Zhang Hongyan, Duan Zhenjuan, Wang Lei, Zhang Yan, Wang Shuo
Tianjin Key Laboratory of Food Nutrition and Safety, Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300222, People's Republic of China.
J Agric Food Chem. 2006 Jun 28;54(13):4499-505. doi: 10.1021/jf060868u.
This paper reports the synthesis of five sulfonamide derivatives, the production of broad-specificity polyclonal antibodies for immunoassay of sulfonamides, and the analysis of milk samples by developed assay. The three-step synthesis procedure reported in most of the literature was adopted and modified in this study. In the procedure, the purification of the intermediate was avoided and the time of synthesis was shortened from >20 to 6-9 h with improved yields. This method is generally applicable to the synthesis of haptens containing the common structure of sulfonamides. Three haptens were coupled to keyhole limpet hemocyanin, and polyclonal antibodies were obtained from rabbits immunized with these conjugates. Using the antibodies obtained, from one of these was developed an enzyme-linked immunosorbent assay (ELISA) based on the competition between free sulfonamides and the hapten-horseradish peroxidase (HRP) conjugates. The hapten-HRP conjugate giving the best competitive results and 11 structurally different sulfonamides showed 50% inhibition at concentrations of <100 ng mL(-1). After removal of the protein with acetone, milk samples were analyzed by ELISA directly; a matrix effect could be avoided when a 1:20 dilution with phosphate-buffered saline was used, and 104-131% recoveries of spiked samples were obtained. The developed immunoassay is suitable to determine sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine, sulfapyridine, and sulfamethizole below the maximum residue limit in milk (100 ng mL(-1) of total sulfonamides) rapidly and reliably.
本文报道了5种磺胺类衍生物的合成、用于磺胺类免疫分析的广谱多克隆抗体的制备以及用所建立的分析方法对牛奶样品进行分析。本研究采用并改进了大多数文献报道的三步合成方法。在该方法中，避免了中间体的纯化，合成时间从>20小时缩短至6 - 9小时，产率提高。该方法一般适用于含有磺胺类共同结构的半抗原的合成。将3种半抗原与钥孔戚血蓝蛋白偶联，并从用这些偶联物免疫的兔中获得多克隆抗体。利用所获得的抗体，基于游离磺胺类与半抗原 - 辣根过氧化物酶（HRP）偶联物之间的竞争，建立了一种酶联免疫吸附测定（ELISA）方法。给出最佳竞争结果的半抗原 - HRP偶联物与11种结构不同的磺胺类在浓度<100 ng mL⁻¹时显示出50%的抑制率。用丙酮去除蛋白质后，直接通过ELISA分析牛奶样品；当用磷酸盐缓冲盐水按1:20稀释时可避免基质效应，加标样品的回收率为104 - 131%。所建立的免疫分析方法适用于快速、可靠地测定牛奶中低于最大残留限量（总磺胺类为100 ng mL⁻¹）的磺胺异恶唑、磺胺噻唑、磺胺米隆、磺胺甲氧嗪、磺胺吡啶和磺胺甲噻二唑。
