| Fumonisins B1 (FB1) occourrs worldwidly in cereal grains and their feeds,especially for the contamination of FB1 in maize and maize-based products, which is also potential threaten to human health and has close association with the development of animal husbandary. Several analytical methods have been used for the analysis of FB1 in food and feeds and the maked typical metheds is high-performance liquid chromatography. This method needs the precolumn derivatisation techniques to make FB1 generate fluorescence to be detected, but it is not commonly used because of its complicated process and expensive equipments. The experiment established indirect competitive Enzyme-linked immunosorbant assay to detect the content of FB1, which is based on monclonal antibodies.Since its simple and rapid advantage, it would satisfy the demand of practical work. This study is the national "863" project "The development of monoclonal antibodies and the study of antibody microarray technology of the main mycotoxin in agricultural products " (Item No.2007 AA10Z429). The purpose of this study was to provide technical support of rapid detection system of detecting the main mycotoxin in agricultural products. TEST ONE:FB1 is a small molecule substances,which is only a semi-antigen having reactionogenicity while with no immunogenicity. It was conjugated with the macromolecules carrier protein so as to draw assistance from T cell epitope of macromolecules protain and then stimulate the body to generate antibodies and specific immune response. Generally the primary amine group of Fumonisin B1 and the carrier protein were conjugated as the synthetic immunogen to prepare relative antibodies. The glutaraldehyde (GA) was used to conjuge FB1 to the carrier protein by one step. The conjugated antigen was proved successful through the methods of Non-denaturing agarose gel electrophoresis and Denaturing polyacrylamide gel electrophoresis, so the conjugated antigen fit the requirements of the experiment.TEST TWO: The conjugating FB1-BSA was immunized 8-week-old BALB/c mice. Through immunization by 5 times, the spleen cells and SP2/0 myeloma cell were fused. After cultured by the HAT selective medium, the positive hybridoma was selected by indirect ELISA method. Hybridoma cell line F3 was gained through three time subcloning,which could stablely secrete monoclonal antibodies against FB1. Ig subclass of the monoclonal antibodies secreted by hybridoma cell line F3 was IgG1. These antibodies was not significant cross-reaction with other structural analogues, and simultaneously had higher specificity.TEST THERE:OVA and FB1 were conjugated by glutaraldehyde (GA) one step. The conjugated protain was coated ELISA plates as the coated antigen, and FB1 as the competitive hapten. Different concentrations of FB1 was reacted with quantitative anti-FB1 monoclonal antibody,then the adoption of HRP and substrate was added. Finally the method of indirect competitive ELISA to detect Fumonisin B1 was established. The experimental results showed that the optimum coating antigen concentration was 20μg mL-1; the optimum concentration of anti-FB1-McAb was 1:800; the work concentration of HRP was 1:10000; the linear equations was y=-0.2389x+0.835(R2= 0.9634), the detection limit concentration of the ELISA was 1.22 ng mL-1.In the reserch of the detection of FB1, the commonly method is to prepare kit based on the use of monoclonal antibody technology. So this study provides technical support of rapid detection system of detecting the main mycotoxin in agricultural products. |
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