Development of immuno PCR method and its biomedical application

以C反应蛋白为模型分子，建立了一种高灵敏度的免疫聚合酶链式反应分析方法。采用聚合酶链式反应(PCR)技术建立检测体系。基于APPC(对氨基苯基磷胆碱)-BSA对磷胆碱的钙依赖性结合特性，将C反应蛋白固定在微板上。封闭后依次加入第一抗体、生物素标记的第二抗体、亲和素和生物素标记的模板DNA。最后用SP6启动子和T7启动子进行聚合酶链式反应。经凝胶电泳法和EtBR染色后的图像分析仪分析表明，扩增产物的量(497bp)依赖于最初添加的C反应蛋白的量。此外，它还会随着一些参数的变化而变化，包括PCR循环次数、温度和退火期。His免疫聚合酶链式反应可检测到低至2.5 ng/ml的C反应蛋白，而夹心式常规酶免疫分析无法检测到C反应蛋白。

A high sensitive assay, which was termed immuno PCR,for C- reactive protein as model molecule was developed. PCR (polymerase chain reaction) was employed in detection system of this assay. C- reactive protein was immobilized on microplate through APPC (p-aminophenylphosphorylcholine) - BSA,based upon its Ca^<2+>-dependent binding property to phosphorylcholine. After blocking, 1st Ab, biotinylated 2nd Ab, avidin and biotinylated template DNA were added in turn. In final, PCR was performed with SP6 promoter primer and T7 promoter primer. As assessed by image analyzer after electrophoresis followed by EtBr staining, the amounts of PCR product to be amplified (497 bp) was shown to be dependent on the amounts of initially added C- reactive protein. In addition, it varied with some parameters, including PCR cycles, temperature and period for annealing. C- reactive protein as little as 2.5 ng/ml could be detected by his immono PCR,while not by sandwich- type conventional enzyme immunoassay.