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

利用免疫PCR开发胰腺癌相关抗原和抑癌基因的超灵敏检测方法。

基本信息

批准号：
07557046
负责人：
IMAI Kohzoh
金额：
$ 3.9万
依托单位：
Sapporo Medical University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557046/
关键词：
Immuno-PCR Pancreas cancer Antigen MUSE 11 ICAM-1

项目摘要

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.

使用两种单克隆抗体 (MoAb) 开发了一种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 (DDI-PCR)，其中抗原夹在中间，并以特定 DNA 分子作为标记。第一个结合循环抗原的 MoAb 被固定，而不是抗原本身。在生物素化的第二MoAb与抗原结合后，使用游离链霉亲和素将生物素化的DNA附着至生物素化的第二MoAb。 PCR扩增抗原-抗体-链霉亲和素复合物的生物素化DNA，琼脂糖凝胶电泳后进行Southern印迹杂交分析PCR产物。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ELISA相比，我们的DDI-PCR的检测限灵敏度提高了10^3倍。在高sICAM-1滴度的胃癌患者的培养基和血清中，与ELISA相比，检测灵敏度提高了约10^3倍。此外，DDI-PCR系统可以检测血清中的抗原低于传统 ELISA 方法的检测限，灵敏度高。因此，DDI-PCR具有显着的优点，即它可以很容易地应用于任何具有两种MoAb的抗原-抗体系统，而不需要制造任何原始分子。