Development of sandwich ELISA for IL-10 quantification in olive flounder (Paralichthys olivaceus) and its application to protein-level analysis during viral hemorrhagic septicemia virus (VHSV) infection.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays a key role in maintaining immune homeostasis. Although studies in teleosts have examined IL-10 gene cloning, mRNA expression, and the functions of recombinant IL-10, analyses at the endogenous protein level remain limited. Considering IL-10 is biologically active only when secreted as a protein, direct detection and quantification of endogenous IL-10 protein are essential to accurately elucidate its immunomodulatory function. In this study, we developed mouse monoclonal and rabbit polyclonal anti-IL-10 antibodies that specifically recognize endogenous IL-10 of the olive flounder (Paralichthys olivaceus). Using these antibodies, we established a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for IL-10 quantification. Endogenous IL-10 was successfully detected in the spleen, serum, and ascitic fluid of fish challenged with viral hemorrhagic septicemia virus (VHSV), and IL-10 levels increased progressively throughout the infection period. Additionally, IL-10 protein-levels in the spleen showed a strong positive correlation with mRNA expression. The analytical platform developed here, along with the protein-level expression data obtained, provides an essential foundation for advancing our understanding of IL-10-mediated immune regulation in fish during pathogen infection.