Development And Application Of Highly Sentivie And Specific Monoclonal Antibodies Against Alfatoxin And Its Precursor Sterigmatocystin

With the sustainable development of economy, the continuous improvement of living standardand more and more higher requirement to food safety, the impact of mycotoxin contamination onagricultural products and food safety has become a social concern and hot spot of the world.Among ofthem, aflatoxin is the most toxic. Aflatoxin and their biosynthetic precursor sterigmatocystincontamination, has become a serious threat to the health and life safety of human and animals andcaused a great loss of economics. Therefore, to establish accurate, sensitive and convenient immuneanalysis technology for aflatoxin and sterigmatocystin is imperative.Antibody is the key point of immunoassay which decides the sensitivity and specificity ofimmunoassay. In our laboratory, anti-aflatoxin B1and anti-aflatoxin M1monoclonal antibodies weredeveloped, which were the most sensitive and specific antibodies in the world. However, monoclonalantibody3G1, which is the most specific antibody towards aflatoxin B1, still has6.4%cross-reactivitywith aflatoxin B2. Anti-aflatoxin M1monoclonal antibody2C9which is the most specific againstaflatoxin M1, but its sensitivity remains to be improved. Sterigmatocystin is the synthesis precursor ofaflatoxin B1, thus could be used as a biomarker of aflatoxin B1occurrence. Development ofimmunoassay towards sterigmatocystin could provide techincal support to aflatoxin B1risk assessmentand prevention of aflatoxin B1contamination. However,there is no related research up to now. As aresult, developing high sensitivity and specificity monoclonal antibody towards aflatoxin B1,M1and itsprecursor has a significant value to the development of determination technology and products, riskassessment of agro-products quality and safety, guarantee commercial safety of agro-products and food,promote agriculture industry and break trade and technology barrier.In order to overcome the difficulties of developing antibody towards aflatoxin B1,M1and itsprecursor with high sensitivity and specificity, the main research contents and innovations are asfollows:1. The hybridoma cell lines and monoclonal antibodies including1B5with high sensitivity andspecificity for aflatoxin B1were achieved. The sensitivity of1B5is more than100times highercompared with monoclonal antibody3G1. The key step of antibody development is antigen synthesis,in which the main point is the design of hapten. In this research, a new type of aflatoxin antigenAFB2-GA-BSA was developed based on the method raised by Christian Cervin and AFB1-BSA wasprepared based on the method by CHU. Balb/C mice were immunized using the newly developedantigen with different immunogen dose of50μg AFB2-GA-BSA and150μg AFB1-BSA every time.Four hybridoma cell lines1F11,2F12,2C7and1B5, were achieved after immunization withAFB1-BSA, cell fusion, incubation with semi-solid medium and two-step screening. The performanceof antibodies was evaluated by indirect competitive ELISA, including sensitivity and cross-reactivity.After comparing with related antibodies reported, the monoclonal antibody B5is the best on sensitivityand specificity. Indirect competitive ELISA was developed based on monoclonal antibody1B5. Theconcentration of coating antigen AFB2-GA-BSA was0.0625μg/mL and antibody1B520000timesdilution. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized.2. The hybridoma cell lines and monoclonal antibodyies including LM13with high sensitivityand specificity for aflatoxin M1was achieved. The sensitivities of the antibodies are more than2timeshigher compared with monoclonal antibody2C9. To obtain AFM1antibody with high sensitivity andhigh specificity, in the early foundation, we increased the immune dose and prolonged the immuneperiod. Nine hybridoma cell lines LM47, LM15, LM16, LM54, LM13, LM32, LM44, LM48andLM46, were achieved after cell fusion, incubation with semi-solid medium and two-step screening.The performance of antibodies was evaluated by indirect competitive ELISA, including sensitivity andcross-reactivity. After comparing with related antibodies reported, the monoclonal antibody LM13isthe best on sensitivity. Indirect competitive ELISA was developed based on monoclonal antibodyLM13. The concentration of coating antigen AFM1-BSA was0.0625μg/mL and antibody LM13120000times dilution. Several physicochemical factors influencing assay performance, such as pH,ionic strength, blocking solution, and diluting solution, were optimized.3. A series of hybridoma cell lines (eg. ST3) were selected and ultra-sensitive sterigmatocystinmonoclonal antibodies with high specificity were prepared. Protein BSA and ST molecules werecoupled to synthesize ST artificial antigen by opening the double bond of double furan ring in themolecular structure of ST. ST artificial antigen was test by UV. The results showed that BSA and STwere successfully coupled because the characteristic absorption peak of complex was different fromthat of the BSA or ST. Three kinds of monoclonal hybridoma cell lines with good performance wereselected with a whole procedure including immunizing the BALB/c mice with the new completeantigen, cell fusion, semi-solid medium, and two-step gradient selection. They were named by ST3，ST4，ST6,respectively. Indirect competitive ELISA was developed based on monoclonal antibodyST3. The concentration of coating antigen ST-GA-BSA was0.5μg/mL and antibody ST380000timesdilution. Several physicochemical factors influencing assay performance, such as pH, ionic strength,blocking solution, and diluting solution, were optimized.