A quantitative enzyme-linked immunosorbent assay for honeybee venom-specific immunoglobulin G

A quantitative enzyme-linked immunosorbent assay (ELISA) for honeybee venom-specific IgG is reported. This ELISA surmounts the problem of poor reproducibility due to nonparallelism of dilution curves in previously reported ELISAs. The assay is performed in polyvinyl "U" microtiter plates in which HBV is physically adsorbed to the wells. The antigen is sequentially overlaid with human serum albumin, unknown serum diluted in 10% normal goat serum (NGS), and peroxidase-labeled anti-human IgG in NGS. o-Phenylenediamine is used a substrate. The addition of stabilizing protein (NGS) is shown to be crucial in producing parallel dilution curves in this heterogeneous antigen-antibody system. Sensitivity is at or below 1 ng/ml. Coefficient of variation (using a midrange serum) is 12.1% for triplicate wells, 15.5% for sequential dilution, and 15.0% for six entirely separate assays. The assay correlates well with other methods, notably with the Staphylococcus aureus protein A solid-phase radioimmunoassay (r = 0.976).