Characterisation of monoclonal antibodies to haemocyte subpopulations of tiger shrimp (Penaeus monodon): immunochemical differentiation of three major haemocyte types

In order to study immune cellular effectors in tiger shrimp (Penaeus monodon), monoclonal antibodies (mAbs) raised from haemocytes could be a potential tool for separating and identifying haemocyte subpopulations. In this study, four mAbs – Z5E10 (IgG2a), Z6A5 (IgG1), Z6A6 (IgG1), and Z6H8 (IgG2b) – were produced by immunising balb/c mouse with a high-density granular cell (GC) suspension prepared from tiger shrimp through negative selection with concanavalin A-linked beads. Haemocyte reactivities were determined by means of immunoenzyme staining, and the molecular masses of antigens were analysed by Western blotting. Z5E10 mAb showed reactivity with both GCs and semigranular cells (SGCs) and recognised a 29 kDa antigen. Z6A6 specifically bound to SGCs while recognising a 163 kDa antigen. The other two mAbs (Z6A5 and Z6H8) separately reacted with SGCs and were able to recognise two antigens which were larger than 205 kDa under non-reducing conditions but could not recognise them under reducing conditions. Data from a periodate oxidation assay revealed that Z6A6 antibody was specific for one glycoprotein, while the antigens recognised by the other three mAbs consisted of protein molecules. According to these results, it was concluded that the Z5E10 antigen and the Z6A6 antigen were different not only from each other, but also from those recognised by the other two mAbs. Furthermore, additivity test results showed that the additivity index value for the mAb pairs Z6H8-Z6A6 (29%) was higher than that of Z6A5-A6A6 (0%). It is suggested that Z6A5 and Z6H8 mAbs could be specific for non-identical epitopes. Following the negative selection with mAb-linked beads, an increase in hyaline cell density in haemocyte suspension was observed which suggests that the epitopes recognised by the four mAbs were located on the surface of haemocytes and that these mAbs may be useful when they are employed to label and separate haemocyte subpopulations for further study of haemocyte functions.