Development and application of IgT monoclonal antibody in Nile tilapia Oreochromis niloticus

Immunoglobulin T (IgT) is a class of Ig unique to teleost, with current research largely supporting its role in mucosal immunity. However, the precise function of IgT in many kinds of teleost remains poorly understood due to a lack of appropriate antibody tools. In this study, we prepared the recombinant protein of Oreochromis niloticus IgT heavy chain (rOn-IgT) and generated a monoclonal antibody (mAb). Through enzyme-linked immunosorbent assay (ELISA) and flow cytometry screening, the 1B3H4 hybridoma was identified as producing an IgG1-type antibody, which can recognize IgT and identify IgT ⁺ B cells in tilapia. The facts that this candidate mAb generates a lymphocyte population distinct with both IgM ⁺ B cells and CD3 ⁺ T cells, and IgT was exclusively expressed in IgT ⁺ but not IgT ^- lymphocytes, validates the specificity of this tilapia IgT mAb. IgT mRNA and protein is widely distributed across various secondary immune and mucosal tissues in tilapia, with IgT ⁺ B cells present in varying proportions. Upon lipopolysaccharide (LPS) stimulation, IgT expression in spleen leukocytes was significantly increased at both the mRNA and protein levels, accompanied by a marked rise in the proportion of IgT ⁺ B cells. In addition, LPS stimulation also led to an increase in the phosphorylation levels of NF-κB and ERK1/2 in IgT ⁺ B cells. Moreover, we suggested that tilapia IgT ⁺ B cells possess robust phagocytic capabilities towards Edwardsiella piscicida, suggesting a potential role in immune response. Overall, we generated an mAb for tilapia IgT, investigated the expression of IgT and distribution of IgT ⁺ B cells both in various tissues and upon LPS stimulation, and revealed the phagocytosis of IgT ⁺ B cells in tilapia. This study would provide value tool and technical support to understand the immune function of IgT ⁺ B cells in teleost.