Study Of Fumonisin B1immunological Rapid Detection Method

At present, the mycotoxin has become an important source of pollution in food,and Fumonisin is one of the more serious pollution of mycotoxin, it is a group ofmetabolic poison mainly produced under certain conditions by Fusarium moniliforme,prolific Fusarium and other Fusarium, The main component is Fumonisin B₁（FB₁） andmost exists in food and feed. Fumonisin were not only has the liver and kidney toxicityand lung toxicity, but also has neurotoxicity, immune system toxicity, carcinogenicityand other strong toxicity, so food and its products polluted by Fumonisin cause seriousthreat on development of animal husbandry and human body health. Recently, themainly method used for detecting FB₁were thin layer chromatography, gaschromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry and so on, such as the need of complicated pretreatment process,need expensive equipment, need to spend a lot of time, need a large amount ofmanpower and material resources and so on. So the establishment immunologicalrapid test method for Fumonisin is particularly important. This study is on the basis ofFB₁artificial antigens preparation, through the monoclonal antibody technologyscreened out of FB₁monoclonal antibodies, established indirect competitive ELISAtest methods for FB₁, prepared the indirect competitive ELISA fast detection kit forFB₁.1. Preparation of Fumonisin B₁artificial antigen and polyclonal antiserumFB₁were coupled with carrier protein by using EDC method, after SDS-PAGEgel electrophoresis for the identification, coupling protein migration rate is far lessthan the carrier protein, it explained the coupling has successed. Then immunized mice,FB₁polyclonal antiserum was prepared, the titer is high up to1:5.12×104, halfinhibition concentration IC50is61.3ng/mL.2. Preparation and characteristics identification of Fumonisin B₁monoclonalantibody After the successful preparation of FB₁artificial antigen and preparation of highsensitivity, strong specificity polyclonal antiserum, the monoclonal antibodytechnology has successfully carried out in the fusion of mice splenic cells and NS0myeloma cells,1G11and2G12cell lins has been screened out by use of indirectELISA method and indirect competitive ELISA method, their cell supernatants titer is1:2.56×10⁴and1:1.28×10⁴. Injected1G11hybridoma cells into the mice’s paraffinedabdomen through intraperitoneal injection, the hybridoma cells proliferated in mice,and secreted specific monoclonal antibodies, the titer of collection ascites can reach1:5.12×10⁵. After identification, the monoclonal antibody subtype is IgG1, affinityconstant is2.7×10¹⁰L/mol, half inhibition concentration IC50can reach6.56ng/mL, andthe cross response rate with other toxins competition is less than1%. That shows theFB₁mAb has more significant on titer, sensitivity, specificity and affinity.3. Preparation of FB₁indirect competitive ELISA detection kitThrough the indirect competitive ELISA method, identify the antigen coatingconcentration of1:1000diluted, the antibody working concentration of1:30000diluted, established the indirect competitive ELISA detection method, and preparationof FB₁indirect competitive ELISA rapid detection kit. Various performance of the kitis good, the correlation coefficient of standard curve linear regression equation is0.9856, the sensitivity is2.1ng/mL, the recovery rate is above80%, the coefficient ofvariation is less than15%, the cross response rate is less than1%, and the results showthat the preparation of FB₁-Kit in this study has good sensitivity, high accuracy, strongspecificity and good stability.