Construction Of Bispecific Antibody Against Sudan ?/Acridine Ester And Study On Its Immunochemiluminescence Method

This research mainly studied the application of bispecific antibodies in chemiluminescent immunoassay methods.Using Sudan Red I as a model analyte,a bispecific antibody against Sudan Red I/anti-luminescent substrate was constructed by using a chemical coupling method and cell fusion method.A chemiluminescence immunological detection method based on the bispecific antibody was established as followed.1.Heterobifunctional cross-linker method to construct bispecific antibodyDithiothreitol?DTT?was used to reduce the disulfide bond between the heavy chain and the heavy chain of the antibody to form free sulfydryl groups.Orthogonal test was used to design the experiment.The experimental results showed that the reaction pH has the most significant effect on the yield of heavy chain and light chain of antibody?HC-LC?,followed by temperature,concentration and reaction time.The optimal reaction pH was between 7 and 8,the concentration was 1 mg/mL,the temperature was 25°C,and the time was 2 hours.Molar ratio of feed n?DTT?/n?IgG?was 30 to 70.heterobifunctional cross-linker 4-?N-maleimidomethyl?cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt?Sulfo-SMCC?was used to link with the anti-acridine monoclonal antibody,and then coupled to the reduced sulfhydryl group of the anti-Sudan red I antibody.The non-reducing SDS-page electrophoresis showed that the coupling reaction was successful.2.Construction of bispecific antibody by hybridization-hybridoma cell fusion method8-azaguanine?8-Ag?was used to induce hybridoma cells that secrete anti-Sudan I antibody,resulting in a hypoxanthine-guanine phosphoribosyl transferase?HGPRT?-deficient strain.Then the splenocytes of acridinium ester-blood Crycombin antigen immunized Balb/c mice were treated with defective cells for electrofusion.After screening with HAT,two positive hybrid-hybridoma cells were selected by ELISA.After subcloning,hybrid-hybridoma cells?G7/A5,A5/D11?secreting bispecific antibody were selected.G7/A5 was injected into the abdominal cavity of mice to produce a large amount of bispecific antibodies against Sudan Red I/acridinum ester.After purification of G7/A5 ascites by octanoic acid-ammonium sulfate method,two kinds of anti-Sudan red I/acridine ester antigen immunoaffinity column were prepared by glutaraldehyde method.The bispecific antibody of anti-Sudan red I/acridine ester was purified by double immunoaffinity column.The optimal purification conditions were as followed:the buffer condition of acridine ester immunoantigen column was 0.01 mol/L PBS at pH 7.0,and the optimal buffer of Sudan red I immunoantigen column was 0.01 mol/L PBS at pH 6.5,thus obtaining pure bispecific antibody.3.Establish chemiluminescence immunoassay based on the bispecific antibodyBased on obtained bispecific antibody against Sudan red I/acridine ester by cell fusion method,the methods of chemiluminescence immunoassay?CLIA?and chemiluminescent enzyme immunoassay?CLEIA?were established respectively.The results showed that CLEIA was more stable and sensitive than CLIA.The competitive CLEIA was used to establish an immunological method for the determination of Sudan I in Chili powder.The antigen antibody concentration,HRP-AE concentration,organic solvent content,pH and ionic strength of indirect competitive CLEIA were optimized,and RLU_max/IC₅₀ maximum value was taken as the optimal optimization condition.After optimization,the concentration of antigen coating was 8?g/m L,the dilution ratio of antibody was 1:500,HRP-AE was 1:500,the buffer conditions were 30%methanol,0.01mol/L PBS pH 6.4,and the ionic strength NaCl was 0mol/L.Under the optimized conditions,CLEIA indirect competition curve was established.The standard curve equation was y=-45.974 x+57.809,R²=0.9919,linear range was 0.2¹0 ng/mL,IC₅₀0 was 1.478 ng/mL.The recovery rate of Sudan I in Chili powder was 71¹03%,and the coefficient of variation was 3.1⁴.7%.