Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA Conjugates

TLDR

Immuno‑PCR links a biotinylated DNA marker to antigen–antibody complexes on microtiter plates using a streptavidin‑protein A chimera, then amplifies the DNA by PCR. The assay detected as few as 580 antigen molecules, achieving roughly 10^5‑fold greater sensitivity than ELISA and potentially enabling single‑molecule detection.

Abstract

An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules (9.6 × 10 -22 moles) to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement (approximately × 10 5 ) in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.