间接竞争酶联免疫吸附测定法快速筛查药酒中双氯芬酸

Indirect competitive enzyme-linked immunosorbent assay for rapid screening of diclofenac in medicinal liquor

目的建立间接竞争酶联免疫吸附测定法快速筛查药酒中双氯芬酸(diclofenac,DCF)。方法采用活性酯法和混合酸酐法分别将DCF与载体蛋白耦合,得到DCF的免疫原和检测抗原。采用DCF-牛血清白蛋白免疫BALB/c小鼠,随后用杂交瘤等技术制备抗DCF单克隆抗体(monoclon alantibody,mAb),基于DCFmAb建立了间接竞争酶联免疫吸附测定法,对该检测方法的性能(准确度、精密度和特异性)进行鉴定。结果紫外扫描结果表明DCF已成功与载体蛋白偶联;获得最优株抗DCF的杂交瘤细胞株(4B9),其IC50值为0.61ng/mL;该检测方法在药酒中的DCF平均添加回收率为85.9%,其批间变异系数(coefficient of variation,CV)(5.3%~9.7%)均大于批内CV(4.9%~9.1%),与类似物(酮洛芬、阿司匹林、吲哚美辛、布洛芬、舒林酸、萘普生、罗非昔布)没有交叉反应。结论本研究建立的间接竞争ELISA法为DCF在药酒中的残留提供了一种新的筛查手段。

Objective To establish an indirect competitive enzyme-linked immunosorbent assay for rapid screening diclofenac(DCF) in medicinal liquor.Methods DCF was coupled to carrier protein using the active ester method and the mixed anhydride method to obtain DCF immunogen and detection antigen.BALB/c mice were immunized with DCF-bovine serum albumin,and then anti-DCF monoclonal antibody(mAb) was prepared by hybridoma technology.An indirect competitive ELISA method was established based on DCF mAb,and the performance(accuracy,precision and specificity) of the detection method was evaluated.Results Ultraviolet scanning results showed that DCF was successfully coupled to the carrier protein;the optimal hybridoma cell line(4B9) against DCF was obtained,with an IC50 value of 0.61 ng/mL;the average recovery rate of DCF added in medicinal liquor by this detection method was 85.9%,and the coefficient of variation(CV) for intra-assay(5.3%-9.7%) was greater than the CV for inter-assay(4.9%-9.1%),and there was no cross-reaction with analogues(ketoprofen,aspirin,indomethacin,ibuprofen,sulindac,naproxen,and rofecoxib).Conclusion The indirect competitive ELISA method established in this study provides a new screening method for DCF residues in medicinal liquor.