Quantification of IgG and IgG4 antibodies to bee venom phospholipase A2 by competitive inhibition in ELISA

Phospholipase A2 (PLA) is the major antigen of bee venom. Individuals often stung by bees, such as bee keepers, show a restricted immune response mainly of anti-PLA IgG4 antibodies. In contrast, patients allergic to bee venom produce high levels of PLA-specific IgE. This isotype restriction, the clinical relevance and the well defined structure of the PLA antigen, provide a useful model for the study of the principles regulating isotype expression in the human antibody response. A fundamental requirement for such studies is the availability of quantitative and sensitive assays to measure PLA-specific antibodies. Here we describe an ELISA method for direct isotype-specific quantification of anti-PLA IgG and IgG4 antibodies. Serum containing anti-PLA IgG antibodies was added at a predetermined dilution to PLA coated microtiter plates. Then mouse monoclonal antibodies to human IgG or IgG4 and different concentrations of purified human IgG and IgG4, respectively, were added simultaneously. The concentration of anti-PLA IgG and IgG4 antibodies in the serum was calculated from the resulting inhibition curve. Additionally, an analytical method to compare unknown antibody samples with a standard in ELISA - avoiding problems of different affinities - is described. Using the technique described here, isotype-specific quantification of anti-PLA antibodies can be performed at a sensitivity of approximately 70 pg/ml with a reproducibility range of 10-15%.