PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SILURUS MERIDIONALIS CHEN SERUM IMMUNOGLOBULIN

Monoclonal antibodies (MAb) to serum immunoglobulins (Ig) of fish are of immense use in understanding on the fish immune system and rapid identification of pathogens. Southern catfish (Silurus meridionalis Chen) is a unique and important culture fish in China. Literature review indicated that MAbs have not been developed against the Ig of Southern catfish. This paper described the purification and characterization of serum Ig on southern catfish, along with the development and characterization of MAbs against Ig on southern catfish. Serum Ig from healthy southern catfish was purified by 9% polyethylene glycol (PEG6000) precipitation and followed by Sepharose-4B gel column chromatography. SDS- PAGE analysis showed that molecular weight of heavy chain and light chain was approximately 77 kD and 27 kD respectively. The purified serum Ig of southern catfish was used as antigen to immunize Balb/c mice. After four times immunization, the spleen of immunized mouse was removed, and the spleenocytes were obtained and fused with myeloma cells by using PEG 4000. The hybrids were plated into 96 well tissue culture plates and observed for colony growth. After two weeks, wells with hybridomas were tested by ELISA for screening the antibody secreting cell. Four hybridoma cell lines which secrete MAbs against the Ig of southern catfish had been established. Culture supernatant of the four positive hybirdomas were collected and tested using the IsoStripTM Mouse Monoclonal Antibody Isotyping Kit, the results showed that two of them were IgG1, one was IgG2a and one belonged to IgG2b. Ascites of four MAbs were obtained and titers ranged from 104 to 107 tested by ELISA. Standard procedures of Western blotting were used to determine the reactivity of the MAbs in hybridoma culture supernatants to reduced southern catfish serum Ig on a 10% gradient gel. Three of four Mabs were able to recognize the heavy chain of the Ig on Western blotting; the last one did have no reaction with reduced southern catfish Ig under low dilution condition, although it showed strong reactivity in ELISA under high dilution condition. Specificity of four MAbs was investigated by ELISA and Western blotting using different fish serum as antigen. Results from ELISA showed that all of them only specifically reacted with serum of Silurus meridionalis Chen and Silurus asotus, and did not have any cross reaction with serum of Ietalurus Punetaus, Peheobagrus nitidus, Cienoyharyngodoni dellus, Carrassius auratus and Oreochromis niloticu. Western blotting further proved that three of four MAbs were able to bind the heavy chain of Ig only from Silurus meridionalis Chen and Silurus asotus, and all MAbs had no reaction with serum Ig of the other fish. The reactivity of MAbs with the fish common bacterial pathogen was tested by ELISA using bacteria, such as Aeromonas, Edwardsiella, Vibrio, Flexibacter columnaris, Salmonella and Escherichia coli, as coating antigen, and all four MAbs had no reaction with those tested bacterial pathogen. The measuring sensitivity of the Mab MaF4-A12 to purified southern catfish Ig was as low as 31 ng. All of these results demonstrated that Mabs were highly specific and sensitive to the Ig of southern catfish, and had the great potential to be used for analyzing the structure of southern catfish Ig, monitoring the immunity level and pathogen diagnosis.