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TERM PAPER: Immunology and

Immunotechnology

Topic:ELISA(Enzyme Linked
ImmunoSorbent Assay)

SUBMITTED BY:-
SUBMITTED TO:-
Name: -Nitish Pathania Mrs.
Sarabjot Kaur
Sec: -K7802-A06
Reg. No. : -10803694

ACKNOWLEDGEMENT
As The Saying goes
“No Work Is Done Without Cooperation”
I am extremely grateful and remain indebted to my friends and my guide Mrs. Sarabjot
Kaur for being a source of inspiration and for their constant support in the Design,
Implementation and Evaluation of this Term Paper. I am thankful to them for their
constant constructive criticism and invaluable suggestions, which benefited me a lot while
developing this paper on topic “ELISA(Enzyme Linked ImmunoSorbent Assay)”.
Also they provide me a constant source of inspiration and motivation for doing hard work
while preparing this term paper. Through this column, it would be my utmost pleasure to
express my warm thanks to them for their encouragement, co-operation and consent
without which I mightn’t be able to accomplish this work of Term Paper.
I also want to express my gratitude to my God and my parents those are a great source for
me of inspiration. I am again very thankful to Mrs. Sarabjot Kaur who gave me this
chance to express my thoughts with the help of this Term paper to make my concepts very
clear and make me aware about the immunotechnology of ELISA and its various
applications in immunology
Thanking You Nitish Pathania
 Abstract:
ELISAs (Enzyme-Linked-Immuno-Sorbent Assays) have resulted in a revolution in diagnostic
procedures in medicine, biochemistry, drug discovery, etc. Enzyme-linked antibodies are used
routinely to detect very small amounts of viruses, hormones, and other antibodies! A variant of
the ELISA procedure is even used these days by the layman in home pregnancy tests.

The ELISA technique is based on enzyme-linked antibodies, that is an antibody (which is a

protein) covalently attached to an enzyme (another protein). In vivo, antibodies (also known as
immunoglobulins) are proteins that specifically bind to molecules, which are referred to as
antigens that are foreign to the host organism. The antigen may be almost any small molecule or
it may be a polypeptide or a portion of a protein or even a carbohydrate. Formation of the
antibody-antigen complex is a key step in the immune response and serves to cause elimination
of small molecules, or viruses, or even whole cells that are foreign to the host organism.
Antigens bind non-covalently and highly specifically to antibodies.

The ELISA procedure takes advantage of the specificity and high affinity of antibodies for a
particular antigen to detect minute quantities of a specific antigen in a complex mixture. The
detection of a specific antibody-antigen complex relies on measuring the activity of an enzyme
covalently linked to the antibody. The enzyme used is usually one that catalyzes a reaction that
produces a colored product. Since the enzyme catalyzes the turnover of thousands of substrate
molecules, and because absorbance of the dye is proportional to the amount of antibody-antigen
complex present in the sample, the ELISA procedure is capable of detecting concentrations of
antigen as low as (10-15 M).
Antibodies are produced in vivo by B-cells, which are one of the major classes of cells
responsible for the immune response. Antibodies can be produced by injecting an animal (e.g., a
goat or a rabbit) with an antigen of interest. Within about one week, the immune system of the
animal launches an immune response and many copies of antibody proteins which bind to the
antigen are produced.
The antibodies can be identified, purified and covalently bound to an enzyme. The enzyme-
linked antibody can now act as a specific probe to detect the presence of the antigen in any
sample. Many antibodies used in the ELISA procedure are commercially available as enzyme-
linked antibodies (also referred to as enzyme linked antibody conjugates). A different enzyme
linked antibody would be required, however, for each antigen to be detected.

Why known as......? Enzyme Linked Immunosorbent Assay(ELISA)

1. Antigen of interest is absorbed on to plastic surface (sorbent).
2 Antigen is recognized by specific antibody (immuno).
3. This antibody is recognized by second antibody (immuno) which has enzyme attached
(enzyme-linked).
4. Substrate reacts with enzyme to produce product, usually coloured.

Introduction:

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme

immunoassay (EIA), is a biochemical technique used mainly in immunology to detect the
presence of an antibody or an antigen in a sample. ELISAs are typically performed in 96-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. Having the
reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound
from nonbound 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 or other tag can be linked directly to the primary antibody or introduced
through a secondary antibody that recognizes the primary antibody. It also can be linked to a
protein such as streptavidin if the primary antibody is biotin labeled. The most commonly used
enzyme labels 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 β-galactosidase, acetylcholinesterase and catalase.
This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme
can convert to some detectable signal, most commonly a colour change in a chemical substrate.

