两种检测百草枯中毒的免疫层析试纸条研制

Development of two immunochromatographic strips for clinical paraquat poisoning

目的 研制可直接对微量生物样本(血液、尿液等)中痕量百草枯进行快速定量检测的免疫试纸条。方法以4,4-联吡啶为原料,通过半抗原制备、动物免疫、抗体筛选和样品垫材料优化,研制两种可对血液、尿液样本中痕量百草枯进行直接检测的胶体金免疫层析试纸条(GICA)和时间分辨荧光免疫层析试纸条(TRFICA),并与高效液相色谱串联质谱和表面增强拉曼光谱技术在样品前处理要求、灵敏度、检测时间和样本适用性等多方面进行比较。结果 筛选的抗体特异性较好,对百草枯的结构类似物1,1’-亚乙基-2,2’-联吡啶二溴盐和4,4-联吡啶均无明显交叉干扰。匹配手持式荧光试纸条读卡仪,TRFICA在一定浓度范围内具有较好的定量能力,对全血样本中的检测限可达0.05 ng/ml,检测时间<10 min。相比液质联用和表面增强拉曼光谱的检测技术,具备造价低廉、操作简便、检测速度快、结果易判别且灵敏度高等优势。结论 研制的GICA和TRFICA为百草枯临床中毒患者的即时检测和预后评估提供了新的筛查确证方法。

Objective To prepare two lateral flow immunochromatography assays(LFIAs) for rapid quantitative detection of paraquat poisoning in trace biological samples( blood or urine). Methods LFIAs were developed through hapten preparation,animal immunity,antibody screening and sample pad material optimization. The performance was compared with high performance liquid chromatography-mass spectrometry(HPLC-MS)and surface enhanced Raman spectroscopy(SERS)in terms of requirements of sample pretreatment,sensitivity,detection time and sample applicability.Results The results indicated that the obtained antibody had a good specificity and no significant cross-interference with the structural analogues of paraquat,such as diquat and,4-bipyridine. In addition,the time-resolved fluorescence immunochromatographic assay(TRFICA)had good quantitative ability in some concentration range when coupled with the handheld TRFICA analyzer. The low detection limit in whole blood samples could reach 0.05 ng/ml,and the detection time was less than 10 min. Compared with HPLC-MS and SERS,the developed colloidal gold immunochromatography assay(GICA)and TRFICA had the advantages of low cost,simple operation,fast detection,easy discrimination and high sensitivity. Conclusion The GICA and TRFICA provide a new screening and confirmation method for point-ofcare testing and prognosis evaluation of paraquat-poisoned patients.