Enzyme Immunoassay First introduced in 1966, as an alternative to fluorescence conjugates In 1971, enzymes labelled antigens and antibodies were

re developed EIAs are of two basics type : Homogenous o There is no need to separate the bound and free fractions o Test can be completed in one step, with all reagent added simultaneously o Can be used only to assay of haptens, such as drug and not microbial antigens and antibodies o Eg. Enzyme multiplied immunoassay technique (EMIT) It is a simple method for small molecules drugs such as opiates, cocaine, barbiturates or amphetamine in serum Heterogeneous o It requires separation of bound and free fractions either by centrifugation or by absorption on solid surfaces and washing o It is a multistep procedure, with reagent added sequentially o Major type of EIA is ELISA Enzyme linked immunosorbent assay (ELISA) Principle: o ELISA test is so named because it involves the use of immunosorbent, an adsorbing material specific for one of the components of the reaction, antigen or antibody. This may be Particulate such as o Cellulose o Agarose Solid phase such as o Polystyrene o Polyvinyl o Polycarbonate tubes o Microwells o Membranes o Disc of polyacrylamide, paper or plastic o A general ELISA is a five step procedure Coat the microtiter plate wells with antigen Block all unbound sites to prevent false positive results Add primary antibody (e.g. rabbit monoclonal antibody) to the wells Add secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG) Reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. Selecting and Coating ELISA Plates Selection o Assay microplate with a minimum protein-binding capacity of 400 ng/cm is used.

o The choice of plate colour depends upon the signal being detected. Clear polystyrene flat bottom plates are used for colorimetric signals while Black or white opaque plates are used for fluorescent and chemiluminescent signals Coating o Plate coating is achieved through passive adsorption of the protein to the plastic of the assay microplate. o This process occurs though hydrophobic interactions between the plastic and non-polar protein residues. o Although individual proteins may require specific conditions or pretreatment for optimal binding, the most common method for coating plates involves adding a 2-10 g/ml solution of protein dissolved in an alkaline buffer such as phosphatebuffered saline (pH 7.4) or carbonate-bicarbonate buffer (pH 9.4). o The plate is left to incubate for several hours to overnight at 4-37C. o Typically, after removing the coating solution, blocking buffer is added to ensure that all remaining available binding surfaces of the plastic well are. o Coated plates can be used immediately or dried and stored at 4C for later use, depending on the stability of the coated protein. With the exception of competition ELISAs, the plates are coated with more capture protein than can actually be bound during the assay in order to facilitate the largest working range of detection possible. Hooking o Some proteins, especially antibodies, are best coated on plates at a concentration lower than the maximum binding capacity in order to prevent nonspecific binding in later steps by a phenomenon called "hooking". o It results from proteins getting trapped between the coating proteins which prevents effective washing and removal of non bound proteins. o When hooking nonspecifically traps detection primary and secondary antibodies, high background signal results lowering the signal to noise ratio and thus sensitivity of an assay. Blocking Buffers o The binding capacity of microplate wells is typically higher than the amount of protein coated in each well. o The remaining surface area must be blocked to prevent antibodies or other proteins from adsorbing to the plate during subsequent steps. o A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. o The blocking buffer is effective if it improves the sensitivity of an assay by reducing background signal and improving the signal-to-noise ratio. o 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.

