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
. 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.
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.
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).
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.
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 50l 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.
50l 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 A10-A12. The plates are sealed and incubated for 1 hour at 370C on a shaker and washed repeatedly in 0.002M PBS pH 7.4.
50l of OPD/H2O2 solution is added to each plate and the plates are incubated at room temperature for 7minutes.
50l of 1M sulphuric acid is added to each plate and the OD read at 490nm on an ELISA plate reader.
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.
A1 – A3
A4 – A6
A7 – A9
A10 – A12
+ = reagent used in well. 0 = no reagent used in wells.