Method for quantitation of IgG subclass antibodies in mouse serum by enzyme-linked immunosorbent assay

One of the methods for calibration of antigen specific antibodies in a serum involves parallel titration of known concentrations of purified normal immunoglobulins (Igs) and a specific antiserum. We evaluated the effect of three methods for capturing known concentrations of purified normal mouse Igs (IgG, IgG subclasses and IgM) on the anti-tetanus toxoid antibody concentrations assigned to a reference mouse serum: (1) direct coating, (2) capture by pre-coated anti-mouse IgG (Fc) specific antibodies, and (3) capture by pre-coated anti-mouse IgG (Fab) specific antibodies. Direct coating of purified Igs onto plastic was the least efficient and would greatly overestimate antigen specific antibody concentrations. Equivalent concentrations of purified IgG subclasses and IgM produced higher absorbances when captured by anti-Fab antibodies pre-coated on plates than by anti-Fc antibodies. As a consequence, the anti-Fc capture method resulted in the assignment of IgG1, IgG2a and IgG2b tetanus specific antibody concentrations which exceeded the total IgG1, IgG2a and IgG2b concentrations. Additionally, the sum of tetanus specific IgG subclass antibodies exceeded the tetanus specific IgG antibody concentration by > 2-fold. In contrast, the anti-Fab capture method resulted in 2-9-fold lower assigned tetanus antibody concentrations which did not exceed the total Ig concentrations. We conclude that when using enzyme-linked immunosorbent assays, parallel titration of purified normal Ig standards captured by anti-Fab antibodies is superior to anti-Fc capture or direct coating for calibrating antigen specific antibodies in a reference serum.