吗啡免疫荧光层析快速定量方法的建立

Establishment of Fluorescence Immunochromatographic Assay for Quantitative Detection of Morphine

目的建立一种高灵敏度的检测方法对生物样本(血液、唾液和尿液)中吗啡进行快速定量。方法采用免疫层析试纸条的反应模式,以稀土元素铕荧光纳米颗粒为标记物,与吗啡单克隆抗体共价偶联,将吗啡抗原和羊抗鼠抗体包被于硝酸纤维素膜上分别作为检测线和质控线,通过竞争性免疫抑制反应原理,结合干式免疫荧光检测仪用于生物样本中吗啡的快速定量。结果通过优化试验条件后制备得到免疫层析试纸条检测,线性范围在3~3 000 ng·m L^(-1);精密度和准确度吗啡质控品试验的RSD<10%;与苯丙胺、甲基苯丙胺和氯胺酮等常见毒品和药品均无交叉反应;试纸条在37℃,7 d的加速试验中,性能没有明显降低。结论本实验初步建立的吗啡免疫荧光层析试纸条定量检测方法具有良好的线性范围和精密度,具有良好的特异性和稳定性。本方法可以快速便捷的对生物样本中吗啡进行定量检测,便于在基层单位推广使用。

OBJECTIVE To establish a high sensitivity method for rapid quantitative detection of morphine in the biological samples including serum,saliva and urine. METHODS With the immunochromatographic lateral flow strip as reaction method,the luminescent lanthanide europium nanoparticles covalently conjugated with morphine monoclonal antibody were adopted as reporters. The morphine antigen and goat anti-mouse antibody were coated at the nitrocellulose membrane separately as the test line and control line.The strip based on competitive inhibition immunoassay principle was detected by the fluorescent reader for rapid quantitative detection of morphine in the biological samples. RESULTS After experiment optimization,the improved strip could provide the line range 3-3 000 ng·m L-1; precise quality morphine control materials verified CV 10%. There were no cross reactions with amphetamine,methylamphetamine,ketamine and so on; keep the strips at 37 ℃ for 7 d,the performance of the strips did not decline markedly.CONCLUSION The preliminary established method shows high linearity,precision,specificity and stability. The method could quantitatively detect the morphine in the biological samples in short time. It is supposed to be applied into the grassroots unit.