Development of an ABO-ELISA for the quantitation of human blood group anti-A and anti-B IgM and IgG antibodies

An indirect ELISA system was designed for quantitation of human blood group A and B IgM and IgG antibodies. The capturing antigens are blood group substance A or B used to sensitize polystyrol microtiter plates. Bound anti-A or anti-B antibodies are revealed either directly, by development with polyclonal anti-human immunoglobulin class-specific conjugate or with more avid mouse monoclonal anti-human isotype antibodies revealed in turn by goat anti-mouse conjugate. Reproducibly, 100 ng specific anti-A IgG provided for a significant above-background signal of 0.2 at OD405 and 15 serum samples had a mean content of 3.98 +/- 8.74 micrograms (mean +/- 2 SD) (range: 0.305-12.62) of specific anti-A IgG/g total IgG. Thus one molecule specific anti-A IgG is found per 7.9 X 10(4)-3.2 X 10(6) total IgG molecules. Statistical correlations were significant between anti-A IgG levels and agglutination titer (P less than 0.05) but non-significant when the specific anti-A IgG levels of individual serum samples were compared to their total IgG content (P greater than 0.05). Dose-response signals were similar for anti-A and anti-B IgM antibodies. Reproducibility of the assay was excellent. Specificity was ascertained by various approaches involving development of primary antibodies with heterospecific antibody conjugate and adsorption of primary antibody from serum using A and B group erythrocytes or soluble A and B substances. Separation of IgM from IgG anti-A antibodies over sizing gel resulted in fractions that were immunosorbed by mouse monoclonal anti-human IgM and IgG respectively but not vice versa.