ISOLATION AND PARTIAL CHARACTERIZATION OF TURBOT (SCOPHTHALMUS-MAXIMUS) IMMUNOGLOBULINS

1. An IgM-like immunoglobulin was isolated from the serum of turbot (Scophthalmus maximus) by both fast protein liquid chromatography (FPLC) on Superose(R) 6 HR columns and affinity chromatography on Mensep(R) protein A cartridges. 2. Molecular weight of the native Ig was estimated to be 835 kD by SDS-PAGE and 820 kD on the basis of gel filtration. 3. Purity was verified by SDS-PAGE (under non-reducing and reducing conditions) and by immunoelectrophoresis and immunoblotting of antisera raised against the isolated Ig fraction in mice: only minor protein contaminants were observed. 4. Both SDS-PAGE under reducing conditions and immunoblotting also indicated that the heavy and light chains of the isolated turbot Ig are of about 78 and 27 kD, respectively. 5. Total circulating Ig level was estimated by single radial immunodiffusion using a polyclonal anti-turbot Ig serum to be about 4.8 mg/ml. 6. The same antiserum was used to verify that the purified proteins were Igs, in an ELISA test using serum from Vibrio anguillarum-immunized turbot as primary antibody and immobilized V. anguillarum cells as antigen. 7. Finally, our data indicate that about 25% of turbot Igs bind to Mem-Sep(R) protein A.