悬液芯片与常规酶联免疫吸附法检测庆大霉素残留的比较

摘要

Objective: A suspension array assay was established for the detection of gentamicin residues and compared with regular enzyme-linked immunosorbent assay (ELISA). Methods: Monoclonal antibody against gentamicin was covalently coupled with polystyrene microspheres with carboxyl groups on their surface by amido bonds to form detection probes. The phycoerythrin (PE)-labeled gentamicin obtained by carbodi mide (EDC) method was al owed to couple with anti-gentamicin monoclonal antibody in an aqueous phase by direct competition method. Then based on the fluorescence signal of the resulting products, a competitive curve for gentamicin was obtained. Meanwhile, a direct ELISA curve for gentamicin was established. Both assays were compared with each other to find variations in detection limit, linear range and spike recovery rate. Results: The IC10 and IC50 of suspension array assay were 0.1 ng/m and 2.8 ng/mL, respectively, and its linear range was 0.1–100 ng/mL; in contrast, the IC10 and IC50 of ELISA assay were 0.27 ng/mL and 1.95 ng/mL, respectively, and its linear range. Each of the assays showed a spike recovery rate varying from 80% to 120% for gentamicin in milk, chicken meat, chicken liver, pork and porcine liver. In addition, suspension array assay had no cross-reactivity with other aminoglycosides, showing a good specificity. Conclusions: Suspension array assay is simple, sensitive and specific and superior to the conventional ELISA assay. As a result, suspension array assay can provide a new technique for the detection of pesticides and veterinary residues.%目的：建立庆大霉素兽药残留的悬液芯片直接竞争检测法，并同时与常规酶联免疫法进行比较。方法：将庆大霉素单克隆抗体与表面带有羧基的聚苯乙烯微球通过酰胺键偶联形成检测探针，利用碳二亚胺法将庆大霉素与藻红蛋白偶联后形成酶标物，采用直接竞争法，在液相体系中庆大霉素与藻红蛋白酶标物竞争性与单克隆抗体相结合，反应后通过荧光信号获得庆大霉素竞争曲线。同时进行庆大霉素直接竞争酶联免疫法曲线的测定。对两种方法在检出限、检测区间以及加样回收率方面进行比较。结果表明：悬液芯片IC10、IC50分别为0.1、2.8ng/mL，检测区间为0.1～100ng/mL，酶联免疫竞争法IC10、IC50为0.27、1.95ng/mL，检测区间为0.1～10ng/mL。两种方法在牛奶、鸡肉、鸡肝、猪肉和猪肝中的回收率均在80%～120%之间。另外，悬液芯片法与其他氨基糖苷类抗生素基本无交叉反应，特异性良好。结论：悬液芯片系统具有操作简单，灵敏度高以及特异性良好等特点，与传统经典的农兽药残留酶联免疫检测法相比，具有一定的优势，从而为农兽药残留检测提供了新的检测技术。