Measurement of the absolute mass concentration of antigen-specific serum IgG via quantitative Western blotting.

The quantitation of antigen-specific antibodies (Ag-specific Abs) is essential for evaluating immune responses after vaccination or infection. Conventional ELISA readouts can be biased by Ab affinity and the choice of reference standard due to the extensive wash steps involved. We present an affinity-independent approach to determine the absolute mass concentration of Ag-specific IgG in animal sera using magnetic bead-based Ag pulldown coupled with quantitative Western blotting without any wash steps. First, the self-consistency of this technique allows us to determine the binding activity of a purified monoclonal Ab of known concentration. Second, this workflow achieves near-complete capture of Ag-specific IgG from animal sera with minimal background, and Western quantification of the captured IgG yields its original serum concentration with good reproducibility. We illustrate the method using mouse sera raised against different protein Ags and directly compare results to ELISA, whose measurements vary with the reference Ab. In the same samples, we quantify both total and Ag-specific IgG and observe that the fraction of Ag-specific IgG increases over time post-immunization. Concentrations obtained by our method typically exceed ELISA values, suggesting the presence of a low-affinity IgG population that ELISA under-detects. This calibration-ready, affinity-agnostic measurement enables accurate and standardized Ag-specific IgG quantitation across studies for the evaluation of immune responses.