o Commonly using blocking buffers are Purified bovine serum albumin in PBS or TBS Purified casein in PBS or TBS Non-fat dry milk proteins in TBS Wash buffers o Washing steps are necessary to remove nonbound reagents and decrease background, thereby increasing the signal:noise ratio o Insufficient washing will allow high background, while excessive washing might result in decreased sensitivity caused by elution of the antibody and/or antigen from the well. o Washing is performed in a physiologic buffer such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) without any additives. o Usually, a detergent such as 0.05% Tween-20 is added to the buffer to help remove nonspecifically bound material. o Another common technique is to use a dilute solution of the blocking buffer along with some added detergent Secondary antibodies Secondary antibodies may be provided in three formats: whole IgG, divalent F(ab')2 fragments and monovalent Fab fragments Whole IgG o Secondary antibodies are typically affinity purified from the pooled serum of immunized hosts. o Whole IgG secondary antibodies produced in the manner are widely applicable, easiest to produce and least expensive for purchase F(ab')2 antibody fragments o While whole immunoglobulins are compatible with most assays, certain methods benefit from removing the Fc portion of the antibody in order to reduce the mass of the antibody or because the probed samples contain active Fc-binding proteins (e.g., Fc receptors, Protein A, Protein G). o The Fc portion can be removed from several species of IgG by digestion with pepsin, leaving the divalent F(ab')2 fragment (~100 kDa) of the antibody intact Fab antibody fragments o The monovalent Fab antibody fragments are useful in blocking applications and other special circumstances where controlled binding ratios and/or the elimination of Fc interactions is required. o Some species of IgG can be enzymatically digested with papain to cleave antibody between the antigen binding domain and hinge region and produce two Fab fragments and an Fc fragment. o Ficin digestion is the recommended treatment to remove the Fc portion of mouse IgG1 antibodies. Enzyme labelling of secondary antibody Enzymes are used to detect target proteins by being conjugated to secondary antibody

The benefits of using enzyme probes (also called reporters) to detect a target protein are three-fold: o High sensitivity The signal output can be easily detected, and therefore low concentrations of target proteins can be identified. Finally, enzyme reporters exhibit rapid turnover, which increases the amount of substrate that a single enzyme converts during a given unit of time. o Long shelf life The enzymes are quite stable when stored properly, and while the enzyme substrate is light-sensitive, the enzyme itself is not sensitive to degradation by ambient light. o Output versatility Substrates that yield either chromogenic, chemiluminescent or fluorescent output are available for the most common enzyme probes. Limitations to their use o Large size Enzyme reporters are considerably larger than organic fluorescent compounds (e.g., FITC, TRITC, AMCA) and therefore may interfere with the biological function of proteins to which they are conjugated. o Substrate requirement Enzyme probes require the addition of a substrate for protein detection, and depending on the substrate used, this reaction can be sensitive to environmental conditions (e.g., light, temperature) and ambient light. o Endogenous interference The enzymes used to detect target proteins in a sample are often expressed in the experimental system used (e.g., tissues, cells), which will also process the substrate and yield nonspecific background signal unless inhibited. Enzyme reporters for probes Horseradish peroxidase o Catalyse the transfer of two electrons from substrate to hydrogen peroxide to produce an oxidised substrate and water o HRP-IgG conjugates are superior to alkaline phosphatase and -galactosidase conjugates due to their o Higher specific enzyme activity (more HRP/mole of antibody) and o Immunological reactivity (less steric hindrance because of the size of HRP) o Drawbacks: Nonspecific staining that results from endogenous peroxide activity in certain tissues Sensitivity to degradation by microorganisms as well as the antibacterial agents used to fight them

Mutagenicity or carcinogenicity of the reaction products of some horseradish peroxidase substrates Inhibitor of HRP are o Sodium azide o Cyanide o Sulphide Substrates are o 3,3'-diaminobenzidine (DAB) o 3,3',5,5'-tetramethylbenzidine (TMB) o 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) o o-phenylenediamine dihydrochloride (OPD) Alkaline phosphatase o Hydrolyze phosphates from nucleotides and proteins o Two form of alkaline phosphatase Endogenous tissue ALP Intestinal ALP o Calf intestinal ALP are ideal in applications o Activity is not inhibited by antibacterial agents such as sodium azide or thiomersal o The endogenous ALP can be inhibited by thiomersal, so it can be used as a markers for different tissues o Substrates are Combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) p -Nitrophenyl Phosphate (PNPP) Glucose oxidase o Isolated from Aspergillus niger that catalyzes the oxidation of -D-glucose to produce hydrogen peroxide and gluconic acid o Inhibitors of glucose oxidase include Ag+, Hg2+, Cu2+ p -chloromercuribenzoate Phenylmercuric acetate. o Label of choice for samples with high endogenous peroxidase or phosphatase activity, as there is no endogenous glucose oxidase activity in mammalian tissues o Substrate Nitro blue tetrazolium chloride (NBT) Beta-galactosidase o Isolated from E. coli that is capable of hydrolyzing a variety of galactopyranoside derivatives, which produce both water-soluble and waterinsoluble products

