呋喃它酮代谢物直接竞争化学发光检测方法的建立

Development of a direct competitive chemiluminescence immunoassay for determining of Furaltadone metabolite(AMOZ)

将呋喃它酮代谢物(AMOZ)与对醛基苯甲酸反应生成CPAMOZ,采用碳二亚胺法将CPAMOZ和HRP偶联得到酶标物CPAMOZ-HRP,在该酶标物和CPAMOZ单克隆抗体的基础上,建立AMOZ的直接竞争化学发光(CLIA)检测方法。结果显示,CPAMOZ-HRP经紫外扫描鉴定偶联成功;以单克隆抗体稀释度1∶8 000为最佳包被浓度,CPAMOZ-HRP稀释浓度1∶64 000为最佳工作浓度建立CLIA方法,其标准曲线呈线性,线性范围0.062 5~8 ng/m L,回归方程为y=-0.397x+0.286(R^2=0.992),IC_(50)为0.289 ng/m L;AMOZ单克隆抗体能特异性识别NPAMOZ(AMOZ的对硝基苯甲醛的衍生物),除AMOZ外与其他参试的化学品或药物无交叉反应;样品最低检出限为0.059 6 g/kg;样品添加回收率在85%~91.93%之间,检测值的变异系数≤10%;实际样品检测结果与进口试剂盒检测结果呈线性相关(y=0.935x+0.556,R^2=0.997),表明本试验建立的方法可靠性等同于进口试剂盒,可满足AMOZ实际检测的需要。

The objective of the study was to develop a direct competitive chemiluminescence im- munoassay for the determination of AMOZ. CPAMOZ was synthesized from AMOZ and 4-Formylbenzoic acid. Then CPAMOZ was coupled to HRP by carbodimiide method in order to compose HRP enzyme-marked conjugates （CPAMOZ-HRP）,which could be successfully identified by UV scanning. The optimal concentration of the coating antibody and CPAMOZ-HRP was 1：8 000 and 1：64 000,respectively. With a linear working range between 0.062 5 and 8 ng/mL, the 50% inhibitory concentration（IC50） was 0.289 ng/mL,and the regression equation was y=-0. 397x＋ 0.286（R2=0. 992）. The specificity of the monoclonal antibody against AMOZ, estimated as the cross-reactivity values with 4-nitrobenzaldehyde derivatized AMOZ（NPAMOZ）andAMOZ, was 100%and 0.16%,respectively. The minimum detection limit of NPAMOZ in the samples was 0.059 6 g/kg. The rates of recovery of sample addition were 85%-91.93%,while the variation coefficient of test values were equal or less than 10%. The CLIA results of the actual sample testing were linearlycorrelated with the results of the imported kit（y=0. 935x@0. 556,R2=0. 997）,which indicated that the reliability of the method was equal to the imported kit and the method could be used for detecting AMOZ.