Recombinant phage-display monoclonal antibody against starry flounder (Platichthys stellatus) IgM enables quantitative ELISA for vaccine-induced humoral responses

Abstract

Species-matched serological reagents are scarce for many aquaculture species, limiting quantitative assessment of vaccine responses. Here, we developed and validated monoclonal antibodies (MAbs) specific to starry flounder (Platichthys stellatus) immunoglobulin M (IgM) using a recombinant phage-display approach. IgM purified from immune serum was used as the antigen for bio-panning, yielding multiple IgM-reactive scFv candidates, all of which recognized the IgM heavy chain by Western blot analysis. A lead clone (1T1A11) was reformatted as an scFv-Fc minibody, expressed in mammalian cells, purified by Protein A affinity chromatography, and verified as a single band of the expected molecular size. The 1T1A11 minibody specifically immunoprecipitated IgM from both purified preparations and serum, confirming its functional specificity. Using this monoclonal antibody, we established an optimized indirect ELISA to quantitatively monitor humoral immune responses following Streptococcus parauberis formalin-killed cell vaccination. The assay revealed clear dose- and time-dependent increases in serum IgM levels, which were further quantified using a purified starry flounder IgM standard curve. Collectively, these results establish 1T1A11 as a specific and scalable reagent that enables standardized, quantitative serological analysis for vaccine evaluation and diagnostic applications in flatfish aquaculture.