间接竞争ELISA法检测呋喃唑酮代谢物

Development of an Indirect Competitive ELISA for Rapid Analysis of Furazolidone Metabolite Residues in Animal Food

摘要:

以人工抗原和特异性抗体为基础，建立了以2-硝基苯甲醛为衍生试剂检测呋喃唑酮代谢物3-氨基-2-恶唑烷酮（AOZ）间接竞争ELISA方法。通过方阵滴定和间接竞争法确定ELISA方法的抗原抗体的最佳工作浓度；得到标准曲线的线性范围为0.25~10 ng/mL，线性关系良好；根据标准曲线计算IC50为0.5881~1.1333 ng/mL。此外，检测了与其他类似物的交叉反应率，显示了很好的特异性；并且通过对虾样品添加回收率的测定，证明了该方法的准确性。高温短时间衍生化处理可达到与37 ℃孵育过夜同样的衍生效果。

Abstract:

Based on artificial antigen and specific polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to detect 3-amino-2-oxazolidone (AOZ), followed by derivatization with 2-nitrobenzaldehyde. The selection of the optimum concentrations of the coating antigen, the polyclonal antibody and HRP-conjugated antibody was performed by ic-ELISA and square titration. Calibration curve was prepared for NPAOZ (3-(2-Nitrobenzylidenamino)-2-oxazolidinone), a 2-nitrobenzaldehyde derivative of AOZ, and good linearity was achieved over the concentration (0.25-10 ng/mL), the obtained IC50 was between 0.5881 ng/mL and 1.1333 ng/mL. In addition, the antibody was high specific for NPAOZ and no cross-reactivity with analog. The accuracy of the assay was validated by determining the recovery in shrimp tissue. High temperature and short time derivative process can be in line with incubating at 37 ℃ overnight.