Study On Rapid And High Sensitive Quantitative Detection Kit For Tenuazonic Acid And Preparation Of Anti-tenuazonic Acid Monoclonal Antibody

Tenuazonic acid?TA?is a kind of alternaria toxin with the most toxic.It is one of the toxic metabolite produced by alternaria fungus and pyricuLaria in specific condition.It has acute toxicity,subacute toxicity,potential carcinogenicity and synergistic toxicity effect.Tenuazonic acid was one of the highest attention and most studied alternaria toixns.At present,the main methods used in TA analyze were instrument detection method depend on chromatography technology,and lack of the rapid immunoassay detection products.The aime of this study was to establish rapid and effective detection method and study on the support kit and to prepare anti-TA monoclonal antibody with high sensitivity and stable properties by hybridoma cell technology,which laid the foundation for the development of more commercial TA immunoassay products.In this study,artificial antigens were synthesized by oximation and active ester method,and anti-TA polyclonal antibody was prepared by animal immunization.Indirect competitive enzyme linked immunosorbent assay?icELISA?and indirect chemiluminescence immunoassay?icCLEIA?,which combines chemiluminescence reaction and immunoreaction,were established by using polyclonal antibody.On the method of important factors,research and development of the assembly kit.In order to obtain higher sensitivity and stable property anti TA antibody,the spleen cells from the mouse capable of producing anti TA polyclonal antibody and mouse myeloma cells?SP0/2?were fused and obtained hybridoma cell lines.After some times of subcloning,5 hybridoma cell lines which stably secreted specific antibody against TA were screened by indirect competitive ELISA.And one of the cell line with the best quality was used to expansion of cultivation in vitro and induced ascites in the abdominal cavity of mice to obtain antibodies.The method of the anti TA monoclonal antibody preparation,provided antibody assurance for the production of immunoassay detection products with stable quality.The detail results in this study are as follows.?1?Established of icELISA detection method and developed of ELISA detection kit.Reserched in the reaction condition of indirect competive enzyme linkedimmunosorbent assay.The optimal working conditions were elected as follow: the coating concentration was 2.5 ?g/mL,the coating time was 1 h in 37?,the the confining liquid was 5% skimmed milk powder,the antiserum was diluted by 64 000 times,and the competitive reaction time was 30 min,the IgG-HRP dilution was 6 000 times,the IgG-HRP reaction time was 60 min,the coloration time was 15 min.The standard curve of the method was y=-21.65x+53.68?R2=0.9941?,the 50% inhibiting concentration(IC₅₀)was 1.48 ng/mL,and the rang of average recovery was81.59%⁹1.52%.The coefficient of variation of intra-assay and inter-assay were2.48% and 9.49%.The detection kit can be kept at least 60 d in 4? when breaked or opened a seal.The cross-reactivity with aflatoxin B1,ochratoxin A,T-2 toxin,patulin,desoxynivalenol,zearalenone and alternariol less than 1%.The results showed that the kit was specificity and no cross reaction with other toxins.?2?Established of icELISA detection method and developed of ELISA detection kit.Reserched in the working condition of icCLEIA.The optimal working conditions were elected as follow: the optimal buffer pH range of 6.4 to 7.4,the concentration of coatigen was 2.0 ?g/mL,the confining liquid was 1.0% OVA,the primary antibody dilution was 1:80 000 and the reaction time was 30 min,the IgG-HRP dilution was1:8000 and the amount of light emitting substrate was 125 ?L/well.Under?optimized?conditions,the regression equation was y=49.82-20.14x?R2=0.9966?,the IC₅₀ of this method was 0.973 ng/mL.The linearity rang was 0.032³0.244 ng/mL.The limit of detection was 0.010 ng/m L.Under the same reagent condition,the specificity and sensitivity were better than icELISA,and it was suitable for the detection of lower TA concentration.The averaged recovery rang of TA from wheat and oat were 81.53%⁹6.09%.The coefficient variation of intra-assay and inter-assay were 4.08% and 8.91%,respectively.The detection kit can be kept at least 90 d in4?when breaked or opened a seal.The cross-reactivity with aflatoxin B1,ochratoxin A,T-2 toxin,patulin,desoxynivalenol,zearalenone and alternariol less than 1%.The results showed that the kit was specificity and no cross reaction with other toxins.?3?Mouse myeloma cells were fused with spleen cells,and 5 strains of positive cell lines were screened by subcloning selection.The cell lines were named TAO-4A1,TAO-4D5,TAO-4F4,TAO-7F11 and TAO-7G11,respectively.The anti-TA monoclonal antibody was prepared by ascites induction though TAO-4F4 cell line,after amplification culture in vitro.Ascites was purified by protein A chromatography and the concentration of monoclonal antibodies was 0.40 mg/mL.The purifiedmonoclonal antibody has heavy and light chains with molecular weights of 50 KDa and 25 KDa,and never hybrid protein be found.The results of icELISA showed that the IC₅₀ of monoclonal antibody was 6.1 ng/mL.Compared with the mouse polyclonal antibody(IC₅₀ was 420 ng/ mL),the IC₅₀ was descreased about 69 times.