Elisa PDF

Enzyme-Linked Immunosorbent Assay
1. Introduction
ELISA is an abbreviation for "enzyme-linked immunosorbent assay." In 1974, P. Perlmann and

E. Engvall developed the test as a substitute for certain radioimmunoassay tests, and eventually,
it replaced the western blot test for HIV confirmation. The ELISA test is versatile and medical
professionals can perform it easily as compared to other more complicated tests; many variations
are available commercially.
2. Definition
The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used

to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some
examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or
soluble receptors in cell supernatant or serum. ELISA assays are generally carried out in 96 well
plates, allowing multiple samples to be measured in a single experiment. These plates need to be
special absorbant plates (e.g. NUNC Immuno plates) to ensure the antibody or antigen sticks to
the surface. Each ELISA measures a specific antigen, and kits for a variety of antigens are
widely available.
Figure1.
The ELISA pictured in Figure 1 is what is known as a sandwich ELISA, here two sets of
antibodies are used to detect secreted products, e.g. cytokines. The method is stepwise in the
order shown. The 1st step is to coat the ELISA plate with capture antibody, any excess,
unbound antibody is then washed from the plate. The capture antibody is an antibody raised
against the antigen of interest.
3. Basic ELISA Principle
In an ELISA assay, the antigen is immobilized to a solid surface. This is done either directly or
via the use of a capture antibody itself immobilized on the surface. The antigen is then
complexed to a detection antibody conjugated with a molecule amenable for detection such as an
enzyme or a fluorophore.
Figure 2. The basic setup of an ELISA assay. A capture antibody on a multi-well plate will
immobilize the antigen of interest. This antigen will be recognized and bound by a detection
antibody conjugated to biotin and streptavidin-HRP.
4. Instrumentation
Depending on the test system used, various laboratory devices may be needed for ELISA
processing. Usual equipment includes pipettes, a device for plate washing, an ELISA reader
(photometer) and a computer with an evaluation software. Some test kits require further devices,
such as an incubator for constant temperature during test implementation.
Microwell Plate Multipipette Washing Device Microplate ELISA reader
washer
5. ELISA step by step
i. Antibody coating
Specific capture antibody is immobilized on high protein-binding plates by overnight incubation.

Plates are blocked with irrelevant protein e.g. albumin.
1. Antibody 2. Protein 3. Detection 4. Streptavidin 5. Addition of 6. Analysis

coating capture antibody -enzyme substrate
conjugate
Figure 4. ELISA procedure
ii. Protein capture
Samples and standard dilutions are added to the wells and will be captured by the bound
antibodies.
iii. Detection antibody
Specific biotinylated detection antibody is added to the wells to enable detection of the captured
protein.
iv. Streptavidin-enzyme conjugate
Streptavidin conjugated with alkaline phosphatase or horseradish peroxidase is added to the

wells and will bind to the biotinylated antibody.
v. Addition of substrate
Colorimetric substrate is added to the wells and will form a colored solution when catalyzed by
the enzyme.
vi. Analysis
Absorbance is measured in an ELISA reader and the amount of protein in the samples is
determined.
6. Choosing the right type of ELISA
There are four main types of ELISA: direct ELISA, indirect ELISA, sandwich ELISA and
competitive ELISA.
Figure 3. The different types of ELISA (direct, indirect, sandwich, and competitive)
Direct ELISA:
Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or
quantify antigen concentration. The specificity of the primary antibody is very important.
PROS: minimum procedure; avoids cross-reactivity from secondary antibody.
CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for
labeling.
Indirect ELISA:
Primary antibodies are not labeled, but detected instead with enzyme-conjugated secondary
antibodies that recognize the primary antibodies.
PROS: secondary antibodies are capable of signal amplification; many available secondary
antibodies can be used for different assays; unlabeled primary antibodies retain maximum
immunoreactivity.
CONS: cross-reactivity may occur.
Sandwich ELISA:
The antigen to be measured is bound between a layer of capture antibodies and a layer of
detection antibodies. The two antibodies must be very critically chosen to prevent cross-
reactivity or competition of binding sites.
PROS: sensitive, high specificity, antigen does not need to be purified prior to use.
CONS: antigens must contain at least two antibody binding sites.
Competitive ELISA:
The antigen of interest from the sample and purified immobilized antigen compete for binding to
the capture antibody. A decrease in signal when compared to assay wells with purified antigen
alone indicates the presence of antigens in the sample.
PROS: crude or impure samples may be used, high reproducibility.
CONS: lower overall sensitivity and specificity.
7. ELISA -advantages and disadvantages
An ELISA assay is typically performed in a multi-well plate (96- or 384-wells), which provides
the solid surface to immobilize the antigen. Immobilization of the analytes facilitates the
separation of the antigen from the rest of the components in the sample. This characteristic
makes ELISA one of the easiest assays to perform on multiple samples simultaneously.
Advantages
 High sensitivity and specificity: it is common for ELISAs to detect antigens at the
picogram level in a very specific manner due to the use of antibodies.
 High throughput: commercial ELISA kits are normally available in a 96-well plate
format. But the assay can be easily adapted to 384-well plates.
 Easy to perform: protocols are easy to follow and involve little hands-on time.
 Quantitative: it can determine the concentration of antigen in a sample.
 Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine,
and saliva among others.
Disadvantages
 Temporary readouts: detection is based on enzyme/substrate reactions and therefore
readout must be obtained in a short time span.
 Limited antigen information: information limited to the amount or presence of the antigen
in the sample.
8. Application of ELISA
 Serum Antibody Concentrations
 Detecting potential food allergens
(milk, peanuts, walnuts, almonds and eggs)

 Disease outbreaks- tracking the spread of disease
e.g. HIV, bird flu, common, colds, cholera, STD etc
 Detections of antigens
e.g. pregnancy hormones, drug allergen, GMO, mad cow disease
 Detection of antibodies in blood sample for past exposure to disease e.g. Lyme
Disease, trichinosis, HIV, bird flu
9. Conclusion
In general, people do not need to prepare for an ELISA test. Medical professionals perform the
test in a lab. If your blood is required, the only hurt is in blood collection. The risks associated
with an ELISA test are rare and associated with blood withdrawal (infection, vessel damage, for
example).

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Enzyme-Linked Immunosorbent Assay

ELISA is an abbreviation for "enzyme-linked immunosorbent assay." In 1974, P. Perlmann and

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used

3. Basic ELISA Principle

5. ELISA step by step

Specific capture antibody is immobilized on high protein-binding plates by overnight incubation.

1. Antibody 2. Protein 3. Detection 4. Streptavidin 5. Addition of 6. Analysis

ii. Protein capture

iii. Detection antibody

iv. Streptavidin-enzyme conjugate

Streptavidin conjugated with alkaline phosphatase or horseradish peroxidase is added to the

PROS: minimum procedure; avoids cross-reactivity from secondary antibody.

CONS: cross-reactivity may occur.

CONS: antigens must contain at least two antibody binding sites.

PROS: crude or impure samples may be used, high reproducibility.

CONS: lower overall sensitivity and specificity.

7. ELISA -advantages and disadvantages

 Serum Antibody Concentrations

 Detecting potential food allergens

(milk, peanuts, walnuts, almonds and eggs)

e.g. HIV, bird flu, common, colds, cholera, STD etc

e.g. pregnancy hormones, drug allergen, GMO, mad cow disease

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