BASIC PRINCIPLE OF ELISA:

• Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).
• The enzyme converts a colorless substrate (chromogen) to a colored product, indicating
the presence of Ag: Ab binding.
• An ELISA can be used to detect either the presence of Antigens or antibodies in a
sample depending how the test is designed.
• ELISA was dveloped in 1970 and became rapidly accepted

ELISA Formats:
ELISAs can be performed with a number of modifications to the basic procedure. The key step,
immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay
plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then
detected either directly (labeled primary antibody) or indirectly (labeled secondary antibody).
The most powerful ELISA assay format is the sandwich assay. This type of capture assay is
called a “sandwich” assay because the analyte to be measured is bound between two primary
antibodies – the capture antibody and the detection antibody.

An ELISA can also be performed as a competitive assay. This is common when the antigen is
small and has only one epitope, or antibody binding site. One variation of this method consists of
labeling purified antigen instead of the antibody. Unlabeled antigen from samples and the labeled
antigen compete for binding to the capture antibody. A decrease in signal from purified antigen
indicates the presence of the antigen in samples when compared to assay wells with labeled
antigen alone. Fluorescent tags and other alternatives to enzyme-based detection can be used for
plate-based assays.

Direct vs. Indirect Detection ELISA Formats:

The 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 staining of tissues and cells.

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 and the primary antibody.
In a sandwich ELISA, it is critical that the secondary antibody be specific for the detection
primary antibody only (and not the capture antibody) or the assay will not be specific for the
antigen.

Selecting and Coating ELISA Plates:

When developing a new ELISA for a specific antigen, the first step is to optimize the plate-
coating conditions for the antigen or capture antibody. Begin by choosing an assay microplate
with a minimum protein-binding capacity of 400 ng/cm². The choice of plate color 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.

Plate coating is achieved through passive adsorption of the protein to the plastic of the assay
microplate. This process occurs though hydrophobic interactions between the plastic and non-
polar protein residues. Although individual proteins may require specific conditions or
pretreatment for optimal binding, the most common method for coating plates involves adding a
2-10 μg/ml solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline
(pH 7.4) or carbonate-bicarbonate buffer (pH 9.4). The plate is left to incubate for several hours
to overnight at 4-37°C. Typically, after removing the coating solution, blocking buffer is added to
ensure that all remaining available binding surfaces of the plastic well are covered. Coated plates
can be used immediately or dried and stored at 4°C for later use, depending on the stability of the
coated protein.
It is important to note that optimal coating conditions can vary with each 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.

Pre-coated ELISA Plates:

For antibodies and proteins, coating plates by passive adsorption usually works well. However,
problems can arise from passive adsorption, including improper orientation, denaturation, poor
immobilization efficiency and binding of contaminants along with the target molecule.
Antibodies can be 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 capability. Fusion
proteins can be attached to a microplate in the proper orientation using glutathione, metal-chelate,
or capture-antibody coated plates.

Antibodies and Probes for ELISA:

Either monoclonal or polyclonal antibodies can 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 different non-overlapping epitopes. When the antigen binds to the
capture antibody, the epitope recognized by the detection antibody must not be obscured or
altered.

Another design consideration in choosing antibodies is cost. A polyclonal antibody is generally

less expensive (~5 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.

Blocking Buffers and Wash Buffers:

The binding capacity of microplate wells is typically higher than the amount of protein coated in
each well. The remaining surface area must be blocked to prevent antibodies or other proteins
from adsorbing to the plate during subsequent steps. 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. The blocking buffer is effective if it improves the sensitivity of an assay by
reducing background signal and improving the signal-to-noise ratio.
In addition to blocking, it is essential to perform thorough washes between each step of the
ELISA. Washing steps are necessary to remove non-bound reagents and decrease background,
thereby increasing the signal : noise ratio. 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. Washing is performed in a physiologic buffer such as Tris-buffered saline
(TBS) or phosphate-buffered saline (PBS) without any additives

Detection Strategies for ELISA:

The final stage in all ELISA systems is a detection step. Unless a radioactive or fluorescent tag
was used, this involves the introduction of an enzyme substrate. The enzyme converts the
substrate to a detectable product. If an ELISA has been constructed and developed properly, then
the intensity of signal produced when the substrate is added will be directly proportional to the
amount of antigen captured in the plate and bound by the detection reagents.

Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA

substrates allow direct visualization and enable kinetic studies to be performed. Fluorescent
ELISA substrates are not as common and require a fluorometer that produces the correct
excitation beam to cause signal emission to be generated from the fluorescent tag.

ELISA Kits, ELISPOT and In-Cell ELISA:

In addition to the individual components and general principles of ELISA discussed in this
article, complete kits are available for detection of specific cytokines and other targets, such
as interferon gamma (IFN gamma) and interleukin. ELISA development kits (Mini Kits and
Screening Sets) for specific targets include only matched pairs of antibodies and a protocol for
coating plates.

