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目的:采用人工偶联方法构建多西环素完全抗原,制备多克隆抗体。方法:分别采用直接偶联法、甲醛一步法、重氮法+混合酸酐法和重氮法+碳二亚胺法等4种方法将多西环素与牛血清白蛋白(bull serum albumin,BSA)或鸡卵清蛋白(ovalbumin,OVA)分别偶联,构建人工完全抗原。选择最好偶联效果制备的抗原免疫BALB/c小鼠,通过直接ELISA法检测多克隆抗体效价,竞争抑制ELISA法分析其灵敏性及特异性。结果:重氮法+碳二亚胺法偶联的多西环素人工完全抗原效果最好,其与BSA的偶联比为8.37∶1,与OVA的偶联比为4.92∶1。采用该方法偶联的抗原免疫小鼠获得的多克隆抗体效价在1∶8 000以上;该抗体对多西环素的半数抑制浓度(IC50)为98.89~120.32μg/L,与其他四环素类药物的交叉反应性较低。结论:重氮法+碳二亚胺法的偶联效率最高,获得的多西环素多克隆抗体效价较高,特异性和灵敏度均较理想。
OBJECTIVE: To construct doxycycline complete antigen by using artificial coupling method to prepare polyclonal antibody. Methods: Four methods of direct coupling, formaldehyde one-step, diazotization + mixed anhydride and diazotization + carbodiimide were used to separate doxycycline and bovine serum albumin (BSA ) Or ovalbumin (OVA) were coupled to construct artificial total antigen. The BALB / c mice were immunized with the antigen prepared by the best conjugation effect. The titer of the polyclonal antibody was measured by direct ELISA and the sensitivity and specificity by competitive inhibition ELISA. Results: The best doxycycline antigens conjugated with diazotization and carbodiimide were the best. The coupling ratio of BSA to BSA was 8.37:1. The coupling ratio to OVA was 4.92:1. The titer of the polyclonal antibody obtained from the mice immunized with this antigen was above 1: 8000. The IC50 of the antibody against doxycycline was 98.89 ~ 120.32μg / L, which was in good agreement with other tetracyclines Drug cross-reactivity is low. CONCLUSION: The diazotization + carbodiimide method has the highest coupling efficiency, and the obtained doxycycline polyclonal antibody has higher potency and better specificity and sensitivity.