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

利用双抗体夹心法和特异性DNA分子作为标记物，建立了一种灵敏的检测血清中抗原的方法，即双决定簇免疫聚合酶链反应（DDI-PCR）。固定化的不是抗原本身，而是结合循环抗原的第一个单克隆抗体。在生物素化的第二MoAb与抗原结合后，使用游离的链霉亲和素将生物素化的DNA连接到生物素化的第二MoAb。将生物素标记的DNA与抗原-抗体-链霉亲和素复合后进行PCR扩增，PCR产物经琼脂糖凝胶电泳后进行Southern印迹杂交分析。与以Panc-1细胞培养上清中可溶性细胞间粘附分子-1（sICAM-1）为抗原的常规ELISA法相比，该方法的灵敏度提高了103倍。对sICAM-1高滴度的胃癌患者血清和培养液的检测灵敏度均比ELISA法提高约103倍，且检测灵敏度高，可在低于传统ELISA法检测限的水平上检测血清中的抗原。因此，DDI-PCR具有显著的优点，即它可以容易地应用于具有两个MoAb的任何抗原-抗体系统，而无需制备任何原始分子。

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.