The neutralization assay, based on inhibition of viral p24 antigen production in an acutely infected cell line, is an alternative to the standard syncytium inhibition assay used in the laboratory. Supernatants are collected from infected cell cultures that have been inoculated with virus stock preincubated with dilutions of HIV antibody-positive sera and control sera. Treated supernatants are then assayed for p24 core antigen concentration using a commercial ELISA. The neutralizing titer is calculated by first determining the mean p24 antigen concentration in cultures preincubated with a negative control serum. The neutralizing titer of the test serum is defined as the serum dilution causing a 50% reduction in the mean p24 antigen concentration of the control. Results obtained by this method show a high degree of correlation to the standard method, and there are a number of advantages. The assay is more sensitive, and results can be obtained after only three days in culture, in contrast to the five day culture required in the standard assay. Supernatants can be collected and stored, to be assayed for p24 in batches, if desired, rather than being assayed at a precise time, as is necessary when quantitating syncytia in the standard assay. The quantity of collected supernatant is sufficient to repeat the p24 antigen assay three times. The test is highly flexible, e.g. it can be used in cultures that do not form sufficient numbers of syncytia to quantitate, so there is a broad choice of cell lines.
