Application of the amplified enzyme-linked immunosorbent assay: comparative quantitation of bovine serum IgG1, IgG2, IgA, and IgM antibodies.

The comparative quantitation of serum antibodies to a defined antigen, using the amplified enzyme-linked immunosorbent assay (a-ELISA), has been demonstrated in a model system in which bovine immunoglobulins (Ig) IgG1, IgG2, IgA, and IgM antibodies to human serum albumin (HSA) were measured. Comparative measurements are facilitated because the same enzyme-antibody complex is used for measuring all 4 isotypes. Serum dilutions from 1:100 to 1:50,000,000 were titrated and, when graphed logarithmically, yielded dose-response plots that contained a linear segment for all but IgA anti-HSA. Data obtained with whole serum and fractions enriched in IgA- and IgM-anti-HSA indicated that dimeric IgA antibodies may compete poorly with those of the IgM and IgG classes and that IgM antibodies are more avid than those of the IgG1 and IgG2 subclasses. Plateauing of complete a-ELISA titrations at optical density (OD)400 nm values lower than those observed for their standard-curve counterparts was interpreted to result from saturation of the antigen. Quantitation was accomplished through the use of standard curves prepared by adsorbing purified, radiolabeled Ig of the 4 isotypes directly to polystyrene. These plots were also valuable in evaluating antiglobulin specificity and potency and for ascertaining linearity in the absence of the primary antibody to be measured, as well as standard curves for determining antibody content in absolute terms. The absolute amounts of IgG1 and IgG2 antibodies to HSA were simliar to those determined by quantitative precipitation and constituted about 25% of the total IgG1 and IgG2 in a hyperimmune bovine serum. Only 5% of the serum IgM was specific antibody to HSA. The specificity of various anti-bovine globulin reagents was further shown by demonstrating the charactristic distribution of IgG1, IgG2, IgM, and IgA anti-HSA in serum fractionated on Sephadex QAE-50 and in sucrose density-gradients. Finally, data on the influence of enzyme-complex concentrations, complex-step incubation times, and the reaction kinetics of soluble antibody-enzyme complexes on a ELISA results are presented.