Preparation and Application of Monoclonal Antibodies Targeting IgM in Pearl Gentian Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

Viral and bacterial pathogen pathogens cause disease outbreaks that challenge the pearl gentian grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) industry’s sustainable development. The lack of monoclonal antibodies (mAbs) targeting serum immunoglobulin M (IgM) in this hybrid grouper impedes the development of non-lethal immunoassays for detecting pathogen infections, as well as research on immune responses following vaccination. We purified serum IgM from hybrid pearl gentian grouper and generated two mAbs—designated 41-H2-E1 and 62-E8-G9—against the purified IgM, finding that mAb 62-E8-G9 specifically recognized the IgM heavy chain, whereas mAb 41-H2-E1 specifically recognized the light chain. In indirect immunofluorescence assays, both mAbs reacted with surface Ig-positive (sIg+) lymphocytes. A double-antibody sandwich ELISA was subsequently established using mAb 62-E8-G9 as the capture antibody and HRP-conjugated mAb 41-H2-E1 as the detection antibody, enabling accurate quantification of serum IgM levels. Significant differences in IgM concentrations were observed between larger and smaller individuals (9.11 μg/mL vs. 3.84 μg/mL, p < 0.05). In immunostimulant administration experiments, both low-and high-dose groups exhibited approximately 2.0-fold higher IgM levels than the control group (p < 0.05). In contrast, vaccination with inactivated vaccines did not result in statistically significant differences in total IgM levels. mAb 41-H2-E1 was further applied to detect Vibrio parahaemolyticus- and Vibrio harveyi-specific immunoglobulins in serum under different vaccination regimens. Collectively, these findings demonstrated that the mAbs developed in this study served as reliable immunological tools for investigating immune function in hybrid pearl gentian grouper.