21724, 1 mg, $150 21624, 1 mg, $157 21627, 1 ml, $130 21629, 0.5 ml, $145
NeutrAvidin Protein Streptavidin
PRIMARY & SECONDARY
ANTIBODIES
39
ANTIBODY
LABELING
Antibody-Modification Sites Antibodies can be easily modified to contain labels such as biotin, fluorescent tags or enzymes to create reagents for ELISA, Western blotting, immunohistochemical staining and in vivo targeting. Pierce offers tools for a variety of antibodymodification strategies. Understanding the functional groups available on an antibody is the key to choosing the proper method for modification. For example: Primary amines (NH2) are found on lysine residues and the N-terminus. These are abundant and distributed over the entire antibody. Sulfhydryl groups (SH) are found on cysteine residues and are formed by selectively reducing disulfide bonds in the hinge region of the antibody.
Fab (Fab')2
Chemical cross-linking reagents have become an invaluable tool in the scientific community. These reagents are used in preparing antibody-enzyme conjugates and other labeled protein reagents. After the protein is conjugated to an appropriate enzyme, it may then be used as a detection reagent in a variety of assays and applications. A number of cross-linking methods have been used to prepare enzyme conjugates. For example, an N -hydroxysuccinimide ester can be prepared from a ligand of interest, then reacted with a primary amine on the surface of the enzyme. While this method is necessary in some applications, such as those in which the ligand does not contain a primary amine, it is not useful as a general-purpose method.
Antigenbinding site VL
SS SS
Light Chains VL
SSS S
VH NH2
CL
CL
SS SS
VH
S-S
SS
S-S
CH1 NH2
CH1
Hinge Region S-S S-S CH2 S S S CH2 S
Carbohydrate residues containing cis-diols can be oxidized (CHO) to create active aldehydes. These are localized to the Fc region on antibodies and are more abundant on polyclonal antibodies.
S-
Carbohydrate FC
Carbohydrate
NH2
CH3 S S
S CH 3 S NH2
Heavy Chains
Amines on lysine residues
Sulfhydryls created when antibody is reduced
40
Fluorescent Labeling EZ-Label Fluorescent Labeling Kits

Make your own fluorescent-labeled antibody in less than two hours!
EZ-Label Kits are designed for labeling any size protein small or large even if you have only a small amount of your protein. Protein sample volumes ranging from 50 l-1 ml can be used, with protein concentration up to 10 mg/ml for each reaction. EZ-Label Kits were specially developed and optimized for the most efficient labeling. EZ-Label Kits contain everything you need to successfully label your antibody or protein: Fluorescent dye provided in individual microtubes, eliminating the need to weigh dye Conveniently packaged dimethylformamide (DMF) to prepare the fluorescent dye solution Pre-made borate and phosphate buffers just add water to the powder and they are ready-to-use Pre-packed, ready-to-use desalting columns for fast buffer exchange when your protein sample volume is greater than 100 l Slide-A-Lyzer MINI Dialysis Units* for easy buffer exchange when your protein sample volume is less than or equal to 100 l Amber reaction tubes no handling in the dark required
EZ-Label Kit Fluorescein Protein Labeling Kit Rhodamine Protein Labeling Kit Fluorescein Isothiocyanate (FITC) Protein Labeling Kit Excitation Emission Wavelength (nm) Wavelength (nm) 491 518 544 576 494 520
U.S. Product # Description Pkg. Size Price 53000 EZ-Label Fluorescein Protein Labeling Kit Kit $237
Sufficient for five coupling reactions. Includes: No-Weigh Pre-Measured Fluorescein Microtubes Dimethylformamide (DMF) BupH Borate Buffer Packs BupH Phosphate Buffered Saline Packs D-Salt Dextran Desalting Columns Slide-A-Lyzer MINI Dialysis Unit Pack Reaction Tubes 6 x 1 mg microtubes 1 ml 5 packs 5 packs 5 columns 5 units 5 tubes
53002
EZ-Label Rhodamine Protein Labeling Kit Kit

Sufficient for five coupling reactions. Includes: No-Weigh Pre-Measured Rhodamine Microtubes Dimethylformamide (DMF) BupH Borate Buffer Packs BupH Phosphate Buffered Saline Packs D-Salt Dextran Desalting Columns Slide-A-Lyzer MINI Dialysis Unit Pack Reaction Tubes 6 x 0.5 mg microtubes 1 ml 5 packs 5 packs 5 columns 5 units 5 tubes
$237
53004
EZ-Label Fluorescein Isothiocyanate (FITC) Protein Labeling Kit
Kit
$237
Sufficient for five coupling reactions. Includes: No-Weigh 6 x 1 mg Pre-Measured FITC Microtubes microtubes Dimethylformamide (DMF) 1 ml 5 packs BupH Borate Buffer Packs BupH Phosphate Buffered Saline Packs 5 packs 5 columns D-Salt Dextran Desalting Columns 5 units Slide-A-Lyzer MINI Dialysis Unit Pack Reaction Tubes 5 tubes * Slide-A-Lyzer MINI Dialysis Unit Technology is protected by U.S. patent # 6,039,871.
These kits contain sufficient reagents to perform five fluorescent labeling reactions, which use up to 10 mg/ml of protein for each reaction (50 l-1 ml volume of protein).
The fluorescent-labeling procedure is easy when you use EZ-Label Kits.

Step 1. Preparation of Protein For salt-free lyophilized protein: Dissolve in borate buffer. Step 2. Labeling Reaction Step 3. Removal of Excess Fluorescent Dye
For sample volume of 100 l or less: Exchange into PBS buffer using Slide-A-Lyzer MINI Dialysis Unit.
For proteins in buffers or salt solutions: a.) Sample volume of 100 l or less: Exchange into borate buffer using Slide-A-Lyzer MINI Dialysis Unit. b.) Sample volume greater than 100 l: Exchange into borate buffer using a D-Salt Dextran Column.
Reconstitute fluorescent dye with DMF.
Add dye to the protein solution. Incubate for 1 hour.
For sample volume greater than 100 l: Exchange into PBS buffer using a D-Salt Dextran Column.
ANTIBODY
LABELING
41
ANTIBODY
LABELING
Maleimide-activated enzymes can be prepared using the heterobifunctional cross-linker Sulfo-SMCC. This reagent contains an N-hydroxy-sulfosuccinimide (Sulfo-NHS) functional group and a maleimide functional group and it is water-soluble due to the presence of the sulfonate (SO3-) group on the N-hydroxysuccinimide ring. The sulfonate group also contributes to the stability of the molecule in aqueous solution. A study of the hydrolysis rate of the maleimide functional group from Sulfo-SMCC showed that it is less prone to hydrolysis to the maleamic acid than the non-sulfonated SMCC. The maleimide groups of Sulfo-SMCC exhibit no decomposition at pH 7 at 30C within 6 hours. The Sulfo-NHS ester group reacts with primary amines on the enzyme surface to form a stable amide bond. After this first step of conjugation, the enzyme will have maleimide groups on its surface that react optimally toward sulfhydryl groups between pH 6.5 and 7.5 to form stable thioether bonds. Maleimidemediated conjugation strategies are summarized in Figure 15.
Enzyme Labeling
Maleimide Activation The heterobifunctional cross-linker SMCC (Product # 22360) and its water-soluble analog Sulfo-SMCC (Product # 22322) have good general utility in preparing immunologically active horseradish peroxidase or alkaline phosphatase conjugates. They are most useful when preparing conjugates of reduced IgG and F(ab)2, because these methods involve the initial step of preparing a maleimide-activated (sulfhydryl-reactive) enzyme derivative. Studies have shown that the two-step maleimide method is superior to glutaraldehyde or meta-periodate methods for enzyme conjugation (Figure 15). The maleimide method gives higher yields with less polymerization, producing a conjugate preparation with superior immunoassay characteristics.
Three methods for free sulfhydryl generation

2
Native protein contains disulfide bonds that can be reduced to generate free sulfhydryls.
Maleimide activation of enzyme