ELISPOT kits (enzyme-linked immunospot assay) for measurement of cytokines in single cells
are available for human IFN gamma, IL-2 and TNF alpha. An alternative ELISPOT for
application to nearly any specific analyte for which antibodies exist is the In-Cell ELISA,
developed for performing ELISA assays on plated cells.

ELISA and its Various Types:

The Enzyme-Linked Immuno Sorbent Assay (ELISA) is a biochemical technique used mainly in
immunology to detect the presence of an antibody or an antigen in a sample using two antibodies.
One antibody is specific to the antigen and the other reacts to antigen-antibody complexes, and is
coupled to an enzyme. This second antibody, which accounts for "enzyme-linked" in the assay's
name, can also cause a chromogenic or fluorogenic substrate to produce a signal.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the
enzyme molecules will produce many signal molecules.
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of
antibody in a sample, it is a useful tool both for determining serum antibody concentrations and
also for detecting the presence of antigen.
1. Direct ELISA
 2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
5. Multiplex ELISA

Figure 1. Types of ELISA.

Shown in diagram generalizing models of different types of ELISA. Further variation of the assay
are there depending upon the labeling and signal detection methodology. The basic approaches
stay the same: fixing either antigen or antibody and detecting antibody-antigen complex.
1. Direct ELISA:

The direct ELISA uses the method of directly labeling the antibody itself. Micro well plates are
coated with a sample containing the target antigen, and the binding of labeled antibody is
quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary
antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of
cross-reactivity of the secondary antibody with components in the antigen sample. However, the
direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming
and expensive proposition. In addition, certain antibodies may be unsuitable for direct labeling.
Direct methods also lack the additional signal amplification that can be achieved with the use of a
secondary antibody.

2. Indirect ELISA:

The indirect, two-step method uses a labeled secondary antibody for detection. 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.

Table 2. Comparison of Direct and Indirect ELISA Detection Methods

Direct Detection
Advantages of Direct Quick methodology since only one antibody is used.
Detection Cross-reactivity of secondary antibody is eliminated.
Disadvantages of Immunoreactivity of the primary antibody may be reduced as a result of
Direct Detection 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.
Indirect Detection
Advantages of Indirect A wide variety of labeled secondary antibodies are available
Detection commercially.
Versatile, since 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 Cross-reactivity may occur with the secondary antibody, resulting in
Indirect Detection nonspecific signal.
An extra incubation step is required in the procedure.

3. Sandwich ELISA:
The sandwich ELISA measures the amount of antigen between two layers of antibodies. The
antigens to be measured must contain at least two antigenic sites, capable of binding to the
antibody, since at least two antibodies act in the sandwich. For this reason, sandwich assays are
restricted to the quantitation of multivalent antigens such as proteins or polysaccharides.
Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of
antigens is low and/or they are contained in high concentrations of contaminating protein.

To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase
typically attached to the bottom of a plate well. Antigen is then added and allowed to complex
with the bound antibody. Unbound products are then removed with a wash, and a labeled second
antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the
“sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody
bound to the matrix, through the use of a colorimetric substrate.
• Major advantages of this technique are that the antigen does not need to be purified prior
to use, and that these assays are very specific.
• However, one disadvantage is that not all antibodies can be used. Monoclonal antibody
combinations must be qualified as “matched pairs”, meaning that they can recognize
separate epitopes on the antigen so they do not hinder each other’s binding.

The sensitivity of the sandwich ELISA is dependent on four factors:

• The number of molecules of the first antibody that are bound to the solid phase.
• The avidity of the first antibody for the antigen.
• The avidity of the second antibody for the antigen.
• The specific activity of the second antibody.
 4. Competitive ELISA:
When two “matched pair” antibodies are not available for a target, another option is the
competitive ELISA. The advantage to the competitive ELISA is that non-purified primary
antibodies may be used. Although there are several different configurations for competitive
ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase.
The enzyme may be linked to either the antigen or the primary antibody. The example shown in
Figure 1 is a labeled antigen as the competitor. In this type of ELISA, there is an inverse
relationship between the signal obtained and the concentration of the analyte in the sample, due
to the competition between the free analyte and the ligand-enzyme conjugate for the antibody
coating the microplate, i.e. the more analyte the lower the signal.
 5. Multiplex ELISA:
A logical progression of the widely used microtiter plate ELISA is toward a protein array format
that allows simultaneous detection of multiple analytes at multiple array addresses within a single
well. There are different types of multiplex ELISA have been developed and in practice. One of
the examples is to measure antigens by coating or printing capture antibodies in an array format
within a single well to allow for the construction of "sandwich" ELISA quantification assays.
Generally, multiplex ELISA can also be achieved through antibody array, where different
primary antibodies can printed on glass plate to capture corresponding antigens in a biological
sample such as plasma, cell lysate, or tissue extract. Detection method can be direct or indirect,
sandwich or competitive, labeling or non-labeling, depending upon antibody array technologies.

