Location: Aquatic Animal Health ResearchTitle: Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus) Author
Submitted to: Fish and Shellfish Immunology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/19/2010
Publication Date: 7/24/2010
Publication URL: http://handle.nal.usda.gov/10113/55556
Citation: Li, Q., Li, Y., Li, H., Wang, Y., Xu, D. 2010. Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus. Fish and Shellfish Immunology. 29:832-38. Interpretive Summary: Monoclonal antibodies (mAbs) have been frequently used as essential probes for identification of cell types and determining functions of immune cells in vertebrates. However, mAbs used in invertebrate immune studies are limited. In this study, one mAb specific for coelomic spherulocytes of A. japonicus was developed and characterized by using indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA), Western blotting, fluorescence-activated cell sorter (FACS)and immunohistochemistry. Monoclonal antibodies (mAb 3F6) was demonstrated to be specific to spherulocytes of A. japonicas. The spherulocytes can be identified and located in different tissues using mAb 3F6 by IIFAT, ICA and immunohistochemistry. Western blotting with mAb 3F6 revealed an antigen of coelomocytes and showed a definite band of molecular weight 136 kDa. The mAb 3F6 will be a useful tool for study on the relationship between haemocytes and spherulocytes, spherulocytes ontogenesis, and functions of spherulocytes.
Technical Abstract: One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3F6 showed specific for spherulocytes of A. japonicus. The mAb 3F6 recognized an antigen of molecular weight 136 kDa in Western blotting. Isotype analysis revealed mAb 3F6 as IgG type. The flow cytometry assay confirmed the microscopy observations and showed coelomocytes positive to mAb 3F6. The antigencity of haemocytes or coelomocytes of Hemicentrotus pulcherrimus, Scapharca subcrenata, Asterina pectinifera, Asterias rollestoni, Ruditapes philipinarum, Patinopecten yessoensis and Mytilus eduliswas compared and the results showed that none of them were positive with mAb 3F6. Most of cells free in polian vesicle were positive with mAb 3F6. The positive cells are in spherical shape, 5-7 µm in diameter, smaller than coelomic spherulocytes of A. japonicus. The result of immunofluorescent staining with cells in hemal vessel showed that there were strong, positive signals on cytoplasm of some spherical cells with diameter of 7-8 µm. Some other cells with higher nucelo-plasmic ratio, about 5-6 µm in diameter showed weak positive signals on membranes. Immunohistochemistry assay revealed that positive signals were mostly observed in the lumen structure of rete mirabile and haemocoel of the respiratory tree. In addition, the outer epidermis of body wall and tentacle also showed positive.