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Background: Mycotoxins as secondary metabolites produced by fungi, carcinogenic, cause mutations, teratogenic and other toxic distributed in wheat, corn, feed, and a variety of food, once poured into the ecological environment, will animal The people and the entire biological cause serious harm to the environment, so much worldwide attention. Mycotoxins detection method of physical and chemical methods and immunoassay methods, physical and chemical methods are high sensitivity, but the operation is complex and expensive instruments, immunological methods can only detect a toxin. Therefore, high-throughput protein chip method will become the development direction of the rapid detection of mycotoxins in food. Methods: This study is first prepared by antigen preparation, antigen identification, immune, cell fusion and screening steps citrinin monoclonal antibodies, then of citrinin (citrinin metabolism. Citrinin was CIT), deoxynivalenol by Fusarium enol (Deoxynivalenol, DON) and fumonisin B1 (fumonisin, B1) for the study, using the principle of indirect competitive immunoassay, fluorescent signal detection signal to determine the CIT, a fixed concentration of DON and FB1 artificial antigen, fixed way, anti-CIT, anti of DON the three anti FB1 monoclonal antibody and IgG-Cy5 secondary antibody concentration to create a separate detection of a toxin protein chip detection method. On this basis, the initial establishment of protein chip method for simultaneous detection of three mycotoxins. Results: (1) After the identification of the three methods, citrinin artificial antigen successfully coupled; (2) through the immunization of mice was prepared hybridoma cell strain, obtained a monoclonal antibody of the anti-citrinin cell strains; (3) citrinin indirect competitive ELISA with an IC50 of 96.2 ng / ml, the detection limit of 10 ng / ml and a linear range of 10-810 ng / ml; (4) barley, corn, feed samples added recovery experiments recoveries were 99-127.1% ,75.4-124 .5% ,78-108%. (5) determine the CIT of DON and FB1 artificial antigen fixed optimal working concentration were 1:100,1:500,1:250; the three mycotoxins monoclonal antibody concentration 1:500,1 : 400,1:4000; IgG-Cy5 secondary antibody concentration of 1:1000 the best artificial antigen fixed conditions of 25 ℃ fixed for 16 h, drawn three mycotoxins competitive inhibition curve, IC50 63.7 ng / mL, 83.5 ng / mL, 54.6 ng / mL, the linear range for :10-810 ng / mL ,10-810 ng / mL ,1.9-125 ng / mL. (6) to draw three mycotoxins detection of competitive inhibition curve, CIT sensitivity (IC50) was 25.7 ng / mL, the linear range of 10-810 ng / mL of DON sensitivity (IC50) was 58.5 ng / mL, linear the range of 10-810 ng / mL, FB1 sensitivity (IC50) to 24.9 ng / mL, the linear range of 1.9-125 ng / mL. Conclusion: by preparing anti citrinin monoclonal antibodies by indirect competitive ELISA add recovery test preliminary validation of the ELISA detection method can be used for real samples; initially established according to the standard of our prescribed limits, the simultaneous detection of three mycotoxins protein chip detection method to meet our limited needs.
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