Serology: Enzyme-linked immunosorbent assay (ELISA)
Serology:
Enzyme-linked immunosorbent assay (ELISA)
Authors: Dr. RW Worthington (Retired)
Licensed under a Creative Commons Attribution license.
COMPETITIVE AND BLOCKING ELISAS
Competitive and blocking ELISAs work on the principle that two antibodies compete for a binding site
on an epitope. Competitive and blocking ELISAs are very similar. Tests in which the serum being
tested and a competing antiserum (or antibody preparation) are reacted simultaneously with the
antigen are called competitive ELISAs. Tests in which the serum being tested and the competing
antiserum (or antibody preparation) are reacted sequentially with the antigen are called blocking
ELISAs (see below).
Competitive and blocking ELISAs have distinct advantages over indirect ELISAs and are very
commonly used. They are done using either monoclonal antibodies (MAbs) or polyclonal sera as the
competing antibody. However, they are best utilised when the test is designed to take advantage of
the high specificity of MAbs. A MAb that recognises an immunodominant epitope can be used for
providing a test of high sensitivity and a MAb against a highly specific epitope can be used in a test
designed to have great specificity. Furthermore, the specificity provided by the MAb allows tests with
high specificity to be done with crude antigens.
In the simplest configuration of a typical blocking test a MAb preparation with specificity for an
immunodominant epitope on the antigen could be used as an enzyme labelled competing antibody.
The antigen would be attached to a plate in the normal manner. The serum to be tested would be
dispensed into the relevant wells on the plate and antibody in the serum allowed to attach to available
matching epitopes, thus blocking these epitopes so that they are not available to interact with other
antibodies. In the next stage of the test the MAb coupled to an enzyme would be dispensed into the
relevant wells. If all the relevant epitopes are already blocked by antibody from the test serum the
MAb will not attach and after washing the wells and adding substrate no colour will develop (see
Figure 1). In practice only some of the relevant epitopes may be blocked if the test serum contains a
low level of antibody. Therefore, results are expressed as the percentage reduction of colour
development compared to a test with a negative control serum in which no blocking occurs. Blocking
ELISAs may be done at a single test serum dilution, or in some tests doubling dilutions of sera are
tested. A cut-off point is established as a percentage of inhibition of colour development. A blocking
ELISA can also be done with a polyclonal serum used as the competing antibody preparation.
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Serology: Enzyme-linked immunosorbent assay (ELISA)
Figure 1: Diagrammatic representation of a blocking ELISA. Y = antibody in test serum. e-Y =
detector antibody conjugated to a marker enzyme.
A competitive ELISA works on the same principle but in this case the test serum and the competing
antibody (MAb or polyclonal serum specific for the antigen) are added as a mixture to the wells on the
plate. They compete for binding sites on the antigen. In the simplest versions of the test the
competing antibody is conjugated to a marker enzyme (
Figure 2). The plate is then washed and substrate added in the normal way. The percentage of
inhibition of colour formation is calculated.
Figure 2: Diagrammatic representation of a competitive ELISA
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Serology: Enzyme-linked immunosorbent assay (ELISA)
Variations of competitive ELISAs
Blocking the binding of antigen to capture antibody
In some blocking ELISAs a capture antibody (Cab) is used on the plate. In this case the
blocking step is at the level of the binding of the antigen to the Cab. The antigen and test serum
are mixed and incubated separately and an aliquot of the serum/antigen mixture is added to the
plate. If the test serum antibody has blocked all the binding sites on the antigen, the antigen will
not attach to the CAb on the plate, and conversely if the serum contained no antibody it will
attach. Attachment of antigen to the CAb is demonstrated using a detector antibody against the
antigen. The detector antibody is either coupled to an enzyme, or its presence is demonstrated
by using a second detector antibody against the first detector antibody in a sandwich test.
Finally the substrate is added and the inhibition of the colour development compared to the
negative control serum indicates the amount of blocking antibody in the test serum.
This system is used for the detection of foot and mouth disease (FMD) antibodies. In this case
a rabbit antibody against the 146S antigen of FMD from a particular type of FMD virus is used
as CAb. A guinea pig antiserum against the 146S antigen of the FMD virus type being tested
for is used as the first detector antibody and a rabbit anti-guinea pig conjugate is used as the
second detector antibody. The antigen is an unpurified supernatant of a tissue culture of FMD
disease virus. In the case of this FMD test all the sera used are polyclonal sera. Some care had
to be taken in designing the system to ensure that the second detector antibody does not react
with CAb. In this case the CAb and second detector are prepared in the same species (rabbit)
and will not interact with each other.
If MAbs are used in the system some additional precautions must be taken because virtually all
MAbs in general use are mouse antibodies. Therefore, if a MAb is chosen as the Cab, a MAb
can only be used as detector antibody if it is conjugated directly to the enzyme, as in
Figure 2. Sandwich ELISAs cannot be used, as in sandwich ELISAs the second detector
antibody, which is an anti-mouse antibody, would react indiscriminately with both the CAb and
first detector antibody. In addition if a conjugated MAb is used as the first detector antibody it
must be demonstrated that the epitopes with which it reacts, have not been blocked by the
CAb. This can usually be assured by choosing a MAb against a different epitope on the antigen
or using a MAb recognising the same epitope as the CAb only when the antigen has multiple
identical epitope sites. Generally large antigens like viruses have many identical epitopes on
their surfaces while purified protein antigens consisting of single protein chains may have only
one epitope of a particular type on each molecule. Polysaccharide antigens have repeating
identical antigens. Even if there are multiple identical epitopes on an antigen they may be
unavailable due to the three dimensional arrangement of the molecules when bound to the
plate and the steric hindrance of other molecules in the vicinity. There is no practical way to
predict all the possible complex interactions and the system must be thoroughly tested using all
the relevant controls.
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Serology: Enzyme-linked immunosorbent assay (ELISA)
In some systems the CAb is a MAb thus providing good specificity and the detector is a
polyclonal antibody that can react with a variety of epitopes on the antigen thereby increasing
the sensitivity.
Blocking the binding of antibody to antigen coated onto the plate.
Antigen can be attached directly to the plate or a CAb can be used. If the CAb is a MAb, it will
provide for a high degree of specificity of binding of antigen to the plate. Test serum is then
added to the plate and incubated followed by the addition of the detector antibody.
The
detector antibody may be conjugated to an enzyme or be unconjugated. If the detector antibody
is unconjugated, it will have to be detected by the addition of a conjugated second detector
antibody to detect the presence of first detector antibody on the plate. This type of test differs
from that described in 2.1.1 in that the blocking step by test sera takes place after antigen is
already attached to the plate and is aimed at blocking the attachment of the detector antibody
to the antigen.
A typical example of this type of test is the blocking ELISA for the detection of swine vesicular
disease virus antibodies in serum. In this test a MAb is used to fix antigen to the plate, followed
by addition of test serum, which if it contains antibody will block the relevant epitope. The
detector antibody is the same MAb that was used as CAb but conjugated to an enzyme. Finally
substrate is added and colour allowed developing. The reduction in the amount of colour
development compared to the negative control provides an indication of the amount of blocking
antibody by the test serum. In this case there are multiple identical epitope sites that can react
with the MAb, on the antigen particles. This allows the same MAb to be used as CAb and
detector antibody.
The usual variations involving the use, avidin/biotin systems, sandwich ELISAs, monoclonal or
polyclonal sera etc. can be used as appropriate.
A competitive ELISA
An example is given below of a competitive ELISA for rinderpest antibody. The protocol has been
adapted from the description of this test given in the OIE Manual of Standards for Diagnostic tests
and Vaccines. In this example all critical reagents can be purchased from the OIE reference
laboratory for Rinderpest.
Reagents

