Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection

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 Abs, 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.

本研究对滚环扩增（rolling circle amplification, RCA）报告系统进行适配改造，以实现蛋白抗原（antigens, Ags）的检测，该改造方案被命名为"免疫滚环扩增（immunoRCA）"。在免疫滚环扩增体系中，寡核苷酸引物（oligonucleotide primer）与抗体（antibody, Ab）以共价键结合；因此，当体系中存在环状DNA、DNA聚合酶与核苷酸时，扩增反应会生成一条长链DNA分子，该分子包含数百份环状DNA序列的拷贝，且始终与抗体结合，可通过多种方式实现检测。借助免疫滚环扩增技术，检测分析物的灵敏度优于酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）与微粒型传统酶联免疫分析方法。免疫滚环扩增所提供的信号放大能力，还可使免疫分析以微斑点（microspot）与微阵列（microarray）形式开展，并具备极高的检测灵敏度。当抗原浓度低至飞摩尔（femtomolar, fM）级别时，可通过计数单个抗原-抗体复合物产生的离散荧光信号，对特异性结合的抗体进行定量。研究还通过玻片上的双色单分子计数实验，对不同比例混合的抗原进行准确定量，从而验证了多重免疫滚环扩增技术的可行性。因此，免疫滚环扩增技术兼具高灵敏度、极宽的动态范围，以及前所未有的单分子检测能力。该抗原检测方法具备普适性，还可拓展至多重免疫分析场景：即使用一组不同的抗体，每种抗体均标记有独特的寡核苷酸引物，通过颜色编码的可视化系统即可区分不同的检测通道。基于多种抗原同时定量的免疫滚环扩增谱分析，可通过利用基于比例的表达分析所提升的信息含量，进一步增强免疫分析的效能。