牛尿液中齐帕特罗残留快速检测免疫胶体金试纸的研制

doi:10.16431/j.cnki.1671-7236.2025.10.044

摘要/Abstract

摘要： 【目的】制备快速检测齐帕特罗(zilpaterol,ZIL)残留的免疫胶体金试纸。【方法】利用过硫酸铵纯化兔源抗ZIL多抗血清，IgG梯度稀释结合NaCl稳定性测试测定胶体金标记IgG最佳浓度、制备金标复合物，并以硝酸纤维素膜为固相载体包被ZIL-BSA偶联物为检测线(T线)、羊抗兔IgG抗体为质控线(C线)，制备检测ZIL残留的免疫胶体金试纸条，并检测其灵敏度、特异性以及在牛尿液中的应用效果。【结果】抗体纯化结果显示，高免血清纯化前后多抗血清的蛋白浓度分别为5.17和2.55 mg/mL；SDS-PAGE结果显示，血清纯化后未检测到非特异性条带，且出现了明显的IgG重链(56 ku)和轻链(23 ku)条带，与预期相符。经IgG梯度稀释结合NaCl稳定性测试测定后，胶体金标记IgG的最佳浓度为1.36 μg/mL(0.17 μg/孔)；灵敏度和特异性检测结果显示，试纸条的裸眼消线值(cut-off value,COV)为1 ng/mL，检测时间为8～10 min，且与克伦特罗(CLB)、沙丁胺醇(SAL)、西马特罗(CIM)、特布他林(TBL)等8种药物均无交叉反应。牛尿液加标试验结果显示，其COV为1 ng/mL，表明试纸条对基质干扰耐受性良好。【结论】本研究成功制备了一种基于免疫胶体金技术的ZIL快速检测试纸条，其具有高灵敏度、高特异性和良好的基质耐受性，可用于畜牧产品中ZIL残留的现场筛查，为食品安全监管提供了一种快速、便捷、低成本的检测工具。

关键词: 齐帕特罗; 残留; 免疫胶体金技术; 快速检测

Abstract: 【Objective】 The purpose of this test was to develop an immunocolloidal gold test strip for the rapid detection of zilpaterol (ZIL) residues.【Method】 A polyclonal antibody against ZIL was purified from rabbit serum using ammonium persulfate precipitation.The optimal concentration of IgG for colloidal gold labeling was determined through a combination of IgG gradient dilution and NaCl-induced aggregation stability testing.Gold-labeled antibody conjugates were then prepared accordingly.A nitrocellulose membrane was employed as the solid-phase carrier,with ZIL-BSA conjugates immobilized as the test line (T line) and goat anti-rabbit IgG antibodies as the control line (C line),to construct a immunocolloidal gold test strip for the detection of ZIL residues.The developed strip was evaluated for its sensitivity,specificity,and applicability in spiked bovine urine samples.【Result】 The results of antibody purification showed that the purified polyclonal antibody concentrations before and after purification were 5.17 and 2.55 mg/mL,respectively.SDS-PAGE results showed that no specific bands were produced after serum purification,and obvious IgG heavy chain (56 ku) and light chain (23 ku) bands appeared,which was consistent with expectations.The optimal IgG concentration for gold conjugation was determined to be 1.36 μg/mL (0.17 μg/well).Sensitivity and specificity assessments showed that the test strip had a visual cut-off value (COV) of 1 ng/mL,with a detection time of 8-10 min.There was no cross-reaction with 8 drugs such as clenbuterol (CLB),salbutamol (SAL),simaterol (CIM),and terbutaline (TBL).The results of the spiked test of bovine urine showed that its COV value was 1 ng/mL,demonstrating good tolerance to matrix interference.【Conclusion】 In this study,a ZIL rapid detection test strip based on immunocolloidal gold technique was successfully prepared,which had high sensitivity,high specificity and good matrix tolerance.It could be used for on-site screening of ZIL residues in livestock products,providing a rapid,convenient and cost-effective tool for food safety supervision.

Key words: zilpaterol; residual; immunecolloidal gold technique; rapid detection

中图分类号:

S859.84

郭东光, 齐越颖, 王兵, 蔡娟, 任雯静, 袁依佳, 陈明艳. 牛尿液中齐帕特罗残留快速检测免疫胶体金试纸的研制[J]. 中国畜牧兽医, 2025, 52(10): 5000-5008.

GUO Dongguang, QI Yueying, WANG Bing, CAI Juan, REN Wenjing, YUAN Yijia, CHEN Mingyan. Development of a Immunocolloidal Gold Test Strip for the Rapid Detection of Zilpaterol Residues in Bovine Urine[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 5000-5008.

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