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人肿瘤坏死因子相关凋亡诱导配体3(TRAIL-R3)检测试剂盒实验操作常见问题分析
异常 | 结果描述 | 原因分析 | 对策 |
白板 | 显色步骤结束后,酶标板所有孔均无颜色。阳性对照不显色。 | 试剂已过效期,或不同试剂盒组分混用 | 检查试剂的组分及批号,确认未过期以及所有组分均属对应的试剂盒。不同试剂盒或不人肿瘤坏死因子相关凋亡诱导配体3(TRAIL-R3)检测试剂盒同批号的试剂不能混用。 |
错加、漏加试剂底物、显色剂 A或B | 严格按说明书的步骤操作,加完试剂后可观察一下液面高度是否一致,以减少漏加现象。 | ||
洗板及加样过程中,酶标受污染失活失去催化显色剂显色的能力 | 确认盛酶标的容器不含酶抑制剂如叠氮钠等,确认配制洗液的容器已洗干净。 | ||
终止液误作洗涤液稀释或当底物缓冲液配制 | 每次配制时都应看清标签标明物质 | ||
蒸馏水有问题 | 确认配制洗液的纯化水达到要求且未污染,与好蒸馏水比较。 |
标本及采集、贮运因素:严重溶血 ,以 HRP 为标记的ELISA 测定中,残留在孔内的血红蛋白具有过氧化物酶样活性,催化底物显色造成假阳性; 混有红细胞的血清 易沉淀或附着在聚乙烯孔内不易洗净;如有 细菌污染 ,菌体中可能含有内源性HRP,也会产生假阳性反应; 标本凝固不全 ,有时为了争取时间快速检测,常在血液还未开始凝固时即强行离心分离血清,使血清中仍残留部分纤维蛋白原,在ELISA测定过程中可以形成肉眼可见的纤维蛋白块,易造成假阳性结果; 采血试管洗涤不彻底 、反复使用易交叉污染; 塑料试管能吸附抗原物质 ,样本久置在塑料管内会使样本内抗原含量下降造成假阴性。
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