Variability in Single Radial Immunodiffusion (SRID) Potency Affected by Influenza Vaccine Reference Antigen/Antiserum Combinations: Relationship Between Dissociation Constant and Robustness of SRID Potency

The potencies of domestic influenza virus reference antigens were initially calibrated with a single radial immunodiffusion (SRID) assay using primarily prepared international reference antigens. The SRID potency should not be affected when using another reference antigen calibrated with the same international antigen. However, the SRID potency of test antigens can vary, although the causes of these discrepancies remain unclear. Here, we calibrated two candidate reference antigens (Lot A and Lot B) in the A(H3N2) subtype with various pairs of reference reagent sets (antigen and antiserum). The potencies of Lot A and Lot B varied depending on the reagent pair used, with a more pronounced effect in Lot A (CV = 5.4% vs. 3.8%). To explore the cause of these divergences, we analyzed the dissociation constant of each reagent pair and scored them based on the hypothesis that pairs exhibiting stronger antigen-antibody binding would have smaller precipitin rings. Comparing these scores with the respective potency scores, we observed a strong correlation between the Binding score (relative BLI-K_D) and potency score in Lot A (r = 0.8464, p = 0.0001) but not in Lot B (r = 0.4000, p = 0.1408). These data suggest that antigen-antibody binding strength is an influencing factor of SRID potency.