期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 97, 期 18, 页码 10113-10119出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.170237197
关键词
prostate-specific antigen; human IgE; IgG; ELISA; immuno-microarrays
We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed immunoRCA. In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide, ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Ags, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据