Enzyme is SMCC-labeled through primary amines to generate a maleimide-activated enzyme for conjugation to free sulfhydryls.
S MEA EDTA
SH Protein
SH
Protein
O N O
O
N
O
H2N
Native protein has a free sulfhydryl on its surface.
Native protein is reacted with SATA. Blocked sulfhydryl groups are introduced on primary amines. Hydroxylamine (H) treatment generates free sulfhydryls.
O SMCC
Protein
NH2 H O
O H
HO N
O
Protein
N C CH2 S C CH3 H O N C CH2 SH
Protein
N Protein SH
O
N
O
Protein
S
O
N
O
Figure 15. Three strategies for maleimide-mediated conjugation of enzymes.
42
Protein
SH
SATA
NHS
O
Two reagents, MercaptoethylamineHCl (Product # 20408) and SATA (Product # 26102), are available to produce free sulfhydryls on macromolecules for conjugation to the maleimide-activated enzymes. For labeling antibody molecules, mild reduction with MercaptoethylamineHCl (MEA) results in two half-antibody fragments containing free sulfhydryl groups in the hinge region. Labeling in this area is advantageous because it directs the modification away from the antigen-binding region. Native proteins lacking a free sulfhydryl on their surface can be reacted with SATA to generate blocked sulfhydryl groups. The SATA molecule reacts with primary amines via its NHS ester end to form stable amide linkages. The acetylated sulfhydryl group (blocked) is stable until treated with hydroxylamine to generate the free sulfhydryls. Pierce offers stable, preactivated enzyme derivatives that are reactive toward sulfhydryl (SH) groups, EZ-Link Maleimide Activated Alkaline Phosphatase (Product # 31486) and Horseradish Peroxidase (Product # 31485). These products eliminate the first step of the two-step maleimide method, simplifying and facilitating the conjugation protocol, while saving several hours. They can be used to prepare enzyme conjugates directly from proteins, peptides or other ligands that contain a free SH group. Two reagents, SATA and mercaptoethylamineHCl, are also included in the kit formats to produce free sulfhydryls on macromolecules for conjugation.
EZ-Link Maleimide Activated Peroxidase References Choi, J.Y., et al. (2002). J. Biol. Chem. 277, 21630-21638. Seo, Y.R., et al. (2002). Proc. Natl. Acad. Sci. 99, 14548-14553. Yoo, J.H., et al. (2004). J. Biol. Chem. 279, 848-858.
Product # Description 31486 EZ-Link Maleimide Activated Alkaline Phosphatase 31493 EZ-Link Maleimide Activated Alkaline Phosphatase Kit
Includes: EZ-Link Maleimide Activated Alkaline Phosphatase Activation/Conjugation Buffer BupH Tris Buffered Saline Pack BupH Phosphate Buffered Saline Pack Polyacrylamide Desalting Column MercaptoethylamineHCl SATA Hydroxylamine DMF Column Extender
U.S. Pkg. Size Price 2 mg $174 Kit

2 mg 20 ml 2 packs 1 pack 1 x 10 ml 6 mg 2 mg 5 mg 1 ml
$359
31485 31494
EZ-Link Maleimide Activated Horseradish Peroxidase EZ-Link Maleimide Activated Horseradish Peroxidase Kit
Includes: EZ-Link Maleimide Activated Horseradish Peroxidase Activated Horseradish Peroxidase Conjugation Buffer 2-MercaptoethylamineHCl SATA Dimethylformamide HydroxylamineHCl Polyacrylamide Desalting Column
5 mg Kit
5 mg 20 ml 6 mg 2 mg 1 ml 5 mg 1 x 10 ml
$132 $317
23460
Protein-Coupling Handle Addition Kit

Includes: SATA HydroxylamineHCl Conjugation Buffer Stock (10X) BupH Pack PBS Dimethylformamide (DMF) D-Salt Dextran Desalting Column Column Extender Ellmans Reagent (DTNB) CysteineHCl H2O
Kit
2 mg 5 mg 20 ml 1 pack 1 ml 1 x 5 ml 1 2 mg 20 mg
$181
ANTIBODY
LABELING
43
ANTIBODY
HO RO
LABELING
HO O O E NalO4 (oxidation) O RO O E O O Enzyme with reactive aldehyde groups
OH HO Enzyme containing polysaccharide chains
using sodium borohydride or sodium cyanoborohydride; however, cyanoborohydride is the better choice because it is more specific for reducing Schiff bases and will not reduce aldehydes. Small blocking agents such as lysine, glycine, ethanolamine or Tris can be added after conjugation to quench any unreacted aldehyde sites. Ethanolamine and Tris are the best choices for blocking agents because they contain hydrophilic hydroxyl groups with no charged functional groups. The pH of the reductive amination reaction can be controlled to affect the efficiency of the cross-linking process and the size of the resultant antibody-enzyme complexes formed. At physiological pH, the initial Schiff base formation is less efficient and conjugates of lower molecular weight result. At more alkaline pH (i.e., pH 9-10), Schiff base formation occurs rapidly and with high efficiency, resulting in conjugates of higher molecular weight and greater incorporation of enzyme when oxidized enzyme is reacted in excess. Low molecular weight conjugates may be more optimal for immunohistochemical staining or blotting techniques in which penetration of the complex through membrane barriers is an important consideration. Washing steps also more effectively remove excess reagent if the conjugate is of low molecular weight, thus maintaining low background in an assay. By contrast, conjugates of high molecular weight are more appropriate for ELISA procedures in a microplate format, where high sensitivity is important and washing off excess conjugate is not a problem. Glutaraldehyde Another method for conjugation uses glutaraldehyde, one of the oldest homobifunctional cross-linking reagents used for protein conjugation. It reacts with amine groups to create cross-links by one of several routes. Under reducing conditions, the aldehydes on both ends of glutaraldehyde will couple with amines to form secondary amine linkages. The reagent is highly efficient at protein conjugation but has a tendency to form various high-molecular weight polymers, making results difficult to reproduce.
EZ-Link Activated Peroxidase References Sandt, C.H. and Hill, C.W. (2001). Infect. Immun. 69, 7293-7303. Turpin, E.A., et al. (2003). J. Clin. Microbiol. 41, 3579-3583. EZ-Link Plus Activated Peroxidase References Glover, L. (2002). Eur. J. Biochem. 269, 4607-4616. Nawa, M., et al. (2000). Clin. Diagn. Lab. Immunol. 7, 774-777. Vlkel, T., et al. (2001). Protein Eng. 14, 815-823.
NaCNBH3 O N RO HO Reductive amination forms stable secondary amine linkage Antibody molecule containing amine groups O O E NH2
Figure 16. Conjugation scheme for periodate oxidation and subsequent reductive amination.
Periodate Glycoproteins such as horseradish peroxidase and glucose oxidase and most antibody molecules can be activated for conjugation by treatment with periodate. Oxidizing polysaccharide residues in a glycoprotein with sodium periodate provides a mild and efficient way of generating reactive aldehyde groups for subsequent conjugation with amine- or hydrazide-containing molecules via reductive amination (Figure 16). Some selectivity of monosaccharide oxidation may be accomplished by regulating the concentration of periodate in the reaction medium. In the presence of 1 mM sodium periodate, sialic acid groups are specifically oxidized at adjacent hydroxyl residues, cleaving off two molecules of formaldehyde and leaving one aldehyde group. At higher concentrations of sodium periodate (10 mM or greater), other sugar residues will be oxidized at points where adjacent carbon atoms contain hydroxyl groups. This reaction should be performed in the dark to prevent periodate breakdown and for a limited period of time (15-30 minutes) to avoid loss of enzymatic activity. Cross-linking with an amine-containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride. Reductive amination has been done
44
Product # Description 31487 EZ-Link Plus Activated Peroxidase
(Periodate Activated)
U.S. Pkg. Size Price 1 mg $ 63 5 x 1 mg Kit

5 x 1 mg 1 x 0.5 ml 25 ml 500 ml 500 ml
Product # Description 31496 EZ-Link Activated Peroxidase

(Glutaraldehyde Activated)
U.S. Pkg. Size Price 1 mg $ 58 5 mg Kit

5 mg 50 ml 250 mg 0.5 ml 200 ml 200 ml
31488 31489
EZ-Link Plus Activated Peroxidase

(Periodate Activated)
$232 $317
31495 31497
EZ-Link Activated Peroxidase

(Glutaraldehyde Activated)
$160 $317
EZ-Link Plus Activated Peroxidase Kit

(Periodate Activated) Includes: EZ-Link Plus Activated Peroxidase Sodium Cyanoborohydride Solution Quenching Buffer BupH Phosphate Buffered Saline Pack BupH Carbonate Buffer Pack
EZ-Link Activated Peroxidase Antibody Labeling Kit

