| The southern catfish(Silurus meridionalis Chen) is an important freshwater food fish in china. In recent years, diseases of southern catfish are increasing due to the expansion of the cultivation scale. Pathogenic bacteria can be resisted by antibiotic in a short time, but it is not able to work in a long time owing to drug resistance of pathogenic bacteria and water environment pollution. Fish, similar to other vertebrates, possess an efficient and developed immune system. Immunoglobulin (Ig) play an important role in humoral immune response of fish. Studying serum Ig of southern catfish will contribute to understanding the process of immune response, which should facilitate maintenance of fish health in aquaculture.Monoclonal antibodies (MAbs) provide ability to enhance immunological, microbiological and cytological analysis due to strong specificity for binding antigen. MAbs specifically reacting with Ig of fish provide a powerful tool for studying fish immune system. MAbs against southern catfish serum Ig are useful tools for monitoring antibody production following infection or vaccination, and immunoblot analysis of antigens recognized by the host immune system. In this study, serum Ig of southern catfish was purified and molecular weight was determined. MAbs against serum Ig of Southern catfish were produced and characterized by ELISA, Western-blotting, flow cytometry and immunohistochemistry. The main results are as follows:1. Serum Ig from southern catfish was purified by euglobulin precipitation, sepharose-4B gel column and affinity chromatography using mannan-binding protein (MBP) staphylococcal or protein A (SpA) as capture ligands. SDS-PAGE showed that serun Ig of southern catfish was successfully isolated by euglobulin precipitation and sepharose-4B gel column chromatography. The H chains were shown to be approximately76kDa in molecular weight while the L chains were26kDa. MBP and Protein A affinity column were not fit for purifying serum Ig of southern catfish. The purified Ig was concentrated by Nanosep Centrifugal Devices. The amount of purified serum Ig from lmL serum sample by sepharose-4B gel column was estimated to about167.7μg, while the amount was about64.2μg by euglobulin precipitation. The yield of euglobulin precipitation was lower than that of sepharose-4B gel column.2. Five monoclonal antibodies (N1-B4-E12, N1-H7-H9, N3-G12-B11, N3-E5-F9and N2-A3-G3) were developed against purified serum immunoglobulins of southern catfish and characterized. All of MAbs did not show any cross reaction on the serum of Ctenopharyngodon idellus, Oreochromis niloticus, myxocyprinus asiaticus, and Ictalurus punctatus by indirect ELISA, but did show cross reaction on the serum of silurusasotus. Western-blotting demonstrated that N1-B4-E12, N1-H7-H9, N3-G12-Blland N3-E5-F9reacted specifically with the heavy chain of Southern catfish Ig, while MAb N2-A3-G3could not recognize any band of serum Ig of Southern catfish.3. Three MAbs, N1-B4-E12, N1-H7-H9and N3-G12-B11, showed positive reaction with peripheral blood leucoytes from Southern catfish in flow cytometry analysis. The percentage of positive cells detected were7.64%,16.25%and8.92%, respectively. The same three MAbs also showed positive reaction in immunohistochemistry test with peripheral blood leucoytes smear. The positive cell presented cytomembrane surface staining and cytoplasmic staining.In this study, four different isolating methods were applied to find the best way to purify serum Ig from southern catfish, which provided basis data for studying the structure of serum form southern catfish. Reactivity of MAbs against Southern catfish Ig developed in this study were determined in ELISA, Western-blotting, flow cytometry and immunohistochemistry, results showed that these MAbs can be useful immunological tool in monitoring health status of Southern catfish and better understanding of teleost immune system. |
PDF Full Text Request