A high throughput serum bactericidal assay for antibodies to Haemophilus influenzae type b

Han Wool Kim, Kyung-Hyo Kim, JiHye Kim, Moon H. Nahm

The protective capacities of antibodies induced with Haemophilus influenzae type b (Hib) vaccines can be directly assessed in vitro with a Hib-specific serum bactericidal assay (SBA). However, the conventional SBA requires several tedious steps including manual counting of bacterial colonies, and therefore, it is seldom used. To overcome these limitations, we have improved the conventional SBA by using frozen target bacteria and by developing an automated colony counting method based on agar plates with the chromogenic dye 2, 3, 5-triphenyl tetrazolium chloride (TTC). These changes enabled us to analyze about 100 serum samples per day per person by SBA. When the intra- and inter-assay precisions were studied, this assay showed a coefficient of variation (CV) ranging from 1 to 38 %. To monitor the long term assay stability for assays involving different bacteria lots, complement lots, and operators, we analyzed bactericidal indices of quality control samples obtained over a 6 year period and found the CV to be about 35-50 %. Lastly, our SBA results were compared with the ELISA results obtained using 90 serum samples from children. We showed that the bactericidal index correlated with IgG anti-Hib antibody levels (r = 0.84), with a bactericidal index of 10 corresponding approximately to 0.15 μg/mL IgG, the widely accepted protective level of antibody. We describe a simple high throughput SBA for anti-Hib antibodies that would be useful for evaluating various Hib vaccines. While additional work will be needed to standardize the assay, this SBA should greatly facilitate studies of Hib vaccines.