(Glutaraldehyde Activated) Includes: EZ-Link Activated Peroxidase Conjugation Buffer Lysine Immobilized Protein A/G Column Gentle Ag/Ab Binding Buffer Gentle Ag/Ab Elution Buffer
Horseradish Peroxidase
High-specific enzyme activity makes it the enzyme of choice.
Highlights: Superior to alkaline phosphatase and -galactosidase conjugates due to the higher specific enzyme activity Small size (40 kDa) allows excellent cellular penetration Variety of substrates available Ideal in blotting and cytochemistry applications Used as the reporter enzyme for SuperSignal Chemiluminescent Substrates
References Cordell, J.L., et al. (1984). J. Histochem. Cytochem. 32, 219-229. Hosoda, H., et al. (1987). Chem. Pharm. Bull. 35, 3336-3342. Passey, R.B., et al. (1977). Clin. Chem. 23(1), 131-139. Porstmann, B., et al. (1985). J. Immunol. Methods. 79, 27-37. Samoszuk, M.K., et al. (1989). Antibody, Immunoconjugates and Radiopharmaceuticals 2, 37-46. Wordinger, R.J., et al. (1987). Manual of Immunoperoxidase Techniques, 2nd Edition. Chicago: American Society of Clinical Pathologists Press, pp. 23-24. Yolken, R.H. (1982). Rev. Infect. Dis. 4(1), 35-68.
Product # Description 31490 ImmunoPure Horseradish Peroxidase 31491 ImmunoPure Horseradish Peroxidase U.S. Pkg. Size Price 10 mg $ 53 100 mg $190
Alkaline Phosphatase
A highly sensitive enzyme for ELISA and immunohistochemical applications.
Highlights: Purified form ready to conjugate without prior dialysis Activity is not affected by exposure to antibacterial agents such as sodium azide or thimerosal Specific activity > 2,000 units/mg One unit is defined as the amount that will hydrolyze 1.0 mole of p -nitrophenyl phosphate per minute at 37C in 1.0 M diethanolamine, 0.5 mM MgCl2, pH 7.8 Specific Activity per mg Protein
Buffer 0.1 M Glycine, 1.0 mM ZnCl2, 1.0 mM MgCl2, 6.0 mM p-Nitrophenyl phosphate, pH 10.4 1.0 M Diethanolamine, 0.5 mM MgCl2, 15 mM p-Nitrophenyl phosphate, pH 9.8 25C > 500 > 1,000 37C > 1,000 > 2,000
References Bulman, A.S. and Heyderman, E. (1981). J. Clin. Pathol. 34, 1349-1351. Cordell, J.L., et al. (1984). J. Histochem. Cytochem. 32, 219-229. Yolken, R.H. (1982). Rev. Infect. Dis. 4, 35-68.
Product # Description 31391 ImmunoPure Alkaline Phosphatase
Calf intestinal. Supplied in Tris Buffer, pH ~7 Triethanolamine, 1 mM MgCl2, 3 M NaCl, pH 7.6
U.S. Pkg. Size Price 20 mg $ 652 100 mg $2,528
31392
ImmunoPure Alkaline Phosphatase
ANTIBODY
LABELING
45
CHOOSING A
SUBSTRATE
ELISA conditions must always be re-optimized when switching to a more sensitive substrate (Table 7). Greater dilutions of an HRP-conjugate should be used with a chemiluminescent substrate than with a colorimetric substrate.
Pierce offers an extensive line of substrates for AP and HRP, depending upon the plate-reading equipment available and the level of sensitivity required in the ELISA. Chemiluminescent and chemifluorescent substrates provide a stronger signal than colorimetric substrates, thus providing greater sensitivity.
Table 7. Properties of ELISA substrates for horseradish peroxidase (HRP) and alkaline phosphatase (AP). Substrate SuperSignal ELISA Femto SuperSignal ELISA Pico QuantaBlu Substrate 1-Step Ultra TMB 1-Step Turbo TMB 1-Step Slow TMB TMB Substrate Kit 1-Step ABTS ABTS OPD Powder OPD Tablets 1-Step PNPP PNPP Kit PNPP Tablets PNPP Powder Product # 37075 37070 15169 34028 34022 34024 34021 37615 34026 34005 34006 37621 37620 34047 34045 Page 50 49 51 53 53 53 53 52 52 52 52 54 54 54 54 Dilution range of Ab Measurement - Color 425 nm chemiluminescent 425 nm chemiluminescent 325 nm/420 nm chemifluorescent 450 nm stopped Yellow 652 nm nonstop Blue 450 nm stopped Yellow 652 nm nonstop Blue 450 nm stopped Yellow 652 nm nonstop Blue 450 nm stopped Yellow 652 nm nonstop Blue 410 nm/650 nm Green 410 nm/650 nm Green 490 nm stopped Green 450 nm nonstop Yellow-Orange 490 nm stopped Green 450 nm nonstop Yellow-Orange 405 nm Yellow 405 nm Yellow 405 nm Yellow 405 nm Yellow Approximate (From 1 mg/ml stock) 1 1:10K 1:20K 2 1:50K 1:100K 1 1:1K 2 1:10K 1 1:10K 1:20K 2 1:50K 1:100K 1 1:500 1:1K 2 1:4K 1:100K 1 1:500 1:1K 2 1:4K 1:100K 1 1:500 1:1K 2 1:4K 1:100K 1 1:500 1:1K 2 1:4K 1:100K 1 1:500 1:1K 2 1:4K 1:50K 1 1:500 1:1K 2 1:4K 1:50K 1 1:500 1:1K 2 1:4K 1:100K 1 1:500 1:1K 2 1:4K 1:100K 1 1:500 2 1:5K 1 1:500 2 1:5K 1 1:500 2 1:5K 1 1:500 2 1:5K Sensitivity* 0.17 pg/well 0.5 pg/well 0.5 pg/well 2 pg/well 7 pg/well 8 pg/well 5.5 pg/well 0.25 ng/well 0.25 ng/well 7 pg/well 7 pg/well 10 ng/well 10 ng/well 10 ng/well 10 ng/well Enzyme HRP HRP HRP HRP HRP HRP HRP HRP HRP HRP HRP AP AP AP AP
* Actual sensitivity is unique to each antibody-antigen pair. The approximate sensitivities listed are conservative amounts that should be easily detectable for most antigens.
46
Enzyme-labeled reagents are detected using chromogenic, chemiluminescent or chemifluorescent substrates. Chromogenic substrates are generally the least expensive, while luminescent or fluorescent substrates are often more sensitive. When performing ELISAs, a soluble substrate is used and converted Choosing a Detection Signal Type
ELISA Colorimetric Substrates Medium/low sensitivity Generally less expensive Many substrates available Slow signal generation Enzyme catalyzed quickly Small linear range/poor low-end linearity Flexible (stopped, nonstopped and kinetic assays) Spectrophotometer provides quantifiable results
to a soluble end product. ELISAs are performed on polystyrene plates, and the enzyme levels are determined by monitoring signal development with a spectrophotometer (a luminometer for luminescence or a fluorometer for fluorescence).
Detection Equipment
Chemifluorescent Substrates High sensitivity Generally more expensive Few substrates available Rapid signal generation Enzyme activity maintained Large linear range/enhanced low-end linearity Flexible (stopped, nonstopped and kinetic assays) Fluorometer provides quantifiable results
Chemiluminescent Substrates High sensitivity Generally more expensive Few substrates available Rapid signal generation Enzyme catalyzed quickly Large linear range/enhanced low-end linearity Nonflexible Luminometer provides quantifiable results
Horseradish Peroxidase Substrates

