Development Of Monoclonal Antibodies To Immunoglobulin Of Japanese Flounder (Paralichthys Olivaceus) And Application In The Study Of Lymphocystis Disease

Japanese flounder (Paralichthys olivaceus) is an economically important fish and recently culturing has become popular in Asian countries such as China, Japan and Korea. However, with the development of intensive culture, significant pathogens have emerged, and cause major losses. Moreover, the drug resistance, residue and diffusion in the environment owing to the drugs used in preventing and curing diseases have made severe effect on food security and public health. Therefore, we need to study the immune system and mechanism of Japanese flounder and establish the evaluation method for immune efficacy of vaccines, in order to prevent and cure diseases by immune defence.Lymphocystis disease (LCD), an important viral disease of Japanese flounder, which has resulted in a great economic loss in marine culture industry. In this study, immunoglobulin (Ig) of Japanese flounder was separated from the antiserum against lymphocystis disease virus (LCDV), monoclonal antibodies (MAbs) against Ig of Japanese flounder were developed and characterized, the distribution of surface membrane Ig-positive lymphocytes was studied, the specific antibody against LCDV was detected, and feasibility of previous exposure of disease and evaluation of immune efficacy of vaccine for Japanese flounder using the MAbs agaist Ig was researched.The followings are the details.(1) Purification of Ig of Japanese flounder. Healthy Japanese flounder was immunised with purified LCDV, and the astisera were separated. The Japanese flounder Ig was primarily separated from serum by precipitation with 50% saturated ammonium sulphate, then further purified by HiTrap Igm Purification Column; HiTrap Protein A Sepharose Column and HiTrap DEAE Sepharose Fast Flow Column using protein purification system (AKTA prime, Amersham), respectively. The protein content was checked by SDS-PAGE. The results indicated that HiTrap Igm Purification Column was not suit to separate Japanese flounder Ig; HiTrap Protein A Sepharose Column could separate a small quantity of Ig specifically with high purity; HiTrap DEAE Sepharose Fast Flow Column colud separate a mass of Ig with a few other proteins, which was finally selected to purify Japanese flounder Ig on the basis of the point of MAbs production in this study. The molecular weight of the heavy and light chains of Japanese flounder Ig was 74kDa and 24kDa, respectively.(2) Production and characterisation of monoclonal antibodies against Japanese flounder Ig. The 4 weeks old Balb\C mice were immunised 4 times within 4 weeks by purified Japanese flounder Ig. Spleen cells were collected from the immunised mouse and fused with myeloma cells, P3-X63-Ag8U1 using 50% polyethylene glycol 4000. The mediums of hybridoma cells were detected by indirect ELISA about two weeks after cultured in HAT-GIT. 10 of positive hybridomas (2D8, 2H1, 1E1, 1C12, 2E6, 1E7, 1B11, 4D1, 2A7, 2E8) were cloned because of secreting high titer antibodies. In isotyping analysis, the results showed that MAb 2D8, 1E1, 1C12, 2E6, 2A7 and 2E8 are IgG isotype, MAb 2H1, 1E7, 1B11 and 4D1 are IgM isotype. Western blot analysis demonstrated that MAb 2D8, 2H1, 1C12, 1E7, 1B11, 2E6, 4D1, 2A7 and 2E8 reacted specifically the heavy chain of Japanese flounder Ig,MAb 1E1 simultaneously reacted the heavy chain and light chain of Japanese flounder Ig, MAb 2E6 could not be determined by western blot analysis. Immunofluorescence results indicated all the MAbs produced could react with the surface membrane Ig of the lymphocytes of Japanese flounder.(3) The distribution of surface membrane Ig-positive lymphocytes. MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytometry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8.(4) Detection of specific antibody in the serum of Japanese flounder against LCDV. Indirect ELISA was used to detect the specific antibody against LCDV in the serum of apparently healthy, LCD-diseased, LCD-recovered Japanese flounder and Japanese flounder immunised with LCDV inactivated vaccine. The results showed that most of the apparently healthy fish showed a low level of ELISA absorbance (OD 0.092), but some of the fish showed high absorbance as the LCD-diseased fish being in the latent period; the fish with remarkable symptom of LCD showed high absorbance (OD 0.165) and the absorbance of fish recovering from LCD was much higher (OD 0.231), which indicated that Japanese flounder recovering from LCD has a certain immunoprotection against the disease again. The level of the specific antibody of Japanese flounder remarkably increased after immunised with LCDV inactivated vaccine (from 0.086 to 0.395), which indicated there is immunoprotection of LCDV inactivated vaccine to flounder.The MAbs against Japanese flounder Ig in this study were demonstrated to specific against the SmIg of lymphocytes and the serum Ig, which provide the method and basis to study the ontogenesis, differentiation and apoptosis of the antibody secreting cells, and provide a powerful tool for studying the production, lasting time and the disappearance regularity of Japanese flounder Ig. More important, MAbs against Japanese flounder Ig provide the foundation to evaluate the immune efficacy of vaccine and previously expose the pathogen-infected Japanese flounder.