o Sensitive and demonstrates no endogenous activity in mammalian cells, and therefore it is useful in applications where endogenous enzyme activity is a persistent problem o One disadvantage of -galactosidase is a lack of substrate variety o Substrate 5-bromo-4-chloro-3-indoyl--D-galactopyranoside (BCIG or X-Gal) Types of substrates Chromogenic substrate o Divide into two group based on the nature of product of enzyme susbtrate reaction o Precipitating substrates It forms insoluble products that precipitate onto the sample or membrane. Commonly used for IHC and Western blotting. Require no more specialized equipment than a light microscope to detect the presence, intensity and localization of the insoluble product Example o DABHRP o BCIPAP o NBTAP and GO o Soluble substrates Forms water-soluble coloured products that dissolve into the test solution Commonly used in ELISA assays. Require a microplate reader is used to measure the absorbance and therefore the amount of soluble product in solution Example: o ABTSHRP o OPDHRP o PNPPAP o Chromogenic substrates are typically used to detect abundant proteins, and reaction development can be monitored visually; this allows greater flexibilty for optimization compared to chemiluminescent or fluorescent blotting systems Chemiluminescent Substrates o Offer several advantages over other detection methods, including a large linear response range that allows the detection and quantitation of a wide range of protein concentrations o Luminol is one of the most commonly used chemiluminescent reagents o Luminol is oxidized by peroxide to form the excited-state 3-aminophthalate, which decays to a lower energy state by releasing photons of light at a wavelength of 425nm o Commonly used for the short-term, rapid detection of protein detection by Western blot analysis using x-ray film, phosphorimagers or CCD cameras

Fluorescent Substrates o These substrates remain non-fluorescent or emit low fluorescence until metabolized by the enzyme probe, after which they emit intense fluorescence. o These substrates offer greater sensitivity and the ability for rapid quantitation because of fluorescent microscopic imaging, fluorescent microplate readers and analytical software Enzyme labeling Several methods have been developed and used routinely to prepare antibody or other protein conjugates with enzymes such as HRP and AP Glutaraldehyde o One of the simplest crosslinking reagents used for protein conjugation. o It reacts with amine groups to create crosslinks by one of several routes. o Under reducing conditions, the aldehydes on both ends of glutaraldehyde couple with amines to form secondary amine linkages. o The reagent is highly efficient at protein conjugation but has a tendency to form various high-molecular weight polymers, making results difficult to reproduce. Periodate o Periodate-activation followed by reductive amination is an excellent strategy for conjugation of HRP and other glycoproteins o Treatment of the glycosylated enzyme with periodate results in oxidation of sugar cis-diol groups resulting in formation of aldehyde groups. o These aldehyde groups will then efficiently react with primary amines of an added antibody or other molecule. o The result is stable conjugation by permanent amide bonds. Sulfo-SMCC or other heterobifunctional crosslinkers o Can be used to conjugate enzyme molecules to antibody or other proteins in single- or two-step methods. o The latter approach enables more precise control of the crosslinking result, because the enzyme can be activated and purified in one step and then conjugated to the target protein in a second step. o This limits the directionality of crosslinking to one specific orientation (e.g., amines on the enzyme to sulfhydryl groups on the antibody) and the possibility of over polymerization.

