Preparation Of Monoclonal Antibody Against Citreoviridin And Establishment Of ELISA For Citreoviridin

Citreoviridin(CIT),a toxic secondary metabolite which is mainly produced by Penicillium citreonigrum is generally detected in cereal grains and grain-based agricultural products worldwide.CIT has a cardiotoxicity,neurotoxicity,genotoxicity and teratogenicity.However,there is no immunoassay for CIT based on anti-CIT monoclonal antibody both at home and abroad.This study aimed to prepare a specific,high-affinity monoclonal antibody against CIT,which was used for establishing a fast and effective immunodetection method.Artificial antigen CIT-KLH and CIT-BSA was successfully prepared via succinic anhydride and carbodiimide two-step methods.CIT-KLH conjugates as immunogen were injected into Balb/c mice,and CIT-BSA conjugates as detection antigen were coated into microtiter plates to detect and screen antibody against CIT.When the titer assay of antiserum were high enough,the spleen cells from Balb/c mice which was immunized with CIT-KLH were fused with SP2/0 myeloma cells.The fusion rates both were 100%and the positive rates were 3.77%and 5.0%,respectively.Eight positive clones were obtained by subcloning,and named as 1A12,3B4,3H6,8D8,8G5,6B8,8D6 and 10C9.Except that the 8D8 mAb was IgG1 subtype,the other seven positive clones were IgM.So 8D8 clone was chosen for further study.The titer of 8D8 mAb reached 1:1.28×10~5 after purified by caprylic/ammonium sulfate precipitation(CA-AS)method,and the concentration of 8D8 mAb was 6.6 mg/mL,the average affinity was 4.57x10~8 L/moL.An indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)for Citreoviridin was established.Under optimal condition,the linear range to detect CIT was 11.02-2370.48 ng/mL and the limit of detection of the ic-ELISA was 11.86 ng/mL.With the mean coefficient of variation lowing 5%,the recovery in intra-assay and inter-assay were 86.83%-92.28%and 85.0%-95.72%,reapectively.The optimal operating conditions of ic-ELISA based monoclonal antibodyagainst CIT were established in this study:The optimal working concentration of coating antigen was 0.5 ?g/mL,The optimal coating time of antigen was 2 h at 37?,or overnight at 4?;the best blocking solutions is 5%PBSM,The optimal blocking time was at 37? incubation 2 h;the optimal dilution of monoclonal antibody and HRP-IgG were 1:40000 and 1:8000,respectively,and the optimal reaction time of substrate was 10 min.The monoclonal antibody obtained in this study provided the solid foundation for a rapid immunoassay method for CIT,and it could be used for establishments of ELISA kit,colloidal gold strips,immune sensor and so on.