Horseradish peroxidase is a 40 kDa protein that catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product or the release of light as a byproduct. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, a high turnover rate, stability, low cost and wide availability of substrates make HRP the enzyme of choice for most applications. When selecting a substrate for HRP-based ELISAs, there are a number of possibilities. The question of sensitivity is often the overriding factor in making a selection. However, consideration should also be given to whether the substrate contains harmful solvents, what detection equipment is available and how the assay will be measured (kinetic or end-point). In many ELISA applications, colorimetric substrates provide a sufficient level of sensitivity and dynamic range. This is evident with the Pierce Endogen Cytokine ELISA Kits, in which a TMB substrate coupled with a streptavidin-HRP detection system results in pg/ml sensitivity. Detection below this level requires a fluorescent or chemiluminescent signal. Characteristics of the HRP ELISA substrates offered by Pierce are summarized in Table 7.
Chromogenic ELISA substrates result in a soluble, colored product. These substrates are used in most ELISAs because detection of a colored product can be performed on a spectrophotometric plate reader. TMB (3,3,5,5-tetramethylbenzidine) is the most common chromogenic substrate for HRP and is available in several formats. 1-Step Ultra TMB (Product # 34028) yields the greatest sensitivity among the TMB substrates, followed by 1-Step Turbo TMB (Product # 34022) and 1-Step Slow TMB (Product # 34024). The 1-Step Substrates are preformulated so no mixing or pre-filtering is required. Although the sensitivity is lower, the 1-Step Slow TMB and 1-Step ABTS (2,2-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) are ideal for kinetic readings. OPD (o-phenylenediamine dihydrochloride, Product # 34005) is another relatively sensitive HRP substrate that produces a yellow-orange color. The greatest sensitivity in ELISA applications is obtained using chemiluminescent or chemifluorescent substrates. These substrates have been steadily gaining in popularity because of their sensitivity (less than 1 pg/ml), large linear range for detection and excellent antibody conservation. Pierce offers the chemiluminescent SuperSignal ELISA Pico (Product # 37070) and SuperSignal ELISA Femto (Product # 37075) Substrates and the chemifluorescent QuantaBlu Substrate (Product # 15169).
CHOOSING A
SUBSTRATE
47
CHOOSING A
SUBSTRATE
90 80 70 60 50 40 30 20 10 0 0 1,000 2,000 3,000 Biotinylated-HRP, pg/well QuantaBlu Substrate TMB ABTS OPD 4,000
over a large range of protein concentrations. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method. Using HRP as the enzyme label and SuperSignal ELISA Femto Chemiluminescent Substrate (Product # 37075), lower detection limits in the upper femtogram range are possible because the enhancers in this substrate greatly intensify the emitted light and extend the signal duration. Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This is in contrast to substrates that produce a stable, colored product; these colors persist in the well after the enzyme-substrate reaction has terminated. In a chemiluminescent ELISA, the substrate is the limiting reagent in the reaction; as it is exhausted, light production decreases and eventually ceases. A welloptimized procedure using the proper antibody dilutions will produce a stable output of light, allowing consistent and sensitive detection of proteins. When the antibody is not diluted sufficiently, too much enzyme is present and the substrate is used up quickly. A stable output of light will never be achieved. This is the single greatest cause of variability in chemiluminescent ELISAs. To avoid this problem, it is crucial to optimize the amount of antibody used for detection. Antibody suppliers typically suggest a dilution range for using their antibody in an ELISA. This dilution range is often appropriate for experiments detected with a relatively insensitive chromogenic substrate, but a much greater dilution is generally required for optimum performance with a sensitive chemiluminescent substrate.
Table 8. Advantages of enhanced chemiluminescence.
@ 425 nm
Figure 17. Comparison of QuantaBlu Substrate to other substrates. QuantaBlu Substrate and the colorimetric substrates were incubated for 30 minutes at RT, followed by addition of a stop solution. QuantaBlu Substrate gave the greatest signal:noise (S/N) ratios and exhibited the lowest detection limit.
When energy in the form of light is released from a substance because of a chemical reaction, the process is called chemiluminescence. Luminol is one of the most widely used chemiluminescent reagents and its oxidation by peroxide results in creation of an excited state product called 3-aminophthalate. This product decays to a lower energy state by releasing photons of light (Figure 18).
O H2O2 O NH HRP NH NH2 O NH2 O O O NH2 O
S/N Ratio
O O O
LIGHT
Sensitive Fast Nonhazardous Stable Large linear response Quantitative
Figure 18. Luminol is oxidized in the presence of horseradish peroxidase and hydrogen peroxide to form an excited state product (3-aminophthalate). The 3-aminophthalate emits light at 425 nm as it decays to the ground state.
Chemiluminescent substrates have steadily gained in popularity throughout the past decade because they offer several advantages over other detection methods. These advantages have allowed chemiluminescence to become the detection method of choice in most protein laboratories. Using chemiluminescence allows multiple exposures to be performed to obtain the best image. A large linear response range allows detection and quantitation
Intense signal with low background Requires less antigen and antibody Rapid substrate processing Signal generated within seconds No health hazards No waste disposal problems Unlike radioisotopes, the shelf life is long Store at 4C Can detect a large range of protein concentrations Results can be scanned using an imaging device, such as a CCD camera
48
HRP Substrates for ELISA

Excellent sensitivity for use in luminometers.
Researchers looking for greater sensitivity in their ELISAs or any other solution-based assay are now able to use chemiluminescent substrates for enzyme detection and quantification. These ELISAs can take place in either a test tube or a microplate and are quantified by measuring relative light units (RLU) in a luminometer. SuperSignal ELISA Pico Chemiluminescent
Table 9. Which substrate is right for you? Substrate SuperSignal ELISA Femto Maximum Sensitivity Substrate SuperSignal ELISA Pico Chemiluminescent Substrate Detection Limits Quantitative to the femtogram level
Substrate was developed for researchers who need high sensitivity at an economical price. SuperSignal ELISA Femto Maximum Sensitivity Chemiluminescent Substrate uses an improved enhancer system that meets the needs of highthroughput screening and diagnostic customers. Both have their own unique features as listed in the table.
Quantitative to the picogram level
Kinetics Immediate light generation 5- to 30-minute stability, depending on HRP concentration Immediate light generation 30-minute stable light output
Working Solution Stability at Room Temperature 6-hour working solution stability
8-hour working solution stability Only 10% loss of activity after 24 hours
SuperSignal ELISA Pico Chemiluminescent Substrate

Experience the same sensitivity in your luminometer that youve come to expect from all Pierce SuperSignal Products.
SuperSignal ELISA Pico Chemiluminescent Substrate is optimized for luminometer-based assays to generate an intense light signal.
Kinetic Analysis of 200 pg of Biotinylated HRP
700 600 500 RLU 400 300 200 100 0 0 10 20 Time (minutes) 30
Highlights: Immediate light generation intense signal is produced immediately at room temperature or at 37C High signal:noise ratio minimal background Low picogram sensitivity detect proteins in your ELISAs down to the picogram levels Room temperature storage a consistent product with ambient shipping and no need to store at 4C 8-hour working solution stability consistent performance of the working solution over an 8-hour period with only a 10% decrease in activity at 24 hours Flexible signal can be read in black or white opaque plates Emits light at 425 nm
References Bradley, K.A., et al. (2004). J. Biol. Chem. 278, 49342-49347. McKevitt, M., et al. (2003). Genome Res. 13, 1665-1674.
SuperSignal ELISA Pico Chemiluminescent Substrate
Luminol-Based, Brand A Dioxetane-Based, Brand B
Figure 19. Immediate light generation with SuperSignal ELISA Pico Chemiluminescent Substrate. 200 pg of biotinylated HRP or biotinylated AP were added to separate wells of Reacti-Bind NeutrAvidin Coated White Polystyrene Plates. The plates were then incubated for 30 minutes at room temperature (RT) on a plate shaker and then each well was washed three times with BupH Tris Buffered Saline. Working solutions of chemiluminescent substrates were prepared according to the manufacturers instructions. For SuperSignal ELISA Pico Chemiluminescent Substrate and another luminol-based system (Brand A), 100 l of each substrate working solution was added to the appropriate plate well. For the dioxetane-based system, wells were washed with 1X Assay Buffer. Next, 100 l of the dioxetane working solution was added to the appropriate plate well. All plates were incubated on a plate shaker at RT for 1 minute. Plates were then read on a plate luminometer with a 0.2 second read time per well. Several readings were taken over a 30-minute period.
Product # Description 37070 SuperSignal ELISA Pico Chemiluminescent Substrate* U.S. Pkg. Size Price 100 ml $195
Includes: Luminol/Enhancer 50 ml Stable Peroxide Buffer 50 ml *SuperSignal Technology is protected by U.S. patent # 6,432,662.
CHOOSING A
SUBSTRATE
49
CHOOSING A
SUBSTRATE
SuperSignal ELISA Femto Maximum Sensitivity Substrate utilizes the SuperSignal System with a new enhancer formulation for superior protein detection and low-end linearity in ELISA applications.
120 100 80 Net RLU 60 40 20 0 0 500 1,000 fg IL-2 1,500
SuperSignal ELISA Femto Maximum Sensitivity Substrate

