Double determinant immuno-polymerase chain reaction for detecting soluble intercellular adhesion molecule-1.
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.
Itoh
F
F
First Department of Internal Medicine, Sapporo Medical University, Japan.
Suzuki
A
A
Hinoda
Y
Y
Imai
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000936:Antigen-Antibody Complex / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D019809:Streptavidin; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; D018799:Intercellular Adhesion Molecule-1; D001710:Biotin; D004247:DNA; D019809:Streptavidin