Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen
Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen
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DOI：
10.1021/pr101070j
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发表时间：
2011-03-01
影响因子：
4.4
通讯作者：
Knemeyert, Jens-Peter
中科院分区：
文献类型：
--
作者：
Schmidt, Ronny;Jacak, Jaroslaw;Knemeyert, Jens-Peter
基于单分子敏感的荧光连接免疫吸附测定，建立了用于检测脂阿拉伯甘露聚糖（LAM）（结核病的脂多糖标志物）的分析平台，其比可比的当前ELISA测定灵敏约3个数量级。不需要扩增步骤。此外，无需进行特定的样品制备。由于检测到单个结合事件，因此仅通过计数单个信号就可以进行真正的定量。利用全内反射配置，可以分析添加LAM的未处理生物样本（人尿液和血浆），而无需在分析期间进行样本纯化或洗涤步骤。每微升含有约600个抗原分子的样品产生明显的信号。所开发的方法可用于存在适当抗体的任何一组靶分子。
Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.