Development and Characterization of a Mouse Monoclonal Antibody Specific to Grass Carp IgM for Immunological Research

The study of immune responses in teleost fish has gained increasing attention due to their ecological and economic significance. Grass carp, as one of the most important freshwater aquaculture species, serves as a valuable model for immunological research. Immunoglobulin M (IgM) is the predominant antibody isotype in teleosts, playing a critical role in adaptive immunity. However, the lack of specific reagents, such as monoclonal antibodies (mAbs), has hindered detailed investigations into grass carp IgM. This study aimed to develop and characterize a mouse monoclonal antibody specific to grass carp IgM to facilitate advanced immunological research.

The production of the monoclonal antibody involved immunizing mice with purified grass carp IgM. Hybridoma technology was employed to generate stable cell lines secreting IgM-specific antibodies. Following fusion and cloning, hybridoma supernatants were screened using enzyme-linked immunosorbent assay (ELISA) to identify clones with high specificity and affinity for grass carp IgM. Positive clones were further characterized for their cross-reactivity with IgM from other fish species to ensure specificity. The selected mAb exhibited no cross-reactivity with non-target proteins, confirming its suitability for grass carp-specific applications.

Western blot analysis demonstrated that the mAb recognized the heavy chain of grass carp IgM under reducing conditions, with an estimated molecular weight of approximately 70 kDa. Immunofluorescence staining confirmed the antibody's ability to bind surface IgM on grass carp lymphocytes, highlighting its utility in flow cytometry and immunohistochemistry. Additionally, the mAb showed no reactivity with IgM from common carp or zebrafish, underscoring its species-specific nature. These findings suggest that the antibody is a reliable tool for detecting grass carp IgM in various experimental settings.

The application of this monoclonal antibody extends to multiple immunological assays. In ELISA, the mAb enabled quantitative measurement of grass carp IgM levels in serum samples, providing insights into humoral immune responses. Flow cytometry experiments revealed distinct populations of IgM-positive B cells in peripheral blood and lymphoid tissues. Furthermore, the antibody facilitated the isolation of IgM-secreting cells for functional studies. Its robustness in diverse techniques underscores its versatility as a research reagent for investigating grass carp immunity.

In conclusion, the successful development and characterization of a mouse monoclonal antibody specific to grass carp IgM represent a significant advancement in teleost immunology. The antibody's high specificity, affinity, and applicability across multiple platforms make it an invaluable tool for studying immune responses in grass carp. Future research could explore its potential in vaccine development and disease resistance studies. This work contributes to the broader understanding of fish immunology and supports efforts to improve aquaculture health management.