Comparison of five calculation modes for antibody ELISA procedures using pertussis serology as a model

During a phase III pertussis vaccine trial, serum antibody responses were measured by two enzyme-linked immunosorbent assays (ELISA) for pertussis toxin and filamentous haemagglutinin. These were used both for studies of antibody levels after vaccination and for diagnostic purposes. Since the absorbance values obtained were not directly proportional to the amount of antibody in the samples, ELISA optical densities were transformed to units by calibration to a reference serum. Five different calculation modes were compared. In four of these modes unit calculations were based on the relationship between dose response curves of the serum sample and a reference serum. In addition, traditional endpoint titres were included in the comparison. The calculation mode using reference line units showed the highest reproducibility, with intrassay coefficients of variation (CV) within the same test plate of 4-7% and interassay CVs of 12-14%. The CVs among the other methods ranged from 6 to 31% for intra-assay comparisons and from 12 to 47% for interassay comparisons. Furthermore, the CV values for intra-assay variations were used to calculate standardized differences between 79 pairs of acute and convalescent sera from cases confirmed by culture. These differences were then used to estimate the 'diagnostic sensitivity' for the different calculation modes. The results indicated that use of the reference line units was the most sensitive, whereas use of the end point titers was the least sensitive of these calculation modes.