二氯喹啉酸人工抗原的合成与鉴定

 以除草剂二氯喹啉酸（quinclorac，Q）、氯化亚砜、氨基己酸和氨基丁酸等为起始原料，经2步化学反应分别合成了2种二氯喹啉酸半抗原：6-(3,7-二氯-8-喹啉甲酰基)氨基己酸（QC）和4-（3,7-二氯-8-喹啉甲酰基）氨基丁酸（QB）。并通过碳化二亚胺法和混合酸酐法将Q、QC和QB分别与载体蛋白（BSA、OVA）偶联制备了二氯喹啉酸的免疫抗原和包被抗原。再将Q-BSA、QC-BSA和QB-BSA分别免疫新西兰大白兔获得了3种抗二氯喹啉酸的多克隆抗体：抗Q-BSA抗体(Q-Ab)、抗QC-BSA抗体(QC-Ab)和抗QB-BSA抗体(QB-Ab)，当相应的包被抗原浓度均为4 ?g·ml-1时，相应的冻干粉效价分别为2×103，1.2×106和1.6×106。由于Q-Ab效价偏低，不适合用于检测。采用同源间接ELISA竞争法测定时，其他2种抗体（QC-Ab、QB-Ab）的抑制中浓度(IC50)均大于50 mg·L-1，检测灵敏度也较低。因此本文对异源间接竞争ELISA法进行了研究，结果表明，异源反应的检测灵敏度高于同源反应，其中QB-Ab的IC50为0.4 mg·L-1；最低检测限为0.006 mg·L-1。

Synthesis and Identification of Artificial Antigen Against Quinclorac

In this paper, two haptens of quinclorac(Q), 6-[(3,7-dichloro-8-quinolino-formacyl) amino-hexanoic acid (QC) and 4-[(3,7-dichloro-8-quinolino-formamino)] butanoic acid (QB), were synthesized by using quinclorac and amino-hexanoic acid or amino-butanoic acid as original materials. These two haptens and quinclorac were separately conjugated to bovine serum albumin (BSA) with the modified active ester method. Three coating antigens, Q-OVA, QC-OVA and QB-OVA were also synthesized with the mixed-anhydride method. As a result of the study, three types of antibody (Q-Ab, QC-Ab and QB-Ab) were produced successfully. Their titers were 2×103，1.2×106 and 1.6×106 respectively, while the concentration of coating antigens were 4 ?g·ml-1. Among these antibodies, the QB-Ab was utilized satisfactorily with coating antigen Q-OVA to detect the herbicide with heterogenous assays; and its IC50 was 0.4 mg·L-1, the limit of detection(LOD) was 0.006 mg·L-1. The difference of the titer between the homologous and heterologous combinations was significant in this study. It was indicated that the spacer length of the coating antigen is important for the recognition by antibodies, and the sensitivity of ELISA can be improved remarkably by heterologous reaction.