AlphaLISA法同时测定动物组织中克伦特罗和莱克多巴胺

Simultaneous determination of clenbuterol and ractopamine in animal tissues by AlphaLISA

摘要:

目的：建立同时测定动物组织中克伦特罗（clenbuterol，CLB）和莱克多巴胺（ractopamine，RAC）的纳米均相时间分辨荧光免疫（amplified luminescent proximity homogeneous assay linked immunosorbent assay，AlphaLISA）分析方法。方法：样品经β-葡萄糖醛酸苷酶/芳基硫酯酶和乙酸铵酶解提取，正己烷脱脂，滤膜过滤，滤液与生物素化抗原、抗体、供体微珠和受体微珠进行酶联免疫，AlphaLISA进行检测。结果：CLB和RAC在0.1～50 ng/mL范围内呈良好的线性关系（R2＞0.98），检测限为0.02 ng/mL；在5、10、20 μg/kg ３个添加水平下CLB + RAC（1∶1）、CLB和RAC的回收率分别为88.8%～102.8%、86.6%～98.3%和84.2%～103.2%；相对标准偏差（relative standard deviation，RSD）均小于12%。结论：该方法快速简单、结果准确可靠，适用于检测和筛选动物组织中的RAC和CLB。

Abstract:

Objective：To develop an amplified luminescent proximity homogeneous assay linked immunosorbent assay（AlphaLISA） method for simultaneous determination of clenbuterol（CLB） and ractopamine（RAC） in animal tissues. Methods：The samples were subjected to enzymatic extraction with β-glucuronidase/sulfatase and ammonium acetate，degreased with n-hexane，and filtrated with a filter membrane. Then the filtrate underwent reactions with biotinylated antigen，antibody，donor beads，and acceptor beads；subsequently AlphaLISA was used for determination. Results：CLB and RAC showed a good linear relationship in the range of 0.1 to 50 ng/mL（R2>0.98），with a limit of detection of 0.02 ng/mL. The recovery rates of CLB + RAC（1∶1），CLB，and RAC at spiking levels of 5，10，and 20 μg/kg were 88.8%-102.8%，86.6%-98.3%，and 84.2%-103.2%，respectively；the relative standard deviations were all less than 12%. Conclusion：The method developed is simple，rapid，accurate，and reliable，thus making it applicable for the determination and screening of CLB and RAC in animal tissues.