Detection of rubella antibodies by hemagglutination inhibition, indirect fluorescent-antibody test, and enzyme-linked immunosorbent assay.

Using hemagglutination inhibition (HAI) as a reference method, 292 (40 nonimmune, 252 immune) human serum samples were tested by indirect fluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) for immune status and quantitation of rubella antibodies. The overall agreement with HAI for immune status was 99.7% (291/292) with IFA and 98.6% (288/292) with ELISA. Two specimens (0.7%, 2/292), negative by HAI, were equivocal by ELISA. Initially a 6.5% (19/292) overall disagreement was obtained for immune status evaluation between HAI and IFA, which was reduced to 0.3% (1/292) upon repeat testing. All of these samples were near the immune/nonimmune cutoff point (95 samples), reflecting an initial disagreement of 20% (19/95) in this category (HAI titers less than 1:20). Likewise, an initial overall disagreement of 4.5% (13/292) was obtained between HAI and ELISA which was reduced to 0.7% (2/292) upon repeated testing. Eleven of the 13 samples were near the immune/nonimmune cutoff point, reflecting an initial disagreement of 11.6% (11/95) with sera having an HAI antibody titer of less than 1:20. Quantitation of rubella antibodies by IFA showed an overall correlation with HAI of 86.6% within less than twofold titer and 99.3% within less than fourfold titers. In testing the ability of ELISA to quantitate antibody, a correlation coefficient (r) of 0.996 was obtained by plotting the measured average optical density (405 nm) of ELISA against the corresponding log of HAI titer. Both IFA and ELISA showed good correlation with HAI for immune status evaluation and for quantitation of rubella antibodies. Technically the HAI was the most cumbersome to perform, whereas IFA was the least technically demanding. Originally, 308 samples were tested; 16 samples (5.2%) could not be evaluated by IFA because of a high level of nonspecific fluorescence. The strict requirement of controlling the temperature range (23 to 24 degrees C) during substrate hydrolysis proved to be a problem with the ELISA test in our laboratory.