Establishment Of Two Sensitive Enzyme Immunoassays For Aflatoxin M₁ And Preparation Of Bispecific Antibody

In this paper,based on the optimization of each reaction system,two enzyme immunoassays for aflatoxin M₁?AFM₁?were established,including Enzyme-linked Immunosorbent Assay?ELISA?and Chemiluminescence enzyme linked immunosorbent assay?CLEIA?,and evaluated by immunoaffinity column-high performance liquid chromatography.In addition,on the basis of AFM₁ monoclonal antibody,the bispecific antibody of anti aflatoxin M₁?AFM₁?-anti horseradish peroxidase?HRP?was prepared by Cell fusion method to establish an AFM₁-ELISA based on bispecific antibodies to improve the sensitivity of the detection method for AFM₁.1.Establishment of enzyme-linked immunosorbent assayAFM₁ was coupled with carrier protein BSA by carbodiimide method,and the characteristic absorption peaks of AFM₁ and BSA were obtained by ultraviolet scanning.AFM₁ and BSA characteristic absorption peaks were obtained by uv scanning,AFM₁-BSA artificial antigen was successfully obtained.By optimizing the ELISA reaction system,we determined that the AFM₁ antigen coating concentration was 0.5?g/mL,the antibody working concentration was 1:30000,enzyme labeled anti-antibody dilution ratio was 1:500,5%skim milk was used as the standard preparation liquid,the sample extract was acetonitrile,the dilution ratios of liquid milk and milk powder were 1:1 and 1:5.Among these conditions,a one-step competitive inhibition curve for AFM₁-ELISA was established with the IC₅₀0 was0.27ng/mL and the limit of detection was 0.043ng/ml of the method.The recoveries was 86.46%-120.12%in liquid milk and yogurt,with the coefficient of variation in the range of 1.42%-6.42%.2.Establishment of chemiluminescence enzyme-linked immunosorbent assayA one-step chemiluminescence enzyme-linked immunosorbent assay for AFM₁was established by comprehensively optimizing the concentration of enzyme labeled anti-antibody,ionic strength,pH and organic solvent in CLEIA reaction system.Its IC₅₀0 was 0.08 ng/mL,the limit of detection was 0.024ng/mL,it only takes 30 minutes.In addition,different levels of spiked recovery were performed in dairy products for methodological evaluation,and the recoveries range from 81.46%to 109.08%with the coefficient of variation was 0.81%to 7.27%in liquid milk.3.Establishment and evaluation of immunoaffinity column-high performance liquid chromatographyThe standard curve of high performance liquid chromatography for AFM₁ was established by optimizing the AFM₁ immunoaffinity column loading buffer under the national standard parameters.The limit of detection and the limit of quantification were 0.075ng/mL and 0.3ng/mL,.The recoveries in milk ranged from 87.50%to99.85%with the coefficient of variation was 0.72%to 12.77%,in milk powder recoveries was 78.76%to 96.14%with the coefficient of variation was 2.95%to7.05%.At the same time,the correlation between ELISA,CLEIA and HPLC was studied?correlation is R²=0.98869 and R²=0.99704?.The AFM₁ content of 10unknown milk samples was detected by the above three methods,the results of ELISA and CLEIA were consistent with the HPLC,indicating that the established method can be used for rapid screening of AFM₁ in actual samples.4.Preparation of anti-aflatoxin M₁/anti-horseradish peroxidase bispecific antibodyHybridoma cells secreting anti-AFM₁ antibody were induced by 8-azaguanine?8-AG?to obtain hypoxanthine-guanine phosphoribosyltransferase?HGPRT?deficient cell lines,then fusion of the AFM₁ deficient cells with HRP immunized spleen cells by cell-electrofusion.Indirect ELISA and bridging ELISA were used as screening systems to select Triple tumor cell.Twenty-eight anti-HRP positive cell lines were screened.one anti-AFM₁/anti-HRP double-positive cell line was obtained by expanded culture.Finally,cloning was lost due to the instability of the cloned cells.