The Synthesis Of Sudan Ⅰ Hapten And The Preparation Of Its Antibody

Sudan Red is often used as a red dye in industry, and researchs have shown that Sudan I has potential carcinogenicity to human. Thus Sudan I is considered as an illegal food additive in China and the European Union, but many criminals used it as the dye in food industry because of its good coloring and cheap. Therefore, it is particularly important to establish a rapid and effective detection method of Sudan I in food.Through the design and synthesis of Sudan I Hapten molecular, the preparation and purification of the artificial antigen, preparation of polyclonal and monoclonal antibody and optimization of a series of detection parameters, we intend to establish a competitive enzyme-linked immunosorbent assay method which can rapid detect the Sudan red in food.This paper can be divided into four parts:(1) Synthesis and identification of Sudan red artificial antigen and coating antigenWe designed and synthesized two Hapten molecules, then purified the Hapten molecules through the column, identified it by mass spectrometry, coupled with BSA and OVA respectively by the DCC/NHS method, BSA conjugate was as artificial antigen while OVA conjugate was used as coating antigen. Finally, conjugates were proved to be successfully coupling by SDS-PAGE and UV spectra, the coupling ratio of carrier protein and Sudan Red Hap ten X was1:9and1:6respectively; to Hapten2was1:30and1:10respectively.(2) Immunization and titer determination of antiserumBSA conjugates were used to immunized three6-8weeks old BALB/c female mice five times, with an interval of14-21days. After the strengthen immune, the spleen cells and myeloma cells were taken from mouse, the antiserum titer from Hapten X immunized mice was significantly higher than the other two Haptens, the titer was about1-.80000; the titer by Hapten2was about1:50000.(3) Preparation and purification of monoclonal antibodyMice with high antiserum titer were chosen, then crudly purified by ammonium sulfate-caprylic acid, the titer of monoclonal antibody cell culture supernatant detected by ELISA was1:800.(4) Optimization of ELISA detection parameterELISA assay would be affected by different testing conditions, thus we optimized the parameters of ELISA detection, including the choice and concentration of the blocking and coating buffer, the concentration of Tween-20, coating antigen and antibody. Ultimately to improve the sensitivity of ELISA.The results obtained from this paper:(1) Sudan Red transformed Hapten molecules were successfully prepared and obtained a Sudan red Hapten X and Hapten2.(2) Hapten and carrier were successfully coupled,we obtained two complete antigen: Hapten-BSA conjugate, Hapten-OVA conjugate.(3) Through cell fusion and subcloning screening, we got the hybridoma cell lines which could secrete specific anti-Hapten X antibody, and the cell culture supernatant titer was1:800.(4) We ultimately got the optimal combinations of detection parameters through the study of different detection condition of ELISA:pH9.6CBS as coating buffer,5%BSA in PBS as the blocking solution, PBST contained0.05%Tween-20, coating antigen concentration is lug/ml and antiserum is diluted20,000times.(5) The space between Hapten and carrier could impact the anti-serum titer, and long arm can induce the generation of high titers of antibody, generally3-5C was more appropriate.(6) For Sudan I molecules, the hydroxyl groups play an important role in the recognition of antibody. In our experiment, we directly use their hydroxyl groups to connect to carrier protein, although the antibody titer is high, the recognition of monoclonal antibody for Sudan I is poor.(7) Using a syringe is much better than ultrasound or muller as emulsifier.(8) Antibody which induced by Hapten2to Sudan IIC50was about0.34ng/ml...