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
). 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.