ELISA TEST (Enzyme-linked immunosorbent assay)

Aim of the experiment

To detect antibodies in the blood.
Principle
ELISA test works on the principle that specific antibodies binding to the target
antigen and detecting the presence and quantity of antigens binding and order to
increase the sensitivity and precision of the assay, the plate must be coated with
antibodies with high affinity.
Introduction
ELISA or EIA, is a test that detects and measures antibodies in your blood. This
test can be used to determine if you have antibodies related to certain infectious
conditions. Antibodies are proteins that your body produces in response to
harmful substances called antigen.
There are four major types of ELISA:
 Direct ELISA (antigen-coated plate; screening antibody)
 Indirect ELISA (antigen-coated plate; screening antigen/antibody)
 Sandwich ELISA (antibody-coated plate; screening antigen)
 Competitive ELISA (screening antibody)
Direct ELISA
Both direct and indirect ELISAs begin with the coating of antigen to the ELISA
plates. The first binding step involves adding antigen to the plates, which is
incubated for one hour at 37 degrees C or can be incubated at 4 degrees C
overnight. Once the incubation step is completed, the next step is to wash the
plates of any potential unbound antibody and block any unbound sites on the
ELISA plate using agents like BSA, ovalbumin, aprotinin, or other animal proteins.
This second step is important because it prevents the binding of any non-specific
antibodies to the plate and minimizes false-positive results. After adding the
buffer, the plate is rewashed, and a selected enzyme-conjugated primary
detection antibody is added. The plate is further incubated for one hour.
In a direct ELISA, the primary detection antibody binds directly to the protein of
interest. Next, the plate is rewashed to remove any unbound antibody and
followed by the addition of a substrate such as alkaline phosphatase or
Horseradish Peroxidase to the plate, which results in a color change. The color
change of the sample occurs by either the hydrolysis of phosphate groups from
the substrate by AP or by the oxidation of substrates by HRP. The advantages of
using direct ELISA include eliminating secondary antibody cross-reactivity, and
due to fewer steps, it is rapid compared to indirect ELISA. Its disadvantages
include: its low sensitivity compared to the other types of ELISA and its high cost
of reaction.
Indirect ELISA
The steps of the indirect ELISA are identical to the direct ELISA, except for an
additional wash step and the types of antibody added after the buffer is removed.
Indirect ELISA requires two antibodies, a primary detection antibody that sticks to
the protein of interest and a secondary enzyme-linked antibody complementary
to the primary antibody. The primary antibody is added first, followed by a wash
step, and then the enzyme-conjugated secondary antibody is added and
incubated. After this, the steps are the same as the direct ELISA, which includes a
wash step, the addition of substrate, and detection of a color change.
The indirect ELISA has a higher sensitivity when compared to the direct ELISA. It is
also less expensive and more flexible due to the many possible primary antibodies
that can be used. The only major disadvantage with this type of ELISA is the risk of
cross-reactivity between the secondary detection antibodies.
Sandwich ELISA
Unlike direct and indirect ELISA, the sandwich ELISA begins with a
capture antibody coated onto the wells of the plate. It is termed a “sandwich”
because the antigens are sandwiched between two layers of antibodies (capture
and detection antibodies). After adding the capture antibody to the plates, the
plates are then covered and incubated overnight at 4°C. Once the coating step is
complete, the plates are washed with PBS, then buffered/blocked with BSA. The
buffer washes are carried out for at least 1-2 hours at room temperature. Finally,
the plate is washed with PBS once again before the addition of the antigen.
The antigen of interest is then added to the plates to bind to the capture antibody
and incubated for 90 min at 37 degrees C. The plate is rewashed, and the primary
detection antibody is then added to the plate and incubated for another 1 to 2
hours at room temperature, followed by a buffer wash. Then the secondary
enzyme-conjugated antibody is added and incubated for another 1 to 2 hours.
The plate is rewashed, and the substrate is added to produce a color change. The
sandwich ELISA has the highest sensitivity among all the ELISA types. The major
disadvantages of this type of ELISA are the time and expense and the necessary
use of “matched pair” and secondary antibodies.
Competitive ELISA
The competitive ELISA tests for the presence of an antibody specific for antigens
in the test serum. This type of ELISA utilizes two specific antibodies, an enzyme-
conjugated antibody and another antibody present in the test serum if the serum
is positive. Combining the two antibodies into the wells will allow for a
competition for binding to antigen. The presence of a color change means that
the test is negative because the enzyme-conjugated antibody bound the antigens.
The absence of color indicates a positive test and the presence of antibodies in
the test serum. The competitive ELISA has a low specificity and cannot be used in
dilute samples. However, the benefits are that there is less sample purification
needed, it can measure a large range of antigens in a given sample, can be used
for small antigens, and has low variability.
Procedure
There was attachment of captured antibody specific to target protein to a
microplate
There was addition of standards and samples containing unknown amount of the
target protein which binds to the capture antibody
To remove unbound substances they were washed.
The was addition of a detection antibody that bond to the immobilized target
protein
Excess detection antibody was washed away and addition of HRP conjugate
Addition of HRP substrate for indirect detection of bound protein
DISCUSSION
False positives and false negatives can occur. A false-positive result indicates you
have a condition when you actually don’t. A false-negative result indicates you
don’t have a condition when you actually do. Because of this, you may be asked
to repeat the ELISA again in a few weeks, or your doctor may order more sensitive
tests to confirm or refute the results.
CONCLUSION
The ELISA test is important and widely used tool in the diagnosis of medical
conditions like hepatitis C, HIV, rubella and lyme fever disease. .It is an effective
accurate tool used to detect the presence of antibodies in the body.
REFRENCES
1.Aydin S. A short history, principles, and types of ELISA, and our laboratory
experience with peptide/protein analyses using ELISA. Peptides. 2015 Oct;72:4-15
.
2.Sherwani S, Chowdhury M, Bugert JJ. ELISA for Molluscum Contagiosum
Virus. Curr Protoc Microbiol. 2017 Nov 09;47:14A.6.1-14A.6.9