Production And Application Of Monoclonal Antibodies Against Serum Immunoglobulin From Chinese Sucker (Myxocyprinus Asiaticus)

The Chinese sucker (Myxocyprinus asiaticus) is an economically important species worldwide. In recent years, with the artificial cultivation technology of Chinese sucker gradually maturity and the breeding scale continually expanding, fish diseases has also become more serious, especially for juvenile Chinese sucker. This resultes in huge financial loss for the fisherman and restricts the development of aquaculture. Studies on the fish immune system and the process of immune response facilitate the prophylaxis of fish diseases. Teleosts are the earliest evolutionary class to possess adaptive immunity and Immunoglobulin (Ig) plays an important role in humoral immune response. Thus, studies on serum Ig of Chinese sucker will contribute to understanding the process of immune response, which can provide theoretical basis for prevention of diseases.Monoclonal antibodies (MAbs) have been studied by the most immunologists because of their strong specificity for binding antigen. MAbs against serum Ig of Chinese sucker were powerful tools for studying of Chinese sucker immune system, purification of Chinese sucker serum Ig is the essential step for preparing of MAb. In this study, Firstly, serum Ig of Chinese sucker was purified. Moreover, mAbs against serum Ig of Chinese sucker were produced and characterizations of mAbs obtained were identified. Lastly, we also researched whether the mAbs could recognise Ig and Ig-positive cells in peripheral blood or not by immunoprecipitation, flow cytometry and immunocytochemistry.The main results are as follows:1) Serum Ig from Chinese sucker was purified by protein A affinity chromatography, MBP affinity chromatography and Sepharose-4B gel chromatography, respectively. SDS-PAGE showed that serun Ig of Chinese sucker was successfully isolated by MBP affinity chromatography and Sepharose-4B gel chromatography, the heavy chains and the light chains in molecular weight were to be approximately76.6kDa and28.5kDa, respectively. Protein A affinity chromatography was not fit for purifying serum Ig of Chinese sucker. The purified Ig was concentrated by Nanosep Centrifugal Devices and the concentration was determined by BCA, the results showed that the protein concentration of Chinese sucker serum1mL after purified by MBP affinity chromatography and Sepharose-4B gel chromatography were about0.6mg/mL and0.523mg/mL, the volume were about180μL and480μL and the quality were about108ug and251.04μg. From the results, we can conclude that the yield of Sepharose-4B gel chromatography was higher than that of MBP affinity chromatography.2) The6-8weeks old Balb/c mice were immunized by the Ig of Chinese sucker, which was purified by Sepharose-4B gel chromatography. Three hybridomas secreting monoclonal (MAbs) against Chinese sucker Ig were obtained by the cell fusion, screening using indirect ELISA and subcloning using limiting dilution method, and named as Y5-B11-C11, Y8-G1-E8and Y9-F12-E9. Characterization of MAbs for mouse Ig class and subclass indicated that Y5-B11-C11was IgGl, Y8-G1-E8and Y9-F12-E9were IgG2b, and the light chains were all K isotype. Titer of the three MAbs was ranged from1:1600to1:3200tested by indirect ELISA. Further experiments proved that all of them only bound to Ig of Chinese sucker specifically, and have not any cross reaction with the Ig of Ctenopharyngodon idellus, Carassius auratus gibelio, Hypophthalmichthys molitrix, Silurus meridionalis, Ictalurus punctatus, Oreochromis niloticus and Lateolabrax japonicus by indirect ELISA. Under reducing conditions in Western-blotting demonstrated that MAb (Y8-G1-E8and Y9-F12-E9) could bind to the light chain of Chinese sucker Ig and did not react the heavy chain of Chinese sucker Ig, Ig of Ctenopharyngodon idellus, Carassius auratus gibelio, Hypophthalmichthys molitrix, Silurus meridionalis and Oreochromis niloticus, while Y5-B11-C11could not be determined by Western-blotting analysis. MAbs (Y8-G1-E8) recognised linear epitope proved by indirect ELISA and were against similar antigen epitope tested by superposable ELISA.3) The application of MAb (Y8-G1-E8) was investigated in immunoprecipitation, flow cytometry and immunocytochemistry. The result of Immunoprecipitation showed that MAb (Y8-G1-E8) could bind to Ig from serum of Chinese sucker. MAb (Y8-G1-E8) was used to identify Ig-positive lymphocytes in the peripheral blood by flow cytometry, and the resulted indicated the percentage of Ig-positive lymphocytes was41.8%. Further experiments proved that MAb (Y8-G1-E8) could recognize Ig-positive lymphocytes and granulocytes in the peripheral blood by immunocytochemistry.The MAbs against Chinese sucker Ig in this study were demonstrated to specific against the serum Ig and Ig-positive lymphocytes in the peripheral blood and provide a powerful tool for studying the structure, function, physicochemical property of Ig and identifying Ig-positive lymphocytes in immune organs. Besides, it also provides materials for studying the production, lasting time and the disappearance regularity of Chinese sucker Ig. More important, MAbs against Chinese sucker Ig provide the foundation to evaluate the immune efficacy vaccine and previously expose the pathogen-infected Chinese sucker.