氟苯尼考胺人工抗原的合成与鉴定

2014-04-29 02:27常超张怡伍金娥
安徽农业科学 2014年13期

常超 张怡 伍金娥

摘要 [目的] 合成并鉴定氟苯尼考胺人工抗原。[方法] 采用水解的方法制备了氟苯尼考胺,并通过紫外、红外、质谱鉴定氟苯尼考胺水解成功。采用戊二醛法合成了氟苯尼考人工抗原,并通过紫外光谱进行鉴定。免疫大白兔制备了氟苯尼考胺高特异性抗体。[结果] 通过紫外、红外、质谱鉴定氟苯尼考胺水解成功,抗体效价达6.4×104。[结论] 氟苯尼考胺人工抗原合成成功,为建立FFA免疫化学方法奠定了技术基础。

关键词 氟苯尼考;氟苯尼考胺;人工抗原

中图分类号 S609.9 文献标识码 A 文章编号 0517-6611(2014)13-04029-03

Abstract [Objective] The research aimed to prepare and identify the artificial antigen of florfenicol amine. [Method] The artificial antigen of florfenicol amine was synthesized by using hydrolysis method. The product was examined by ultraviolet spectrum, infrared spectrum and mass. FFA was covalently attached to carrier protein as immunogen by glutaraldehyde method. The conjugates was identified with ultraviolet spectrum. FFABSA was used to immune New Zealand white rabbits and prepare polyclonal antibody of FFA with high affinity and high specificity.[Result] The results showed these products were synthesized successfully. The antibodies produced with FFABSA conjugate showed titer of 6.4×104. [Conclusion] The synthesis route of FFA artificial antigen was feasible. This research could lay the foundation for further establishment of the immune detection method for FFA.

Key words Florfenicol; Florfenicol amine; Artifical antigen

氟苯尼考(Florfenicol,结构式见图1)是替代禁用药物氯霉素的一种理想的药物,现已广泛应用于水产养殖的防病抗病上[1-3]。氟苯尼考在动物组织中的主要代谢产物为氟苯尼考胺(florfenicol amine,FFA,结构式见图1),许多国家以氟苯尼考胺作为使用氟苯尼考的残留标示物[4]。尽管氟苯尼考毒性低于氯霉素,但由于不正确或超量的使用,势必造成氟苯尼考在畜禽水产品中的残留,特别是现已出现了对氟苯尼考耐药的菌株,而且氟苯尼考的用量呈上升的趋势,这对人类的身体健康造成了潜在的威胁。因此,研究其残留监控技术对保障我国人民身体健康和水产品食用安全具有重要意义。目前国内外对氟苯尼考的检测主要有HPLC、LCMS

等方法[5-10],仪器方法虽然灵敏度高、准确性好,但需要昂

贵的仪器,检测成本较高,不适合大量样品的筛选。酶联免疫吸附测定法(ELISA)因具有快速、灵敏、高效等优点,用于大批量药物残留筛选越来越广泛。国内外生物公司已经开始研发氟苯尼考检测试剂盒,但这些试剂盒检测的靶物质为氟苯尼考原型药,不能完成其代谢产物氟苯尼考胺的检测[11]。到目前为止我国没有自行合成的氟苯尼考胺标准品,这制约了我国研究氟苯尼考胺,也未见有关氟苯尼考胺ELISA试剂盒面市。笔者拟采用化学方法水解氟苯尼考制备氟苯尼考胺标准品,合成氟苯尼考胺人工抗原,以期为制备氟苯尼考胺的特异性抗体和建立氟苯尼考胺ELISA检测方法提供技术支撑。

3 结论

该研究采用水解的方法,制备了氟苯尼考胺,合成了氟苯尼考胺人工抗原,为制备针对FFA特异性抗体和建立氟苯尼考残留标示物FFA检测方法提供了技术技术支撑。武汉轻工大学食品科学与工程学院实验室目前采用最灵敏的特异性抗体(R5)已建立了检测FFA间接竞争ELISA 方法(成果另文发表),为我国开发FFA免疫检测试剂盒打下坚实的基础,具有一定的创新性和经济价值。

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