Chen Yiqiang, Li Xiangmei, He Lidong, Tang Shuheng, Xiao Xilong
China Agricultural University, College of Veterinary Medicine, Department of Pharmacology and Toxicology, Beijing 100193, People's Republic of China.
J AOAC Int. 2010 Jan-Feb;93(1):335-42.
A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2%) and slight or negligible crossreactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50% inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25-200 microg/kg ranged from 73 to 91%, with intraday CVs of 9-16% and interday CVs of 8-15%. For MIN, the average recoveries ranged from 108 to 131%, with intraday CVs of 10-16% and interday CVs of 8-15%. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 microg/kg in muscle (n=24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.
本研究报道了一种基于单克隆抗体（mAb）的庆大霉素（GEN）及其类似物小诺米星（MIN）的酶联免疫吸附测定（ELISA）和试纸条检测方法。以庆大霉素-戊二醛-牛血清白蛋白（GEN-GDA-BSA）偶联物作为免疫原。所制备的抗GEN单克隆抗体与小诺米星表现出高交叉反应性（131.2%），与其他氨基糖苷类药物的交叉反应性轻微或可忽略不计。基于该单克隆抗体，开发并评估了用于GEN和MIN检测的ELISA和试纸条检测方法。ELISA对GEN的50%抑制浓度（IC50）为0.75 ng/mL，对MIN为0.58 ng/mL。对于GEN，在25 - 200 μg/kg范围内的平均回收率为73%至91%，日内变异系数（CV）为9%至16%，日间CV为8%至15%。对于MIN，平均回收率为108%至131%，日内CV为10%至16%，日间CV为8%至15%。相比之下，GEN或MIN的试纸条检测在磷酸盐缓冲盐水中的检测限为5 ng/mL，在肌肉中的检测限为50 μg/kg（n = 24），结果可在10分钟内判断。通过试纸条检测、ELISA和高效液相色谱法（HPLC）对实际样品的检测结果表明，这两种免疫测定方法与HPLC方法相关性良好，可作为快速筛查猪肌肉中GEN和MIN残留的便捷工具。
