新型喹乙醇残留标志物人工半抗原的设计及其在动物组织残留检测中的应用

A Novel Hapten Design for Marker Residue of Olaquindox and Its Application on the Veterinary Residue Detection in Animal Tissues

关键词:  喹乙醇残留标志物  半抗原设计  酶联免疫分析  残留检测

Key words:  maker residue of Olaquindox  Hapten Design  ELISA  residue detection

摘要:

为提高酶联免疫方法测定喹乙醇残留标志物（MQCA）的检测性能，设计了一种新型MQCA人工半抗原，该半抗原既完整保留MQCA分子特征性基团和结构，又具有便于和蛋白质载体偶联的基团-NH2。通过三步化学反应合成该半抗原，将其与蛋白质载体偶联成为免疫抗原后，进一步制备特异性抗体,研制了MQCA酶联免疫试剂盒，灵敏度高，特异性好，IC50为1.94 μg/L。建立了测定动物性产品中MQCA残留量的直接竞争酶联免疫法，测定猪肝、猪肉、鸡肉、鸡蛋中MQCA的检测限分别为0.42、0.23、0.28、0.28 μg/kg。猪肉中不同添加浓度MQCA（1, 2, 4 μg/kg）的回收率为70%~97%；批内、批间变异系数均低于12%。本试剂盒可同时快速检测大批样品，有望在MQCA残留检测中发挥重要作用。研究有助于指导小分子药物半抗原的合理设计，为现有的半抗原设计方法提供新的理念和思路。

Abstract:

In order to improve the detection performance of enzyme-linked immunosorbent assay (ELISA) for maker residue of Olaquindox (MQCA), a novel hapten of MQCA was designed. This hapten keeps the characteristic structure and goups of MQCA, additionally, it can be easily coupled with protein carrier according to its –NH2. The hapten was synthesized involving in three steps. Based on its coupling with protein to become the immunogen, polyclonal antibodies were prepared. A rapid direct competitive ELISA was established to detect MQCA in animal tissues. The antibody showed high sensitivity and high specificity, with the IC50 being 1.94 μg/L. The limits of detection of MQCA in liver, pork, chicken, and egg were 0.42, 0.23, 0.28 and 0.28 μg/kg, respectively. The average recoveries ranged from 70% to 97% at different spiked levels (1, 2 and 4 μg/kg) in pork for MQCA, and the intra/inter-day coefficients of variation were below 12%. The prototype kit could be advantageously used for the screening of large groups of samples, and has potential to play important role in the detection of MQCA. In addition, this study may help to guide the antigen design of small molecule medicine more reasonably, and supply new concept for the hapten design.