In practice, the ELISA tests previously described are being replaced by 'sandwich' tests, also known as 'third and fourth generation assays'.

These make use of the fact that antibodies each have more than one binding site. IgG antibodies have two binding sites and the IgM antibodies which appear early in the course of infection each have ten sites. This means that if one site binds to a fixed antigen, the other(s) can bind to a labelled antigen, holding them together in an 'immune complex'. This immune complex can be detected using the label on the antigen in the way previously described for detecting labelled enzymes. These tests are particularly good at detecting low levels of IgM antibodies, and can therefore detect antibodies earlier in the course of HIV infection than previous tests.

Another advantage of this system is that the sample can be added at the same time as the labelled antigen, reducing the number of washing steps involved. Technicians therefore have to spend less time on them and they are easier to automate, reducing costs.

Combined antibody and p24 antigen tests

Yet another advantage is that the same test system can include both an antibody test and a test for the HIV protein called ‘p24’ (Gag protein), which is the main component of the core of the virus. Testing for p24 detects some cases of HIV infection before antibodies are produced, further shortening the 'window period' in which people may test negative despite being able to transmit the virus. This is especially useful in blood transfusion services, although it adds costs if separate p24 or PCR (viral load) tests are done on samples which are positive in the initial screening test but antibody-negative in the Western Blot or IFA tests (described later), used to confirm positive results.

The p24 antigen test is a mirror-image of the double antigen sandwich just described. Here, part of the test surface is coated with carefully selected monoclonal antibodies that recognise p24.

The sample is added to the test system together with a labelled antibody that recognises another part of the p24 molecule. If p24 is present in the sample, then a complex is formed which can be revealed in the same way as the label on the antigen in the antigen-sandwich.

Using the same label on the antibodies and on the antigens in the combined system makes the test very simple to perform.

These combined antigen/antibody tests will NOT detect antibodies directed against p24. Fixed p24 antigens, needed to detect antibodies directed against p24, must be excluded, because they would also bind the labelled anti-p24 antibodies, making the test unusable.

One consequence is that if any vaccine were to be based on the p24 protein, then antibodies directed against that vaccine could not cause vaccine recipients to test positive in these tests