Suzuki A, Itoh F, Hinoda Y, Imai K
First Department of Internal Medicine, Sapporo Medical University School of Medicine.
Jpn J Cancer Res. 1995 Sep;86(9):885-9. doi: 10.1111/j.1349-7006.1995.tb03101.x.
A sensitive method for the detection of antigens in sera, termed double determinant immunopolymerase chain reaction (double determinant immuno-PCR) was developed, using two monoclonal antibodies (MoAbs), in which the antigens are sandwiched, and a specific DNA molecule is used 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, and the PCR products were analyzed by Southern blot hybridization after agarose gel electrophoresis. Compared with the conventional enzyme linked immunosorbent assay (ELISA) using soluble intercellular adhesion molecule-1 (sICAM-1) in the supernatant of cultured Panc-1 cells as an antigen, our double determinant immuno-PCR was 10(3) times more sensitive in terms of the detection limit. Not only in culture medium, but also in 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, this system can detect the antigen in sera at a level below the detection limit of traditional ELISA methods with high sensitivity. Thus, double determinant immuno-PCR has the significant advantage that it can be readily applied to any antigen-antibody system for which two MoAbs are available.
开发了一种用于检测血清中抗原的灵敏方法，称为双决定簇免疫聚合酶链反应（双决定簇免疫PCR），该方法使用两种单克隆抗体（MoAb），其中抗原被夹心，并且使用特定的DNA分子作为标记。固定的是与循环抗原结合的第一种MoAb，而不是抗原本身。生物素化的第二种MoAb与抗原结合后，使用游离抗生物素蛋白链菌素将生物素化的DNA连接到生物素化的第二种MoAb上。与抗原-抗体-抗生物素蛋白链菌素复合的生物素化DNA通过PCR进行扩增，PCR产物在琼脂糖凝胶电泳后通过Southern印迹杂交进行分析。与使用培养的Panc-1细胞上清液中的可溶性细胞间粘附分子-1（sICAM-1）作为抗原的传统酶联免疫吸附测定（ELISA）相比，我们的双决定簇免疫PCR在检测限方面灵敏度高10³倍。不仅在培养基中，而且在sICAM-1滴度高的胃癌患者血清中，与ELISA相比，检测灵敏度提高了约10³倍。此外，该系统能够以高灵敏度检测血清中低于传统ELISA方法检测限水平的抗原。因此，双决定簇免疫PCR具有显著优势，即它可以很容易地应用于任何可获得两种MoAb的抗原-抗体系统。
