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PCR检测方法.检测体系采用聚合酶链反应（PCR）。利用C-反应蛋白与磷酸胆碱的Ca^2+依赖性结合特性，将C-反应蛋白固定在微孔板上，并将固定化的C-反应蛋白用APPC（p-aminophenylphosphorylcholine）- BSA固定。封闭后，依次加入第一抗体、生物素化的第二抗体、亲和素和生物素化的模板DNA。最后，用SP 6启动子引物和T7启动子引物进行PCR。如在电泳后接着EtBr染色后通过图像分析仪评估的，待扩增的PCR产物（497 bp）的量显示依赖于最初加入的C-反应蛋白的量。此外，它还受一些参数的影响，包括PCR循环次数、温度和退火时间。his-immono-PCR可检测到低至2.5ng/ml的C-反应蛋白，而夹心式常规酶免疫法则不能检测到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.