| Aflatoxin is a class of mycotoxin, which are toxic secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus. Many kinds of AFT, such as B1、B2、G1、G2、M1、M2were well known. AFT belongs to the carcinogenic, teratogenic, mutagenic substance. AFT was identified as class I of the most toxic carcinogen. And is seriously hazard to human and animal health. AFB1is the most toxic in all kinds of AFT. AFB1is one of the key indicators for monitoring in the field of food safety and animal feed. AFT is mainly metabolized in the liver of animal body. Relative research shows that AFT was indirect carcinogens.AFB1-8,9epoxide is the middle metabolites of theAFB1has a strong activity which will form adducts with DNA, RNA and proteins and blocked the normal metabolism of nucleic acids, in the progress, the oxidases CYP450has played a important role. Most of food and ingredients are easy to be contaminated by AFT. Peanuts, peanut oil, dried fruit, milk had been reported frequently. In recent years, with the awareness of people’s food safety, most countries have developed strictly limits on the content of AFT in a variety of food and crop products. There are a variety of AFB1detection methods, commonly used such as thin layer chromatography, high performance liquid chromatography, and immunological methods.Immunological detection method compared to other methods which has many advantages, for example, rapid, sensitive, and specificity, low cost, batch testing samples, lesser harm to the operator. There is good prospect of application.The molecular weight of AFB1is312.27, which is no immunogenicity of small molecules, and belongs to the hapten. Preparation of the complete antigen of AFB1and gain of the monoclonal antibody of AFB1is the prerequisite for the establishment of immunological rapidly test method of AFB1. In this study, the carboxyl group was introduced in the Aflatoxin B1(AFB1) molecule by Succinimide ester method in order to produce AFB1O. Then, AFB1-BSA and AFB1-OVA were synthesized by EDC and DCC methods. Thin layer chromatography technology, UV spectroscopy, SDS-PAGE and immunological methods were used to identify the complete antigen. Balb/C mice were immunized with AFB1-BSA. The titer, sensibility and specificity of the antiserum was determined by indirect ELISA and competitive ELISA. The antiserum titer of all the Balb/C mice were over1:3200. The No.6mouse reached the highest of1.28×10-4and the sensitivity is good with half inhibitory concentration IC50of22.268ng·mL-1, low cross-reactivity.In this study, the cell fusion of conventional chemical method was used to develop the clone of hybridoma. After cultivating by selection medium screening by ELISA. continuous20generations subculture in vitro and repeated frozen three times, as a result, gaining a cell line6D9was gained with stably secreting monoclonal antibodies against AFB1.After expansion of cultivation in vitro and induced ascites in the abdominal cavity of mice to obtain antibodies, After detecting by ELISA. The titer of the culture supernatant was1:200and the antibody titer of ascites was1:6400by ELISA. Monoclonal antibody subtype of6D9hybridoma is IgG1through antibody identification. In conclusion, the complete antigen of AFB1was synthesized successfully. The monoclonal antibodies of AFB1has been developed in this study, which laid a foundation for the establishment of a rapid detection method against AFB1. |
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