Development of a supersensitive detection method for pancreas cancer associated antigen and tumor suppressor gene by using immuno-PCR.

本文建立了一种检测血清中抗原的灵敏方法，即双决定簇免疫聚合酶链式反应(DDI-PCR)。第一个结合循环抗原的摩押被固定起来，而不是抗原本身。在生物素标记的第二单抗与抗原结合后，使用游离链霉亲和素将生物素化的DNA连接到生物素化的第二单抗上。用聚合酶链式反应扩增与抗原-抗体-链霉亲和素结合的生物素化DNA，扩增产物经琼脂糖凝胶电泳后进行Southern杂交分析。与传统的以PANC-1细胞培养上清液中的可溶性细胞间黏附分子-1(sICAM-1)为抗原的酶联免疫吸附试验相比，本方法的灵敏度提高了103倍。在高sICAM-1滴度的胃癌患者血清和培养上清液中，检测灵敏度均较ELISA提高约103倍，且DDI-PCR系统可在低于传统ELISA法检测下限的条件下检测血清中的抗原，且灵敏度高。因此，DDI-PCR具有一个显著的优点，即它可以很容易地应用于任何含有两个MoAbs的抗原-抗体系统，而不需要制造任何原始分子。

A sensitive method for the detection of antigens in sera, termed double determinant immuno-polymerase chain reaction (DDI-PCR), was developed using two monoclonal antibodies (MoAbs) in which the antigens are sandwiched and a specific DNA molecule as a marker. Instead of the antigen itself, the first MoAb to bind the circulating antigens was immobilized. After the biotinylated second MoAb was bound to the antigen, free streptavidin was used to attach a biotinylated DNA to the biotinylated second MoAb. The biotinylated DNA complexed with antigen-antibody-streptavidin was amplified by PCR.The PCR products were analyzed by Southern blot hybridization after agarose gel electrophoresis. Compared with the conventional ELISA using soluble intercellular adhesion molecule-1 (sICAM-1) in the supernatant of cultured Panc-1 cells as an antigen, our DDI-PCR was 10^3 times more sensitive in detection limit. In both the culture medium and sera from gastric cancer patients of high sICAM-1 titer, an approximately 10^3 -fold enhancement in detection sensitivity was obtained compared with ELISA.In addition, the DDI-PCR system can detect the antigen in sera at a level below the detection limit of traditional ELISA methods with high sensitivity. Thus, DDI-PCR has the significant advantage that it can be readily applied to any antigen-antibody system with two MoAbs without making any original molecules.