Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA Conjugates

分子生物学 生物素化 溴化乙锭 抗原 化学 链霉亲和素 DNA 单克隆抗体 抗体 琼脂糖 聚合酶链反应 碱性磷酸酶 生物 生物化学 生物素 基因 遗传学
作者
Takeshi Sano,Cassandra L. Smith,Charles R. Cantor
出处
期刊:Science [American Association for the Advancement of Science (AAAS)]
卷期号:258 (5079): 120-122 被引量:810
标识
DOI:10.1126/science.1439758
摘要

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 × 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 × 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.
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