Establishent Of Method On Detection Of BTV Antibody With Evanescent Wave Fiber Optic Immunosensor

Bluetongue, as a kind of the world organization for animal health (OIE) must be reported to a violent animal infectious disease, the main harm of cattle, sheep and other domestic and wild ruminants, in the world scope widely popular. International organizations and governments are to be bluetongue animal epidemic prevention and control and monitoring as the key, in animals and animal products international trade has very important influence. At present, all countries in the world, there are no effective bluetongue vaccines and prevention and control measures, can with immune monitoring primarily, therefore, establishing a rapid, sensitive, convenient, specific strong BTV detection method is particularly important.Evanescent wave fiber optic immunosensor (EWFI) is evanescent wave principle, fluorescence analysis principle and immune optical testing combined in one of the detection device, be specific to the strong, high sensitivity, the detection speed, simple operation, etc. Therefore, in the environmental monitoring, clinical disease monitoring, food hygiene inspection and the biological testing and other fields have a broad prospect of application.This research use BTV VP7monoclonal antibodies and BTV VP7protein (both for the preparation of laboratory), conduct BTV antibody evanescent wave fiber optic immunosensor (EWFI) detection methods, BTV VP7protein as the antigen was immobilized to optical fiber, VP7monoclonal antibodies as testing samples, Cy3markers of the sheep of rat IgG antibodies as testing antibody and evanescent wave fiber optic immunosensor (EWFI) detection method of BTV antibody to the established. This study of the method of commonly used for reference ELISA board, to optical fiber bag was VP7protein concentration and Cy3marking of antibody is optimized, and the results prove VP7protein best bag was concentration for150μg/ml, Cy3mark testing two of the optimal concentration of20μg/ml. After the initial optimization, get VP7monoclonal antibody detection limit lOng/ml. In addition, the research of this detection method of experimental steps are optimized, has no impact on the experimental results, the influenza virus antibody and Cy3mark antibody blends rejoin after the reaction tank not only simplify the experimental procedure, also shorten testing time, make this testing method is simple, convenient, more quickly.Evanescent wave fiber optic immunosensor (EWFI) detection experiments, optical fiber regeneration has been difficult to solve, this experiment to this problem is studied. This research use glycine-HCl buffer (pH2.0), TEA solution (pH11.5) and0.5%of SDS (pH1.9) solution to regenerate the optical fiber, and the results showed that the TEA solution (pH11.5) regeneration effect is best, can be recycled at least ten circulation; Glycine-HCl buffer (pH2.0) can regenerate six circulation; And0.5%of the SDS (pH1.9) solution can only renewable two cycle. Therefore, in this experiment choose TEA solution as optical fiber regeneration fluid.Finally, the detection method and the common BTV antibody test method, ELISA comparison method, the method of performance evaluation.