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 Immunopolymerase 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.