ELISA: pros and cons:

 Pros:
 No radioactivity needed: safe and inexpensive
 Less expensive equipment
 Huge capacity through standard 96-microwell, pattern: miniaturization and
automation.
 Equal or even better sensitivity than RIA
 Versatility: same principle allows for use of different labels, incl. enzymes
(coloured end product or light) and fluorochromes.

 Cons:
 Somewhat more difficult to develop, control and standardize than RIA.
Sigma ELISA (Enzyme Linked Immunosorbent Assay):

An ELISA or enzyme linked immunosorbent assay is a laboratory technique used to detect the
presence of antibody in a sample. Medical professionals can use an ELISA test to determine the
degree to which a person is allergic to a given antigen.
The first step of an ELISA protocol calls for an ELISA antigen to bind to the inside of a well
(96 well plates are commonly used).
A primary antibody is then incubated in the plate. The plate is washed with a buffer before it is
incubated with a secondary antibody conjugated to an enzyme. This enzyme will induce a color
change in the solution when the appropriate substrate is added. The strength of this color change
is measured in a microplate reader and compared to a control sample. The research can then
determine how much antibody is present in their sample

When the body's immune system encounters a particular antigen (for example, a characteristic
protein on the surface of a virus or bacterium), antibodies that are specific for that antigen
intercept it, physically binding to it in a "lock and key"-like fashion, thereby neutralizing the
organism.
Enzyme-Linked Immunosorbent Assay (ELISA) utilizes an antibody labeled with an enzyme
marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an
immunosorbent substrate, they both retain their biological activity; the change in enzyme activity
as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the
antigen and can be measured spectrophotometrically or with the naked eye.
Sigma ELISAs Protocol:
1. A monoclonal or polyclonal antibody (capture antibody) specific for protein is coated into
the wells of a multi well plate (96 wells in a plate).
2. Antigen in standards, unknown samples and controls is added and incubated in order to
facilitate binding of antigen and antibody.
3. Several washes are performed after each incubation in order to remove excess of unbound
reagents.
4. A second (detection) antibody specific for the whole protein at specific site is added and
incubated 1 hour at RT.
5. During the second incubation, this antibody serves as a detection antibody by binding to
another epitope of the immobilized antibody.
6. After removal of excess detection antibody, HRP-IgG is added and binds to the detection
antibody to complete the four-membrane sandwich.
7. Substrate solution is added, which is acted upon by the bound enzyme to produce color.
The intensity of this colored product is directly proportional to the concentration of antigen
present in the original sample.
8. The optical density measured at 450 nm in the multiwell plate reader is used to calculate
the concentration of protein in question.
Applications:
• Because the ELISA can be performed to evaluate either the presence of antigen or the
presence of antibody in a sample, it is a useful tool for determining serum antibody
concentrations (such as with the HIV test or West Nile Virus).

• It has also found applications in the food industry in detecting potential food
allergens such as milk, peanuts, walnuts, almonds, and eggs.

• ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of
drugs.
• Enzyme-Linked Immunosorbent Assays (ELISA) is widely used in research, diagnosis,
and testing because of they are affordable and sensitive to tiny amounts of material.
• Usually, the assay involves multiple binding and rinsing steps, followed by
spectrophotometry. However, in some cases, properly engineered assays can be done in a
single step giving qualitative results readable by eye.
• The pregnancy tests commonly available in drug stores use ELISA to detect the human
chorionic gonadotrophin hormone in urine.

ELISA test for HIV detection:

• The enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA), was
the first screening test commonly employed for HIV. It has a high sensitivity.

In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV
antigens have been attached. If antibodies to HIV are present in the serum, they may bind to
these HIV antigens. The plate is then washed to remove all other components of the serum. A
specially prepared "secondary antibody" — an antibody that binds to human antibodies — is
then applied to the plate, followed by another wash. This secondary antibody is chemically
linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the
amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and
catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported
as a number; the most controversial aspect of this test is determining the "cut-off" point
between a positive and negative result.
References:
• http://www.piercenet.com/Proteomics/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403
• http://en.wikipedia.org/wiki/ELISA
• www.biosupply.co.uk/doc.php?id=2601
• http://www.bio.umass.edu/micro/immunology/elisa/elisa0.htm
• http://thefutureofthings.com/articles/37/biopen-senses-biothreats.html
• http://www.genwaybio.com/gw_file.php?fid=6056
• G.L. Anderson and L. A. McNellis, J. Chem. Educ., 75, 1275 (1998)