Hapten labeling of nucleic acids for immuno-polymerase chain reaction applications.
A method for the ultrasensitive protein detection in the range of 0.01 to 10,000 amol of the model antibody anti-mouse-IgG from rabbit is described, using a combination of Immuno-polymerase chain reaction (PCR) and PCR-enzyme-linked immunosorbent assay (PCR-ELISA). The antibody was first immobilized on antigen-coated microplates; in a second step, a commercially available DNA-labeled species-specific antibody was added; and finally the deoxyribonucleic marker was amplified in a PCR step, including twofold labeling with biotinylated primer and a hapten-coupled nucleotide during PCR. Subsequently, the labeled PCR product was immobilized on streptavidin-coated microplates and detected with an antibody-enzyme conjugate. The protocol could easily be adapted to the detection of other antibodies or antigens by exchanging the antigen-specific antibody. Several modifications of the method as well as optimization steps, potential error sources, and countermeasures are discussed.
Adler
Michael
M
Chimera Biotech GmbH, Dortmund, Germany.
D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000941:Antigens / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006241:Haptens; D009696:Nucleic Acids; D016133:Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D000906:Antibodies; D000941:Antigens; D006241:Haptens; D009696:Nucleic Acids