IMMUNO-PCR - VERY SENSITIVE ANTIGEN-DETECTION BY MEANS OF SPECIFIC ANTIBODY-DNA CONJUGATES
IMMUNO-PCR - VERY SENSITIVE ANTIGEN-DETECTION BY MEANS OF SPECIFIC ANTIBODY-DNA CONJUGATES
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DOI：
10.1126/science.1439758
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发表时间：
1992-10-02
期刊：
影响因子：
56.9
通讯作者：
CANTOR, CR
中科院分区：
文献类型：
--
作者：
SANO, T;SMITH, CL;CANTOR, CR
一个抗原检测系统，称为免疫聚合酶链反应（免疫PCR），开发了一个特定的DNA分子被用作标记。使用对生物素和免疫球蛋白G均具有紧密和特异性结合亲和力的链霉亲和素-蛋白A嵌合体将生物素化DNA特异性地附着到已固定在微量滴定板威尔斯孔上的抗原-单克隆抗体复合物上。然后，通过PCR扩增连接的DNA的片段。用溴化乙锭染色后，通过琼脂糖凝胶电泳对PCR产物进行分析，可以容易且可重复地检测到少至580个抗原分子（9.6 × 10（-22）摩尔）。与使用嵌合体-碱性磷酸酶偶联物的酶联免疫吸附试验的直接比较表明，使用免疫PCR获得了检测灵敏度的增强（约x 10（5））。鉴于PCR的巨大扩增能力和特异性，这种免疫PCR技术具有比任何现有抗原检测系统更高的灵敏度，并且原则上可以应用于检测单个抗原分子。
An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules (9.6 x 10(-22) moles) to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement (approximately x 10(5)) in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.