The most powerful substrate for high-throughput screening/diagnostic applications with high sensitivity and superior low-end linearity.
Net RLU
SuperSignal ELISA Femto Substrates rapid signal generation has the added benefit of decreasing the substrate incubation period. SuperSignal ELISA Femto Substrate generates detectable light within 1 minute of addition to trace amounts of soluble HRP, saving up to 30 minutes per assay. This feature makes SuperSignal ELISA Femto Substrate ideal for high-throughput screening (HTS) applications in which as many as 100,000 assays may be run daily on robotic equipment. As incubation periods are often a limiting factor in the development of rapid automated assays, SuperSignal ELISA Femto Substrate is the logical choice for automated HTS applications. Highlights: Immediate light generation intense signal generated immediately at both room temperature and 37C Improved low-end linearity easy detection of low quantities of proteins with high signal:noise ratios and low-end linearity of dose response curves High sensitivity femtogram-level detection of target proteins in an ELISA Reduction in assay time high sensitivity allows for reduction in ELISA incubation steps Stability storage for six months at room temperature or a minimum of 12 months at 4C with a six-hour working solution stability Emits light at 425 nm
Incubation Time Required to Reach Maximum Signal SuperSignal ELISA Femto 1 minute at room temperature Maximum Sensitivity Substrate SuperSignal ELISA Pico 1 minute at room temperature Chemiluminescent Substrate Dioxetane-based Substrate System 30 minutes at room temperature Luminol-based Substrate System 1 minute at room temperature Acridan-based Substrate System 5 minutes at room temperature TMB Colorimetric Substrate 15 minutes at room temperature
Figure 20. Femtogram detection of target protein and superior low-end linearity. The dose response curve generated from an IL-2 ELISA illustrates the exceptional low-end linearity achieved with SuperSignal ELISA Femto Substrate and the incredible sensitivity attainable. The SuperSignal ELISA Femto Substrate detected down to 168 fg of IL-2. The R2 value of the curve was calculated to be 1.00 for signal generated at less than 1,600 fg of IL-2.
1,500 1,200 900 600 300 0 0 5 10 pg IL-2 SuperSignal ELISA Femto Substrate SuperSignal ELISA Pico Substrate Dioxetane-based Substrate Luminol-based Substrate Acridan-based Substrate 15 20 25
Figure 21. Signal intensity and kinetics comparison of chemiluminescent substrates. The dose response curve using SuperSignal ELISA Femto Maximum Sensitivity Substrate in an IL-2 ELISA was compared to curves generated with a dioxetane-based substrate, another luminol-based substrate, an acridan-based system and TMB. SuperSignal ELISA Femto Maximum Sensitivity Substrate demonstrated immediate light generation with maximum peak intensity and high RLU values.
References Brandt, E.B., et al. (2003). J. Clin. Invest. 112, 1666-1677. Hanley, N.R.S. and Hensler, J.G. (2002). J. Pharmacol. Exp. Ther. 300, 468-477. Masri, H.P. and Cornellissen, C.N. (2002). Infect. Immun. 70, 732-740. Su, S.V., et al. (2004). J. Biol. Chem. In press.
Product # Description 37075 SuperSignal ELISA Femto Maximum Sensitivity Substrate* U.S. Pkg. Size Price 100 ml $205
50
Includes: Luminol/Enhancer 50 ml Stable Peroxide Solution 50 ml *SuperSignal Technology is protected by U.S. patent # 6,432,662.
QuantaBlu Fluorogenic Peroxidase Substrates

The ideal fluorescent substrates for use with peroxidase enzymes.
A variety of substrates are available for detecting peroxidase activity in ELISA-based assays. Colorimetric substrates (e.g., TMB, OPD and ABTS) have been used widely for years. Each of these substrates varies greatly with respect to its performance characteristics such as detection sensitivity, working range and attainable signal:noise ratios. Substrate flexibility is also a key issue that affects an assay. Stability, development time requirements and the capacity to perform stopped and/or kinetic assays vary significantly among these substrates. Ideally, a substrate is stable, is very sensitive, has high attainable signal:noise ratios, possesses a broad dynamic range, and allows the user to perform stopped, nonstopped and kinetic assays. QuantaBlu Fluorogenic Peroxidase Substrate meets all the requirements of an ideal substrate for use in peroxidase detection. QuantaBlu Substrate generates a blue fluorescent product upon reaction with peroxidase. The fluorescent product does not photobleach. Fluorometric-based detection overcomes the limitations of colorimetric substrate detection, which does not allow for quantitation of greater than four optical density units. QuantaBlu Substrate allows for stopped, nonstopped and kinetic assays to be performed. Incubation times with QuantaBlu Substrate for stopped and nonstopped assays may be varied between 1 to 90 minutes at either room temperature or 37C. QuantaBlu Substrate exhibits a flat baseline in assays, which facilitates low-level detection sensitivity and allows for high signal:noise ratios. QuantaBlu Fluorogenic Peroxidase Substrate provides the ability to rapidly detect peroxidase at very low concentrations in short periods of time. Figure 23 illustrates the detection of peroxidase from 0-10 pg per well from 1.5 to 6.5 minutes of substrate incubation time. At cycle 1 (1.5 minute incubation) as little as 2.5 pg of peroxidase could be detected, while at cycle 6 (6.5 minute incubation) 0.625 pg of peroxidase could be detected. Highlights: More sensitive than TMB, OPD or ABTS substrates Flexible stopped, nonstopped or kinetic assays possible Large dynamic range (4 log peroxidase concentration range) Excellent stability working solution is stable for 24 hours Large Stokes shift; excitation/emission maxima of 325/420; range of 315-345/370-460 Does not photobleach
90 80 70 60 S/N Ratio 50 40 30 20 10 0 0 1,000 2,000 3,000 Biotinylated-HRP, pg/well QuantaBlu Substrate TMB ABTS OPD 4,000
Figure 22. Comparison of QuantaBlu Substrate to other substrates. QuantaBlu Substrate and the colorimetric substrates were incubated for 30 minutes at RT, followed by addition of a stop solution. QuantaBlu Substrate gave the greatest signal:noise (S/N) ratios and exhibited the lowest detection limit.
4.0 3.5 S/N Ratio 3.0 2.5 2.0 1.5 1.0 0 2 4 6 Biotinylated-HRP, pg/well 8 10 6 minutes 5 minutes 4 minutes 3 minutes 2 minutes 1 minutes
Figure 23. Rapid and sensitive detection with QuantaBlu Fluorogenic Peroxidase Substrate. Detection of bound biotinylated peroxidase at 0-10 pg/well using QuantaBlu Substrate. QuantaBlu Fluorogenic Peroxidase Substrate was read in a nonstopped mode using a 1-minute instrument cycle time between reads.
References Atamna, H., et al. (2000). Proc. Natl. Acad. Sci. 97, 686-691. Ayala, P., et al. (2002). Infect. Immun. 70, 5965-5971. Jefcoat, A.M., et al. (2001). Am J Physiol Lung Cell Mol Physiol. 281, L704-712. Savage, M.D., et al. (1998). Previews 2(1), 6-9. Savage, M.D., et al. (1998). Previews 2(2), 18-19.
Product # Description 15169 QuantaBlu Fluorogenic Peroxidase Substrate*
Includes: QuantaBlu Substrate QuantaBlu Stable Peroxide Solution QuantaBlu Stop Solution
U.S. Pkg. Size Price Kit $169

250 ml 30 ml 275 ml
15162
QuantaBlu NS/K Substrate (for nonstopped and kinetic assays)
Kit
$140
Includes: QuantaBlu Substrate 250 ml QuantaBlu Stable Peroxide Solution 30 ml * QuantaBlu Fluorogenic Peroxidase Assay Technology is protected by U.S. patent # 6,040,150.
51
CHOOSING A
SUBSTRATE
Highlights One component Ready to use Excellent choice when maximum sensitivities are not required Slow reaction that can be easily followed with a kinetic reader OPD can be easily dissolved in a substrate buffer such as Stable Peroxide Substrate Buffer (10X) (Product # 34062) Sensitivity Low Kinetic Measurement Possible Yes Absorbance Maximum 405 nm Color Green Product # 37615 Low High High Highest sensitivity Easy to use Results in seconds No DMF or DMSO in reagent One component Ready to use Highest sensitivity No DMF or DMSO in the reagent One component Ready to use No DMF or DMSO in the reagent One component Ready to use No DMF or DMSO in the reagent Very High Yes No No No 405 nm 492 nm 492 nm 450 nm Green Yelloworange Yelloworange Yellow 34026 34006 34005 34021
Colorimetric Substrates for HRP

