DEVELOPMENT OF POLYCLONAL ANTIBODY AGAINST TO SPERM PROTEIN OF BLACK LIP PEARL OYSTER (Pinctada margaritifera, LINNAEUS 1758) IN CHUUK STATE, FEDERATED STATE OF MICRONESIA

Abstract

Polyclonal antibody specific to the sperm protein of black lip pearl (BLP) oyster, Pinctada margaritifera was developed in this study to localize and quantify the protein. To obtain mature sperm, ripe male BLP oysters ranging 120-140 mm in shell length (the longest axis of the shell) were collected from Chuuk State, Federated State of Micronesia in December 2008. The testes containing ripe perms were first excised and placed on a petri-dish and squeezed to extract the sperms. The crude sperm fluid was then sieved using 50 and 100 µm meshes to remove the debris. The purified sperms were washed several times with filtered seawater in low speed centrifuge and stored at -70ºC until used. To develop antibody, the purified sperm was homogenized using an ultrasonifier and the protein level adjusted 1 mg/ml to be served as antigens. After 12 weeks period of immunization, specificity of the rabbit anti-BLP oyster sperm protein serum developed was tested using ELISA and Western blotting. Western blot assay showed that the rabbit antiserum developed in this study cross-reacts with the somatic tissues including mantle and gills. Consequently, the cross-reacting IgG in the antiserum was removed using an immunoadsorbent prepared by polymerizing the somatic tissue proteins. In ELISA, the rabbit anti-BLP oyster sperm IgG detected 0.024-6.250 µg of BLP oyster sperm protein, indicating that