Isotype-specific optimization of antigen-specific ELISA reveals differential effects of blocking reagents.

Accurate detection of antigen-specific antibodies by ELISA requires minimizing nonspecific background, yet blocking conditions are rarely optimized for antibody isotype. Here, we systematically evaluated the impact of blocking reagents on isotype-specific detection of antibodies against influenza A/PR8 hemagglutinin (HA) in mice using an antigen-specific indirect ELISA with isotype-specific detection. Sera from naïve and infected animals were analyzed for HA-specific IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, and IgM across multiple blocking conditions. No single reagent was universally optimal: 1.5% bovine serum albumin (BSA) yielded the highest signal-to-noise ratios for IgG1, IgG2a, IgG2b, IgG2c, and IgA, whereas IgG3 required 20% fetal bovine serum (FBS) to reduce background. In contrast, IgM detection exhibited persistently high background with all conventional blocking reagents tested, including BSA, FBS, and non-fat dry milk. Screening of alternative protein sources identified soy milk as uniquely effective for IgM detection. Soy milk markedly reduced background in naïve serum while preserving strong antigen-specific signal in immune samples, resulting in a substantial improvement in signal-to-noise ratio. These findings demonstrate that ELISA blocking conditions are strongly isotype-dependent and highlight the use of unconventional protein blockers as an effective strategy for improving detection of challenging antibody classes.