High-Throughput BLI for One-Step Anti-IgM Detection inSerum.

Abstract

Accurate measurement of antigen-specific antibody responses is essential for evaluating antibody avidity and quantification. Traditional. Enzyme-Linked Immunosorbent Assay (ELISA), while widely used, is limited by lengthy procedures, dependence on secondary antibodies, and inconsistent reproducibility. In this study, biolayer interferometry (BLI) was established and validated for simultaneous quantification and avidity assessment of specific IgM in serum of(Large yellow croaker) usingouter membrane protein Omp-H as antigen. Sera from immunized and control fish were analyzed by both BLI and ELISA, with systematic comparison between platforms. Optimal serum dilutions were 1:128 for BLI and 1:1024 for ELISA. Validation with another outer membrane protein, Omp-W, confirmed the method's broad applicability. BLI association signals and avidity indices correlated strongly with ELISA values, yielding consistent results for both antigens. BLI successfully captured specific antibody responses in infected sera and demonstrated superior inter-plate reproducibility compared to ELISA, which exhibited significant inter-plate variation. However, BLI required lower serum dilutions (hence larger volumes) to achieve comparable sensitivity. These findings establish BLI as a rapid, single-step method providing reliable quantitative and avidity data for teleost IgM, offering a reproducible alternative to ELISA with potential applications in vaccine evaluation and aquaculture infection detection.