Double determinant immuno-polymerase chain reaction: a sensitive method for detecting circulating antigens in human sera.

Abstract

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.