Description 1-Step ABTS
ABTS Tablets OPD Tablets OPD Powder TMB Substrate Kit
1-Step Ultra TMB-ELISA
Very High
No
450 nm
Yellow
34028
1-Step Turbo TMB
High
No
450 nm
Yellow
34022
1-Step Slow TMB
Medium
Yes
450 nm
Yellow
34024
ABTS
ABTS (2,2-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt) is a water-soluble HRP substrate that yields a green end product upon reaction with peroxidase. The green product has two major absorbance peaks, 405 nm and 650 nm. ABTS is less sensitive than OPD and TMB in ELISA applications. It is less readily oxidized, and its color development is slower (approximately 20 minutes). This may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities. Highlights: One component Ready-to-use Excellent choice when maximum sensitivities are not required Slow reaction that can easily be followed with a kinetic reader
References Paing, M.M., et al. (2002). J. Biol. Chem. 277, 1292-1300. Sau-Ching Wu, S.-C., et al. (2002). Appl. Envir. Microbiol. 68, 3261-3269.
Product # Description 34026 ABTS 37615 1-Step ABTS U.S. Pkg. Size Price 50 tablets (10 mg/tablet) $ 81 250 ml (Ready-to-use) $ 70
OPD
OPD yields a water-soluble yellow-orange product when reacted with peroxidase with an absorbance maximum of 492. OPD can easily be dissolved in a substrate buffer such as Stable Peroxide Substrate Buffer (Product # 34062).
Product # Description 34005 OPD 34006 OPD Tablets Pkg. Size 25 g powder 50 tablets (5 mg/tablet) U.S. Price $ 45 $104
52
TMB
TMB is a chromogen that yields a blue color (measurable at 370 nm or 652 nm) when oxidized with hydrogen peroxide (catalyzed by HRP). The color then changes to yellow (measured at 450 nm) upon addition of sulfuric or phosphoric acid to stop the reaction. TMB is very sensitive and more quickly oxidized than other HRP substrates, resulting in faster color development. The 1-Step TMB Substrates are one-component substrates that require no preparation before use. Unlike other commercially available substrates, these products contain no DMF or DMSO. There are three formulations that differ primarily in their sensitivities. 1-Step Slow TMB is intermediate in sensitivity ideal for kinetic readings. The sensitivity of the 1-Step Turbo TMB compares to that of OPD used at approximately 1 mg/ml. 1-Step Ultra TMB-ELISA produces the highest signal:noise ratio and sensitivity in the picogram range. Highlights: Ready-to-use single component No hydrogen peroxide required No filtering required Noncarcinogenic Various sensitivities to suit any assay
Absorbance at 450 nm (TMB) Absorbance at 410 nm (ABTS) Turbo TMB Slow TMB ABTS 1.4 Absorbance at 450 nm 1.2 1.0 0.8 0.6 0.4 0.2 00 50 100 150 200 250 300 350 400 450 500 Concentration of Human IFN Gamma (pg/ml) Pierce 1-Step Ultra TMB - ELISA Competitor B Competitor D Competitor I Competitor K Competitor M Competitor N
Figure 25. 1-Step Ultra TMB-ELISA provides more signal than other TMB substrates.
9.0 7.5 S/N Ratio 6.0 4.5 3.0 1.5 0
Ul Pie tra rce TM 1B Ste p EL IS Co A m pe tit or Co B m pe tit or Co D m pe tit or Co I m pe tit or Co K m pe tit or Co M m pe tit or N
1.5
TMB Sources Used
1.0
Figure 26. 1-Step Ultra TMB-ELISA produces higher signal:noise (S/N) ratios than other TMB substrates.
References Hong, P.W., et al. (2002). J. Virol. 76, 12855-12865. Murphy, M.B., et al. (2003). Nucleic Acids Res. 31, e110. Su, S.V., et al. (2004). J. Biol. Chem. In press. Tek, V. and Zolkiewski, M. (2002). Protein Sci. 11, 1192-1198. Thomas, P.E., et al. (1976). Anal. Biochem. 75, 168-176. Weimer, B.C., et al, (2001). Appl. Envir. Microbiol. 67, 1300-1307. Wu, S.-C. and Wong, S.-L. (2002). Appl. Envir. Microbiol. 68, 1102-1108.
0.5
0.0 1.56 x 10-5 6.25 x 10-5 1.25 x 10-4 3.13 x 10-5 Dilution of HRP Conjugate (from 1 mg/ml stock)
2.5 x 10-4
Product # 34024 34022 34028 34021 Description 1-Step Slow TMB 1-Step Turbo TMB 1-Step Ultra TMB-ELISA TMB Substrate Kit
Includes: Peroxidase Substrate (TMB) Peroxide Solution (H202)
Figure 24. Comparison of sensitivities of Turbo TMB, Slow TMB and ABTS. Pkg. Size 250 ml 250 ml 250 ml Kit
200 ml 200 ml
U.S. Price $ 66 $ 91 $101 $102
CHOOSING A
SUBSTRATE
53
CHOOSING A
SUBSTRATE
The most common ELISA substrate for alkaline phosphatase is the chromogen p -nitrophenyl phosphate (PNPP), which is available in several formats. PNPP yields a yellow reaction product that is water-soluble and absorbs light at 405 nm. The 1-Step PNPP Substrate (Product # 37621) offers the convenience of a ready-to-use reagent with similar sensitivity to the two-component kit. The Phosphatase Substrate Kit (Product # 37620) includes PNPP tablets and diethanolamine buffer (5X). PNPP is also available as 25 g of powder (Product # 34045) or in tablet form (Product # 34047). Diethanolamine Substrate Buffer (Product # 34064) is a convenient, ready-to-use formulation sold as a 5X concentrate with an optimal and consistent pH. Soluble ELISA substrates for AP are summarized in Table 7, on page 46.
Kinetic Measurement Possible Yes Absorbance Maximum 405 nm Color Yellow Product # 37621
Alkaline Phosphatase Substrates

Alkaline phosphatase, a 140 kDa protein that is generally isolated from calf intestine, catalyzes the hydrolysis of phosphate groups from a substrate molecule, resulting in a colored or fluorescent product or the release of light as a byproduct. AP has optimal enzymatic activity at a basic pH (pH 8-10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. As a label for ELISA, AP offers a distinct advantage over other enzymes. Since its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period. Due to the large size of AP, it is very sensitive to freeze/thaw and should be aliquotted and stored at 4C.
Description 1-Step PNPP
Phosphatase Substrate Kit
Features/Benefits One component Ready to use Stable for 12 months The easiest-to-use PNPP substrate available Ideal for ELISA Easy to use Minimal assay-to-assay variability Low background
Sensitivity High
High
Yes
405 nm
Yellow
37620
PNPP Tablets PNPP Powder
High High
Yes Yes
405 nm 405 nm
Yellow Yellow
34047 34045
PNPP
Detection of alkaline phosphatase label in ELISA applications.
PNPP (p -Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications.1 When alkaline phosphatase and PNPP are reacted, a yellow water-soluble reaction product is formed. This product absorbs light at 405 nm. Pierce offers PNPP in four formats. PNPP is available either as a crystalline powder or 5 mg tablets. Also available is the Phosphatase Substrate Kit, which contains PNPP tablets and a Diethanolamine Buffer to yield more than one liter of substrate. The Diethanolamine Substrate Buffer (Product # 34064) is also provided individually as a 5X concentrate. The Pierce formulation has an optimal and consistent pH and it is stable even at a 1X concentration. Until recently, the method for preparing PNPP solutions required dissolving the powder or tablets in buffer and then diluting the solution to the desired concentration. Substrate instability made it necessary to prepare it on a daily basis. Pierce 1-Step PNPP circumvents this time-consuming and variable-introducing step by providing a single-component PNPP substrate. This substrate is stable for 12 months at 2-8C and can be stopped with conventional methods.
Reference 1. Snyder, S.L., et al. (1972). Biochim. Biophys. Acta 258, 178-187. Bosque, P.J., et al. (2002) Proc. Natl. Acad. Sci. 99, 3812-3817. Jan, J.-T., et al. (2000) J. Virol. 74, 8680-8691.
Product # Description 34045 PNPP 34047 PNPP Tablets 37620 Phosphatase Substrate Kit
Sufficient reagents for 1.05 liters of substrate. Includes: Diethanolamine Buffer PNPP Tablets
U.S. Pkg. Size Price 25 g powder $100 105 tablets $102 (5 mg/tablet) Kit $135
225 ml 105 tablets (5 mg/tablet)
37621
1-Step PNPP
100 ml
$ 77
54
ONPG Colorimetric -Galactosidase Soluble Substrate

For detection of -Galactosidase label in ELISA applications.
-Galactosidase Soluble Colorimetric Substrate

Description Highlights ONPG Forms a completely soluble product when reacted with -Galactosidase High extinction coefficient at 405 nm Yields a yellow product easily detectable in the visual range Sensitivity High Kinetic Measurement Possible Yes Absorbance Maximum 420 nm Color Yellow Product # 34055
When using -Galactosidase as the label for proteins in ELISA studies, a wide variety of substrates are available, including o -nitrophenyl--D-galactopyranoside (ONPG), naphthol-AS-BI-D-galactopyranoside (Nap-Gal) and 4-Methyl-umbelliferyl-D-galactopyranoside (Mum-Gal). However, it is important to choose a substrate with adequate solubility, that uses readily available equipment and that gives a significant reading over the background. ONPG is a superior -Galactosidase substrate option.1 The product formed is completely soluble and has a high extinction coefficient at 405 nm. The substrate yields a yellow product that is easily detectable in the visual range after stopping the reaction with 1 M sodium carbonate.
Reference 1. Craven, G.R., et al. (1965). J. Biol. Chem. 240, 2468-2477.
Product # Description 34055 ONPG U.S. Pkg. Size Price 5 g powder $ 63
Substrate Buffers
Substrate buffers for alkaline phosphatase and horseradish peroxidase.
Buffered stable peroxide is used by researchers who prefer to make their own HRP-ELISA substrates. This is done easily by adding a chromogen of choice to the buffer. The Stable Peroxide Substrate Buffer available from Pierce has a long shelf life (12 months after receipt), and is provided as a 10X concentrate. A total volume of 1,000 ml can be made from one 100 ml bottle of concentrate. If you use 10 ml of substrate per plate, you can make substrate for 100 plates from only one bottle of Stable Peroxide Substrate Buffer. Diethanolamine Substrate Buffer is used with soluble alkaline phosphatase substrates such as PNPP. Our formulation is convenient, ready-to-use and reduces the possibility of assay-to-assay variability. Diethanolamine Substrate Buffer is sold as 5X concentrate with an optimal, consistent pH. It is stable even at a 1X concentration.
Highlights: Ready-to-use Minimize assay-to-assay variability

