A double-antibody radioimmunoprecipitation (RIP) assay has been developed to provide a sensitive and specific measure of antibody to hemagglutinins of H3N2 influenza viruses. Chloramine T was used to radiolabel purified hemagglutinins to high specific activity without loss of antigenicity. The purity of the labeled hemagglutinin was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which also established that both the HA1 and HA2 polypeptides were iodinated. Radiolabeled hemagglutinins with a specific activity that did not exceed 12 μCi/μg of protein could be maintained for up to 30 days at −70°C in the presence of supplemental protein. The RIP assay was compared with conventional methods, hemagglutination inhibition and viral neutralization tests, using H3N equine 1 hybrid viruses for determining serum antihemagglutinin antibody titers. The geometric mean titers for human convalescent sera after infection with A/England/72 virus were 118, 161, and 18,822 for hemagglutination inhibition, viral neutralization, and RIP tests, respectively, and the three tests demonstrated significant rises in antihemagglutinin antibody titers with equal efficiency. In general, a positive correlation existed between antihemagglutinin antibody titers determined by these three procedures; however, the antibody level determined by RIP assay for each individual could not be related to hemagglutination inhibition or viral neutralization titers by a constant factor. A similar lack of a constant relationship was found by using hyperimmune guinea pig antisera, which suggests that the RIP assay can detect antibody populations that exhibit differing efficiencies for inhibition of viral hemagglutination and replication
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