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Hapten Labeling of Nucleic Acids for Immuno-Polymerase Chain Reaction Applications

  • Michael Adler
Part of the Methods in Molecular Biology™ book series (MIMB, volume 283)

Abstract

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.

Key Words

Immuno-PCR ELISA PCR-ELISA indirect assay secondary antibody 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Michael Adler
    • 1
  1. 1.Chimera Biotec GmbHDortmundGermany

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