References Louis, H., et al. (2000) J. Histochem. Cytochem. 48, 499-508. Neisewander, J.L., et al. (2000) J. Neurosci. 20, 798-805. Pifer, J., et al. (2002) J. Immunol. 169, 1372-1378.
Product # Description 34062 Stable Peroxide Substrate Buffer (10X) 34064 Diethanolamine Substrate Buffer (5X) U.S. Pkg. Size Price 100 ml $ 40 225 ml $ 47
CHOOSING A
SUBSTRATE
55
ELIFA SYSTEM
Easy-Titer ELIFA System
The Easy-Titer ELIFA System offers faster ELISA results by overriding limiting diffusion, which is the rate limiting step in ELISA.1,2 Limiting diffusion is a phenomenon in which soluble reactants are depleted near the reaction surface, and the rate of reaction is limited by diffusion of new reactants to the site.3 The enzyme-linked immunoflow assay (ELIFA) system actually reduces one-hour incubations to just a five-minute filtration step. A vacuum pulls substrates through a transfer membrane that has complementary ligands or enzymes bound to it. Cannulas precisely and quantitatively transfer unbound substrate to the collection chamber and transfer colored product into corresponding microwells (Figure 27) for analysis in an automated microplate reader. Assay times required for the Easy-Titer ELIFA System and a traditional ELISA are compared in Table 8. Highlights: Allows complete ELISA-type assays to be performed in just 25 minutes Offers greater sensitivity because transfer membranes bind more protein than plastic surfaces 96-well arrangement is compatible with 96-well microplates and multi-channel pipettes Most wash steps are eliminated, further reducing assay time No cross-contamination between samples because of tight seals Results are easily quantitated
Sample well Sample application plate Membrane Gaskets Transfer cannula Microplate Collection chamber Figure 27. Easy-Titer ELIFA System. Colored product is transferred to microplate wells for an ELISA or precipitated on the membrane for a dot blot. Table 8. Comparison of assay times for Easy-Titer ELIFA System and standard ELISA. Faster Than ELISA Adsorb antibody Block empty sites Bind antigen Antibody-enzyme Produce color Total Assay Time Easy-Titer System 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 25 minutes ELISA 1 hour 30-60 minutes 1 hour 1 hour 10-30 minutes 3.6-4.5 hours
References 1. Engvall, E. and Perlmann, P.O. (1971). Immunochemistry 8, 871-875. 2. Palomki, P. (1991). J. Immunol. Method 145, 55-63. 3. Rao, P.N. and Taraporewala, I.B. (1992). Steroids 57, 154-161. 4. Coons, A.A., et al. (1942). J. Immunol. 45, 159-170. 5. Weller, T.H. and Coons, A.H. (1954). Proc. Soc. Exp. Biol. (New York) 86, 789-794. 6. Schwab, C. and Bosshard, H.R. (1992). J. Immunol. Method 147, 125-134. 7. Stenberg, M. and Nygren, H. (1988). J. Immunol. Method 113, 3-15. 8. Valkirs, G.E. and Barton, R. (1985). Clin. Chem. 31, 1427-1431. 9. Ijsselmuiden, O.E., et al. (1989). J. Immunol. Method 119, 35-43.
56
Easy-Titer ELIFA System

Reduces each step to a five-minute filtration!
Highlights: Effective ELISA device allows immunoassays to be completed in just 25 minutes (each step is reduced to a 5-minute filtration) Ideal for ELISA-type immunoassays, dot blots and nucleic acid blots Binding kinetics of any ELISA step performed using Easy-Titer System overrides limiting diffusion Greater sensitivity because transfer membranes bind more protein than plastic surfaces 96-well arrangement is compatible with 96-well microplates and multichannel pipettes Most wash steps are eliminated, further reducing assay time Cross-talk between samples is eliminated due to the use of tight seals Results are quantitated easily by simply drawing color solution into a microplate and reading using a standard ELISA reader Use insoluble or precipitating substrates to create dot blot results
Primary Antibody Dilution Labeled Secondary Antibody Dilution

1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:1 1:2 1:4 1:8 1:16 1:32 1:64 A B C D E F G H 1 2 2 3 4 6 7 8 9 10 11 12
Figure 28. A checkerboard titration is a single experiment in which the concentration of two components is varied in a way to visualize the best signal:noise ratio conditions.
References 1. Paffard, S.M., et al. (1996). J. Immunol. Method 192, 133-136. Liu, Z.-H., et al. (2001). Proc. Natl. Acad. Sci. 98, 7289-7294. Menalled, L.B., et al. (2002). J. Neurosci. 22, 8266-8276.
Product # Description 77000 Easy-Titer ELIFA System*
Includes: Sample application plate Top silicone gasket Membrane support plate with bottom silicone gasket and 96 cannulas Vacuum chamber with o-ring Thumbscrews One-way valves Valve/tubing adapters Tubing Extra cannulas
U.S. Pkg. Size Price $1,011
4 4 4 4 ft 10
77015 77016 77070 77071
Biodyne A Nylon Membranes Biodyne B Nylon Membranes Easy-Titer Replacement Cannulas Easy-Titer Replacement 96-Well Clear Silicone Gaskets
25/pkg. 25/pkg. 100/pkg. 4/pkg.
$ 265 $ 190 $ 57 $ 104
* The Easy-Titer ELIFA System is protected by U.S. patent # 5,219,528.
ELIFA SYSTEM
57
BULK AND CUSTOM OFFERINGS

Need more product than our catalog package size offers?
Contact the Bulk & Custom Products Department at Pierce or your local distributor to request a price quote that will satisfy your large-scale needs. All of our popular ELISA products are available in bulk quantities* and in special package sizes. Delivery can be arranged to meet your schedule. Pierce can bring all of these critical components together to help you develop your own ELISAs on a grand scale:
Coated Microplates Blocking Buffers

Serum-based Blockers Milk-based Buffers
(96-well or 384-well)
Non-serum Blockers
Immunoassay Buffers
Sample Preparation Buffers Assay Reagent Diluents Wash Buffers Stop Solutions
Immunoassay Conjugates
Antibody-enzyme Conjugates Biotinylated Antibody Conjugates Streptavidin-enzyme Conjugates NeutrAvidin-enzyme Conjugates Protein A, G, L or A/G-enzyme Conjugates Nickel-chelate Enzyme Conjugate
Enzyme Conjugate Stabilizer Soluble Substrates

Colorimetric Fluorescent Chemiluminescent Partner with Pierce! Pierce can also help you commercialize your ELISA kit. We have the expertise, manufacturing capability and ISO 9001 certification to help you launch a new dimension to your business. Our scientific staff will work with your research team to implement world-class project management and meet your deadlines for a new product launch. Partner with Pierce to reach the next level of commercial success for your company.
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* Minimum quantities apply based on largest catalog package size available. Call for more information.
The choice is up to you!

The ChoiceCoat Custom Microplate-coating Service from Pierce Pierce has been coating plates for more than 10 years. Put this experience and expertise to work for you! Pierce can process up to 7,500 microplates per day and has developed novel chemistries and coatings for a number of applications. Following are just a few of the applications in which Pierce custom-coated microplates have been effective in generating valuable results. Competitive Ligand Binding Assays Oligonucleotide Binding Assays Protease Assays Kinase and Phosphatase Assays Sandwich ELISAs Pull-down Assays and Immunoprecipitation
ChoiceCoat Microplate Coating Options Available Microplate Type/Manufacturer Black (96- or 384-well) White (96- or 384-well) Clear (96- or 384-well) Clear bottom, Black Clear bottom, White Your Microplate
Note: Covalent chemistries also an option!
Coat Protein/Ligand Antibodies Bacterial Fusion Proteins Oligonucleotides or Peptides Biological Polymers Metal Chelates Your Ligand
Blocking Buffer SuperBlock Blocker StartingBlock Blocker Purified Casein BSA Serum Your Blocker
ChoiceCoat Custom Microplate Packaging Options Pierce can provide the appropriate packaging for your custom microplates, based upon your usage. Coated microplates can be packaged for large-screening applications (i.e., packaged in 25 plates ready for stacking) or for inclusion in a final kit for resale (i.e., single-pouch packages). Stability testing for custom microplates can be determined if long-term storage is critical for your application. Scheduled shipments can be established easily to make the microplates available when you need them most.
Minimum Quantities for ChoiceCoat Custom Microplate-coating Service Get started creating your own custom microplate with as few as 500 (96-well) microplates or 200 (384-well) microplates. Pierce will provide five coated microplates at no charge for testing purposes before we ship any lot of ChoiceCoat Custom Coated Plates to you. Pierce will work closely with you to develop the appropriate quality assurance tests so that we provide the most consistent product to meet your needs. In the United States, contact the Pierce Bulk & Custom Sales Department at 1-800-874-3723 or 815-968-0747. Outside the United States, contact your local Perbio Science office or Pierce distributor for a FREE ChoiceCoat Consultation.
Partner with Pierce to discover the best choices!
BULK AND CUSTOM OFFERINGS
59
RECOMMENDED READING
Antibodies: A Laboratory Manual
More than 700 pages of valuable information about antibodies and antibody production.
This valuable manual contains 726 pages of information about the theory, production and use of monoclonal and polyclonal antibodies. Oversized print and colorful graphics make it easy to read and understand.
Product # Description 15051 Antibodies: A Laboratory Manual
Harlow, E. and Lane, D., Published by Cold Spring Harbor Laboratory, 1988, 726 pages, Softcover
U.S. Price $132
Protein Methods
Treats practical protein-related issues that most other books do not address.
This expanded second edition provides step-by-step explanations of basic methods for extracting, handling, storing, measuring, solubilizing and concentrating proteins.
Product # Description 20001 Protein Methods
Bollag, D.M., et al., Published by Wiley-Liss, Inc., 1996, 400 pages, Comb-bound
U.S. Price $ 78
ELISA Theory and Practice

