Design and Synthesis of Immunoconjugates and Development of an Indirect ELISA for Rapid Detection of 3, 5-Dinitrosalicyclic Acid Hydrazide
作者:Yu-Dong Shen、Shi-Wei Zhang、Hong-Tao Lei、Hong Wang、Zhi-Li Xiao、Yue-Ming Jiang、Yuan-Ming Sun
DOI:10.3390/molecules13092238
日期:——
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), IC50 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.
在这项研究中,我们设计、合成了新型免疫结合剂,然后利用这种结合剂开发了一种快速、特异和灵敏的间接 ELISA 方法,用于直接检测鸡组织中存在的 3,5-二硝基水杨酸酰肼(DNSH)残留物,DNSH 是硝化纤维素的一种有毒代谢物。首先设计了单体 DNSHA,并将其与 BSA 共价结合形成免疫原,免疫家兔后可产生针对 DNSH 的多克隆抗体。此外,还设计了一种结构上不同于 DNSHA-OVA 的新型 3,5- 二硝基水杨酸戊白蛋白(DNSA-OVA)免疫共轭物,并将其用作 "亚结构包被抗原",以提高间接 ELISA 分析法直接检测 DNSH 的灵敏度。基于 "亚结构包被抗原 "概念,建立了一种优化的间接 ELISA 方法,该方法检测 DNSH 具有良好的特异性和高灵敏度,交叉反应小于 0.1%(不包括母体化合物 nifursol),IC50 为 0.217 nmol/mL,检测限为 0.018 nmol/mL。最后,对鸡组织中的 DNSH 样品进行简单有效的分析表明,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 残留的潜力。