Types of ELISA Direct Indirect Sandwich Competitive Capture ELISPOT Cylinder or Cassette

Direct ELISA Direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct detection can be performed with antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection is not widely used in ELISA but is quite common for immunohistochemical staining of tissues and cells Advantages o Quick because only one antibody and fewer steps are used. o Cross-reactivity of secondary antibody is eliminated. Disadvantages o Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. o Labeling primary antibodies for each specific ELISA system is time-consuming and expensive. o No flexibility in choice of primary antibody label from one experiment to another. o Minimal signal amplification.

Indirect ELISA The indirect detection method uses a labeled secondary antibody for detection and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody In this method antigen is coated in the well and then the patient serum containing the specific antibody (primary antibody) is added in the well After sufficient incubation the well are washed to remove the unbound the antibody Then the secondary antibody which is enzyme labeled is added to the well, this will bind specifically to the primary antibody After incubation wash to remove the unbound secondary antibody and add the substrate which is converted by the enzyme into a coloured product Advantages o A wide variety of labeled secondary antibodies are available commercially.

o Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. o Maximum immunoreactivity of the primary antibody is retained because it is not labeled. o Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. o Different visualization markers can be used with the same primary antibody. Disadvantages o Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal o An extra incubation step is required in the procedure Competitive ELISA o Competitive binding process executed by original antigen (sample antigen) and add-in antigen o A simplized procedure list is as follow: Primary antibody (unlabeled) is incubated with sample antigen. Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the same antigen. Unbound antibody is removed by washing the plate. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.") The secondary antibody that is specific to the primary antibody and conjugated with an enzyme is added. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. o For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. o Advantages High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected Suitable for complex samples, since the antigen does not require purification prior to measurement Flexibility and sensitivity, since both direct and indirect detection methods can be used Sandwich ELISA o Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection o The sandwich ELISA quantifies antigens between two layers of antibodies (i.e. capture and detection antibody). o The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich o Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies

o The steps are as follows: Prepare a surface to which a known quantity of capture antibody is bound. Block any nonspecific binding sites on the surface. Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed. A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is stuck between two antibodies); Apply enzyme-linked secondary antibodies as detection antibodies that also bind specifically to the antibody's Fc region (non-specific). Wash the plate, so that the unbound antibody-enzyme conjugates are removed. Apply the substrate Measure the absorbency of the plate wells to determine the presence and quantity of antigen o Advantages The sample does not have to be purified before analysis Assay can be very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA), but lower than ELISpot IgM antibody capture ELISA (MAC ELISA) o The MAC-ELISA is the ideal test because it is both simple and sensitive (i.e., highly likely to find true-positives) and it can be used with both serum and cerebrospinal fluid (CSF) specimens. As the IgM does not cross the BBB, +ve MAC-ELISA of the CSF implies CNS infection o Important in the detection of those conditions requiring rapid diagnosis: Maternal Rubella Japanese Encephalitis St. Loius Encephalitis o Principle Serum antibodies of the IgM class, when present, combine with antihuman IgM antibodies attached to the polystyrene surface of the microwell test strips. Antigen is then added to the microwell HRP Conjugated Monoclonal Antibody (MAb) is added to antigen, which allows the formation of antigen-MAb complexes. Residual serum is removed from the assay plate by washing After incubation, the microwells are washed and a colourless substrate system is added. The substrate is hydrolysed by the enzyme and colour is produced Colour development is indicative of the presence of IgM antibodies in the test sample ELISPOT (enzyme-linked immunospot assay) o Refers to ELISA-like capture and measurement of proteins secreted by cells that are plated in PVDF-membrane-backed microplate wells.

o It is a "sandwich" assay in which the proteins (cytokines) are captured locally as they are secreted by the plated cells such as T-cells o After cells are washed off, captured cytokines are bond by biotin-conjugated secondary antibodies. o Enzyme-avidin combines with biotin and reveals colors with certain substrate. o So far there will be visual spots on membranes. o Every spot represents single cell that secrets certain cytokine. These cells are called sports forming cells, SFCs. o Positive ratio can be calculated by a divide of total cell number by sports number o ELISPOT is like a Western blot in that the result is spots on a membrane surface

(d) MAC ELISA