Basic principles and techniques.
The ELISA Guidebook presents the principles of the assay technique called enzyme linked immunosorbent assay. The book gives detailed descriptions of the basic ELISA principles and techniques that will aid both novice and experienced researchers in developing their own rapid and accurate assays. This 421-page book features the following range of topics:
Direct, indirect and competitive ELISA Capture ELISA on solid-phase Basic immunology Stages in ELISA Theoretical considerations Practical exercises Immunochemical techniques
Product # Description 15056 The ELISA Guidebook
Crowther, J.R., Published by Humana Press, Inc., 2001, 421 pages, Softcover
U.S. Price $100
60
Protein Assay Technical Handbook

The 38-page Protein Assay Technical Handbook is a helpful guide to choosing the best protein assay and standard for a sample. It contains abundant information on the most widely used protein assay methods (BCA, Lowry and Bradford Assays), including the principle behind each protein assay and its unique advantages and disadvantages. The handbook also includes methods for detection specific protein types such as histidine-tagged proteins, antibodies and proteases.
Product # Description 1601004 Protein Assay Technical Handbook U.S. Price FREE
Western Blotting Handbook and Troubleshooting Guide

This 52-page handbook contains full-length and abbreviated Western blotting protocols, tips on antibody and blocking buffer optimization, a chemiluminescent substrate selection guide, a procedure for stripping and reprobing Western blots, tips on clearer film development, and other information that will be useful to experts and first-timers alike. Learn more about four new Western blotting products that can improve your blots signal:noise ratio: Restore Western Blot Stripping Buffer, Qentix Western Blot Signal Enhancer, Erase-It Background Eliminator and StartingBlock Blocking Buffers.
Product # Description 1600990 Western Blotting Brochure U.S. Price FREE
Antibody Technical Handbook

This new 68-page handbook helps you choose the best methods to produce, purify, fragment and label antibodies. Topics include basic immunology, carrier proteins, adjuvants, antibody purification methods, antibody fragmentation with proteases, and labeling antibodies with a variety of tags (e.g., biotin, fluorophores, enzymes, iodine) for purification or detection.
Product # Description 1601092 Antibody Technical Handbook U.S. Price FREE
Sorry, books are nonreturnable.
RECOMMENDEDREADING
61
G r a s p t h e P r o t e o m e
ELISA
Pump up the volume!
Three ways to get the highest signal:noise ratio possible.

SuperSignal ELISA Substrates
Strong, sensitive signal clear detection in the femtogram range Low noise no auto-generated background like many fluorescent systems Immediate signal generation no waiting for detection Stable light output no drop in signal for up to 30 minutes
Turn down the noise!

StartingBlock Buffers
Limits background when others dont guaranteed serum protein- and biotinfree formulations Does not cross-react with rabbit antibodies a common problem with other blockers No-wait blocking just pipette and remove Convenient buffers are available with or without Tween-20 Detergent added
Reacti-Bind Coated Plates

Pre-coated plates for binding, antibodies, fusion proteins and biotin or for covalently binding targets Selective plates bind only the target ligand, not unwanted, noise-producing molecules Convenient normal- and high-binding versions are available
# 1601158 11/04
Switzerland: Tel 0800 56 31 40 euromarketing@perbio.com
www.piercenet.com
Belgium & Dist.: Tel +32 (0)53 83 44 04 euromarketing@perbio.com
Tel: 815-968-0747 or 800-874-3723 Fax: 815-968-7316 Customer Assistance E-mail: CS@piercenet.com Outside the United States, visit our web site or call 815-968-0747 to locate your local Perbio Science branch office (below) or distributor China: France: Germany: Hong Kong: The Netherlands: United Kingdom: Tel: (8610)8048 9552 Tel 0800 50 82 15 Tel 0228 9125650 Tel : 852 2753 0686 Tel 076 50 31 880 Tel 0800 252185 support@perbio.com.cn euromarketing@perbio.com de.info@perbio.com salesHK@perbio.com euromarketing@perbio.com uk.info@perbio.com
Pierce Biotechnology, Inc., 2004. Printed in the U.S.A. Pierce products are supplied for laboratory or manufacturing applications only. Prices listed herein are accurate at time of printing. CyDye, Cy3 and Cy5 are registered trademarks of Amersham Biosciences. Tween and Brij are registered trademarks of ICI Americas. Kathon and Triton are registered trademarks of Rohm & Hass Co. Biodyne is a trademark of Pall Corporation. Texas Red is a trademark of Molecular Probes, Inc.

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G r a s p t h e P r o t e o m e

ELISA AND ELISPOT PRODUCTS

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Enzyme-linked immunosorbent assays (ELISAs) are designed for detecting

ELISA AND ELISPOT PRODUCTS

ELISA THE PIERCE WAY

Use a pre-coated plate for efficiency and consistency.

Block unoccupied sites.

STEP 3. Add sample and incubate

STEP 4. Formulate wash buffers

Add detergent to buffers

STEP 5. Primary and Secondary Antibodies

STEP 6. Enzyme Substrates for Detection

Typical ELISA Protocol

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Direct Assay Figure 1. Immunoassay formats.

Capture Assay Sandwich or ELISPOT

Direct vs. Indirect Detection Techniques for ELISA

Primary Antibody Dilution

Figure 2. A checkerboard titration using a precipitating substrate.

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(Coating Antibody at 2 g/ml)

HRP-Conjugated Streptavidin Titration Curves (Biotin-Labeled Antibody at 60 ng/ml)

SELECTING AN ELISA PLATE

ImmunoWare Reagent Reservoirs

U.S. Pkg. Size Price 200/pkg. $ 99

Sealing Tape for 96-Well Plates

U.S. Pkg. Size Price 100/pkg. $ 48

Reacti-Bind 96-Well Plates

Reference Foster, B.A., et al. (1999). Science 286, 2507-2510.

Reacti-Bind 8-Well Strip Plates

U.S. Pkg. Size Price 100/pkg. $462

Accessory 15033 Reacti-Bind Strip Well Ejector 1/pkg. $ 29

Reacti-Bind DNA Coating Solution

U.S. Pkg. Size Price 100 ml $ 63

SELECTING AN ELISA PLATE

Figure 4. Coupling reactions of Reacti-Bind Activated Plates.

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Phosphopeptide Detection Assay Comparison of Biotin-Binding Protein Coated Plates

0 0 5 10 15 Biotinylated Phosphopeptide (pM/well)

0.8 Pierce Absorbance at 490 nm 0.6 Competitor B

0.0 0 10,000 20,000 Cells/Well 30,000 40,000

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Reacti-Bind Protein A, G and A/G Coated Plates

Figure 7. Properly oriented antibodies retain higher activity.

Reacti-Bind Secondary Antibody Coated Plates

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Reacti-Bind Anti-GFP Coated Plates

Reacti-Bind NeutrAvidin Coated Plates

NeutrAvidin Coated Plate Characteristics

1.5 Absorbance at 450 nm

0.05 0.10 Units src kinase

Reacti-Bind NeutrAvidin HBC Coated Plate Characteristics

* U.S. patent pending on high-binding capacity plate-coating technology.

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Reacti-Bind Streptavidin Coated Plates

Reacti-Bind Biotin Coated Plates

* U.S. patent pending on high-binding capacity plate-coating technology.

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HisGrab Nickel Coated Plates

Figure 11. Binding comparison of histidine-tagged ATF fusion protein.

* U.S. patent pending on high-binding capacity plate-coating technology.

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