Beloglazova Natalia V, Eremin Sergei A
Faculty of Pharmaceutical Sciences, Laboratory of Food Analysis, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium.
Faculty of Chemistry, Department of Chemical Enzymology, M.V. Lomonosov Moscow State University, Leninsky Gory 1, 119991, Moscow, Russia.
Anal Bioanal Chem. 2015 Nov;407(28):8525-32. doi: 10.1007/s00216-015-9006-6. Epub 2015 Sep 28.
In this paper we describe the development of a sensitive, fast, and easily performed fluorescence polarization immunoassay for determination of cephalexin in milk. The experimental work was performed to increase sensitivity and specificity. Therefore, the structures of the tracers were varied by synthesis of both cephalexin (CEX) and cephalotin (CET) conjugates with a variety of fluorescent labels. Two rabbit antisera containing antibodies against cephalexin and cephalotin were tested in homologous and heterologous combinations with the tracers. For every working antibody-tracer combination, the analytical conditions and cross-reactivity for structural analogues-cephalosporins and other antibiotics that could also be present in milk-were determined. It was found that the highest sensitivity was achieved by use of the homologous pair CET-EDF-anti-CET antibody (limit of detection (LOD) 0.4 μg kg(-1) for standard solutions prepared in buffer), but this combination was not appropriate because of high cross-reactivity with CET. For subsequent experiments, therefore, CEX- EDF-anti-CEX antibody were chosen (LOD 0.8 μg kg(-1) for standard solutions prepared in buffer). Part of this manuscript is devoted to the variation of precipitation agents for pretreatment of milk before analysis; milk is an extremely complicated matrix. The optimum protein precipitation agent was methanol. This technique for cephalexin determination was characterized by a limit of detection of 1 μg kg(-1). The method was validated by using naturally contaminated and spiked milk samples. The results obtained corresponded very well with those obtained by HPLC, which was used as confirmation method.
在本文中，我们描述了一种用于测定牛奶中头孢氨苄的灵敏、快速且易于操作的荧光偏振免疫分析法的开发。开展实验工作以提高灵敏度和特异性。因此，通过合成带有多种荧光标记的头孢氨苄（CEX）和头孢噻啶（CET）缀合物来改变示踪剂的结构。测试了两种分别含有抗头孢氨苄和抗头孢噻啶抗体的兔抗血清，使其与示踪剂进行同源和异源组合。对于每一种工作抗体 - 示踪剂组合，测定了牛奶中可能存在的结构类似物（头孢菌素和其他抗生素）的分析条件和交叉反应性。结果发现，使用同源组合CET - EDF - 抗CET抗体可实现最高灵敏度（缓冲液中制备的标准溶液的检测限（LOD）为0.4 μg kg⁻¹），但由于与CET的交叉反应性高，该组合不合适。因此，在后续实验中选择了CEX - EDF - 抗CEX抗体（缓冲液中制备的标准溶液的LOD为0.8 μg kg⁻¹）。本手稿的一部分致力于分析前牛奶预处理沉淀剂的变化；牛奶是一种极其复杂的基质。最佳蛋白质沉淀剂是甲醇。这种测定头孢氨苄的技术检测限为1 μg kg⁻¹。通过使用天然污染和加标的牛奶样品对该方法进行了验证。获得的结果与用作确认方法的高效液相色谱法获得的结果非常吻合。
