Shen Yu-Dong, Zhang Shi-Wei, Lei Hong-Tao, Wang Hong, Xiao Zhi-Li, Jiang Yue-Ming, Sun Yuan-Ming
College of Food Science, South China Agricultural University, Guangzhou 510642, P. R. China.
Molecules. 2008 Sep 23;13(9):2238-48. doi: 10.3390/molecules13092238.
In this study novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to directly detect residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol present in chicken tissues. The hapten DNSHA was first designed and used to covalently couple to BSA to form an immunogen which was immunized to rabbits to produce a polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acidovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was designed and used as a "substructural coating antigen" to improve the sensitivity of an indirect ELISA analysis for a direct DNSH detection. Based on the "substructural coating antigen" concept, an optimized indirect ELISA method was established that exhibited good specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than 0.1% (excluding the parent compound nifursol), IC(50) of 0.217 nmol/mL and detection limit of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%, with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of DNSH residues in tissue.
在本研究中，设计并合成了新型免疫缀合物，然后用于开发一种快速、特异且灵敏的间接ELISA方法，以直接检测鸡组织中存在的硝呋索尔有毒代谢物3,5 - 二硝基水杨酸酰肼（DNSH）的残留量。首先设计半抗原DNSHA，并将其与牛血清白蛋白（BSA）共价偶联形成免疫原，将该免疫原免疫兔子以产生抗DNSH的多克隆抗体。此外，设计了一种结构不同于DNSHA - OVA的新型3,5 - 二硝基水杨酸卵清蛋白（DNSA - OVA）免疫缀合物，并将其用作“亚结构包被抗原”，以提高间接ELISA分析直接检测DNSH的灵敏度。基于“亚结构包被抗原”的概念，建立了一种优化的间接ELISA方法，该方法在检测DNSH时表现出良好的特异性和高灵敏度，交叉反应率小于0.1%（不包括母体化合物硝呋索尔），半数抑制浓度（IC50）为0.217 nmol/mL，检测限为0.018 nmol/mL。最后，对鸡组织中DNSH样品进行的简单高效分析表明，间接ELISA分析的平均回收率为82.3%，平均变异系数为15.9%。因此，所开发的间接ELISA方法具有快速检测组织中DNSH残留量的潜力。