Phosphate buffered saline (PBS) is 0.01M phosphate buffered saline pH 7.4 unless
otherwise specified.

Blocking buffer is 0.01M PBS with 0.1% Tween 20 and 0.3% normal bovine serum.

Antigen is freeze-dried Kabete O strain, an attenuated strain of rinderpest virus. It is
reconstituted in distilled water and diluted according to the supplier’s instructions in PBS.

The competing antibody is a MAb specific for the H antigen of the rinderpest virus (MAb
C1).
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Serology: Enzyme-linked immunosorbent assay (ELISA)

A rabbit anti-mouse antibody conjugated to horseradish peroxidase used to detect MAb
C1.

Substrate is OPD/H2O2 solution as described in Section 1.4.1.

Negative control sera consist of six sera from animals from a disease free and
unvaccinated population of animals.

The positive control serum is a positive control obtained from the reference laboratory or a
positive control serum that has been standardised against a reference serum. The positive
control serum should be tested in suitable dilutions expected to give a strong, medium and
weak positive result.
Assay protocol

The antigen is reconstituted in 0.01M PBS according to the supplier’s instructions and 50 l
dispensed into all wells of a microtitre plate. The plate is sealed and incubated for 1 hour at
370C on a shaker and washed repeatedly in 0.002M PBS pH 7.4.

40 l of blocking buffer is added to all wells in the horizontal rows B – H and 50 l of
blocking buffer is added to the wells in horizontal row A (Figure 3).

10 l of each test serum is added to two wells on the plate horizontal rows B – H. The six
negative control sera and the positive control sera are also tested.

50 l of MAb C1, diluted according to the supplier’s instructions in blocking buffer (or
previous titration), is added to all wells except wells A1-A6, which receive 50l of PBS.

The plates are sealed and incubated for 1 hour at 370C on a shaker and then washed
repeatedly in 0.002 M phosphate buffer.

50l of rabbit anti-mouse conjugate suitably diluted in blocking buffer, is added to each well
except wells A1-A3 and A10-A12. 50 l of blocking buffer is added to wells A1-A3 and A10A12. The plates are sealed and incubated for 1 hour at 370C on a shaker and washed
repeatedly in 0.002M PBS pH 7.4.

50l of OPD/H2O2 solution is added to each plate and the plates are incubated at room
temperature for 7minutes.

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50l of 1M sulphuric acid is added to each plate and the OD read at 490nm on an ELISA
plate reader.
Serology: Enzyme-linked immunosorbent assay (ELISA)
Figure 3: Plate set-up for competitive ELISA. Dispensing blocking buffer. B50 = 50 l blocking
buffer. B40 = 40 l blocking buffer.
The results are expressed as percentage inhibition of colour development compared to the
average of the negative control sera. According to the method description 50% inhibition is
considered to be positive and <50% is considered negative. The cut-off point should be
checked against the result of the low positive serum that should be just positive. The control
wells in Horizontal row A received reagents as indicated Table 1 and should yields the
expected result as shown in the table.
Table 1: Contents and expected result of control wells in row A.
Reagents used
Wells
A1 – A3
A4 – A6
A7 – A9
A10
–
A12
Antige
n
+
+
+
+
MAb, C1
Conjugate
OPD/H2O2
0
0
+
+
0
+
+
0
+
+
+
+
+ = reagent used in well. 0 = no reagent used in wells.
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Expected
result
no colour
no colour
dark colour
no colour