中文摘要：

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。

英文摘要：

Objective:To conjugate the doxycycline (Dox) with bull serum albumin (BSA)/ovalbumin (OVA) and generate polyclonal antibody against the Dox.Methods:The Dox was coupled with BSA/OVA via direct,one-step formaldehyde,diazo plus mixed anhydride and diazo plus carbodiimide conjugation method,respectively.The BALB/c mice were immunized with Dox-BSA which conjugated by most efficient method.The titer,sensitivity and specificity of antibody were detected and assayed by direct or indirect competitive inhibition ELISA.Results:It had been declared that the diazo plus carbodiimide conjugation method was most efficient,and the coupling ratio of Dox ∶ BSA was 8.37 ∶ 1,and Dox ∶ OVA was 4.92 ∶ 1.The polyclone antibody (pAb) titer was more than 1 ∶ 8 000 by indirect ELISA.The half maximal inhibitory concentration (IC50) was 98.89-120.32 μg/L.There was less the cross reactivity with other tetracyclines.Conclusion:The conjugation method of diazo plus carbodiimide was the most efficient and the pAb has high titer,better sensitivity and specificity.