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IL-4 is a pleiotropic cytokine of the T cell source, by acting on the different cell types to regulate host defense and host immune. IL-4 in the regulation of T and B cell differentiation and the development of Th2-type immune response is required. In addition, IL-4 is also a single adjustment of nucleated cells, and macrophage activation factor. Already extensive literature study of genes and their biological activity of IL-4 in humans, mice, rats, pigs and other mammals. Poultry, due to lack of poultry related specific reagents hindered the poultry studies of IL-4, this study aimed to prepared chicken IL-4-specific monoclonal antibody based, initially established chicken IL-4 dual monoclonal antibody sandwich ELISA method, and provide the conditions for poultry immunoassay immune cell function and immune regulation. Chicken anti-IL-4 monoclonal antibody preparation and identification applications lymphocyte hybridoma technique immunization with purified recombinant protein rGST-ChIL-4, 8-week-old BALB / c mice, abdominal subcutaneous and intraperitoneal immunization, 100μg / . First free antigen with an equal volume of Freund's complete adjuvant mixed emulsion abdominal subcutaneous immunization; two free antigen with an equal amount of complete Freund's adjuvant emulsified by abdominal subcutaneous immune; three free antigen by intraperitoneal immunization; The interval of two weeks; tail vein to enhance the immune antigen without adjuvant, 100 μg / PCS, 3d, take immune mouse spleen cells and myeloma SP2/0-Ag-14 cell fusion. Purified rHis-ChIL-4 as the detection of antigen positive clones were screened by indirect ELISA method. Stable secretion ChIL-4 monoclonal antibody cell lines obtained nine named 16D8, 17D7, 18F7, 18F12, 19F1, 20C9, 20F2, 20G3, 6A8, ascites potency order 1,280,000,1,280,000,640,000,640,000 640,000,640,000,640,000,640,000,160,000,160,000 addition to IgG2a subclasses 16D8 and 20F2 McAb, the rest are IgG1. The tests showed that the resultant mAb with the corresponding fusion protein response, and other fusion protein and control bacteria does not react, display good specificity. Western-blot trials have shown that the resulting monoclonal antibody can react with the corresponding fusion protein specific band appears. Chicken in vivo detection of IL-4 levels, as certain disease detection indicators for the prevention and control of disease, and study the interactions between the mechanism of cell-mediated immunity and cytokines useful material. Chicken IL-4 double monoclonal antibody sandwich ELISA method initially established by the determination of the initial screening of 6 mAb antibody saturation determination and additive index, 20C9 monoclonal antibody concentration 10μg/ml as coating antibody enzyme bio-16D8, labeled antibody as a detection antibody to create antibody sandwich ELISA, and fumble the blocking solution, dilution optimum reaction time of the antigen, the enzyme-labeled antibody concentration, reaction time, and HRP-streptavidin. optimal working concentration, reaction time, The optimum reaction time and other conditions of the substrate, comparative test the final choice of phosphate buffer containing 10% fetal calf serum as a blocking solution containing 4% PEG, 0.05% Tween-20 in phosphate buffered solution as a diluent, antigen reaction time 2h, the concentration of the enzyme labeled antibody 2μg/ml, the optimum reaction time of 30 min, the concentration of HRP-streptavidin 1:2,000, the best reaction time of 30 min, the substrate reaction time of 5min. This method can be detected ChIL-4 0.039μg/ml.
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