四环素类药物多残留酶联免疫检测方法

 【目的】构建一种四环素类药物多残留酶联免疫检测试剂盒。【方法】将四环素类药物与载体蛋白相连作为抗原免疫动物，获得了抗四环素类药物的抗体，建立了动物性食品中四环素类药物残留的ELISA检测方法，并将该试剂盒的检测性能与进口试剂盒比较，对实际样品的测定结果与高效液相-串联质谱法比较。【结果】人工抗原中四环素与蛋白质分子的结合比约为15﹕1，血清效价达到1﹕2 000倍以上，50%抑制浓度(IC50)为3 μg?L-1左右，7种四环素类药物交叉反应率在58%～100%之间，牛奶和鸡肉中四环素类药物的检测限分别为6.6 μg?L-1和6.5 μg?kg-1, 在鸡肉中四环素、土霉素和金霉素的回收率在40%～120%之间。【结论】试剂盒的最低检测限为0.3 μg?L-1，检测范围为0.3～30 μg?L-1，可同时测定兽医常用的四环素、金霉素、土霉素和多西环素4种药物残留。灵敏度、精密度和准确度均能满足兽药残留检测要求。

Development of an ELISA Method for Multi-Residue Detecting of Tetracyclines

【Objective】 Immunoassay kit for detecting of tetracyclines multi-residues was developed. 【Method】 Anti-tetracyclines antibody was gotten by immunizing animals with tetracycline-protein combination. An ELISA method for detecting tetracyclines residues in animal tissues was developed. This method was compared with R-biopharm Company Immunoassay Kit and LC-MS/MS method. 【Result】 The conjugate rate was 15 tetracycline mole to one protein mole in artificial antigen. Antiserum valence was more than 1﹕2 000 and the IC50 was about 3 μg?L-1. The cross-reactive rate range for 7 kinds of tetracyclines was from 58% to 100%. The limit of detection were 6.6 μg?L-1and 6.5 μg?kg-1 in milk and chicken muscle, respectively. The recovery rate range was from 40% to 120% in chicken muscle. 【Conclusion】 The lowest detection limit of this test kit was 0.3 μg?L-1 and the measuring range was from 0.3 to 30 μg?L-1. It can determine tetracycline, chlortetracycline, oxytetracycline and deoxycycline usually used in veterinary clinic at the same time. The sensibility, precision and accuracy of the method can satisfy the need of detecting veterinary residue.