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

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 x 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 x 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.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…Immuno-PCR is an ELISA-type immunoassay that uses PCR for exponential amplification of ELISA signal [84]. As in the classical sandwich ELISA described above, analyte (BoNT) is captured by the adsorbed antibody and detected by reporter antibody binding to preformed capture antibody/analyte complex.…”

Protein detection via direct enzymatic amplification of short DNA aptamers

“…(26)(27)(28)(29)(30) Using antibody-DNA hybrid constructs, the antibody's binding affinity was complemented by the sensitive detection achievable with PCR. In addition, immuno-DNA detection strategies have been extended to use rolling circle amplification (RCA), an isothermal technique that generates a long ssDNA oligomer tethered to the immuno-DNA conjugate.…”

Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood

“…Immuno-PCR is a method in which PCR techniques are teamed up with immunodetection methods [6]. The method uses a linker molecule, with affinity for both DNA and antibody, which specifically binds a DNA template to an antibody-antigen complex.…”

“…The template is then amplified by PCR using the proper primers and a set number of cycles. The presence of the PCR product, through gel electrophoresis or threshold cycle analysis by real-time PCR, reveals the existence of the complexes and thus is used to calculate the presence of the antigen [6]. One advantage of the immuno-PCR method is that it is approximately 10 5 -fold more sensitive than alkaline phosphatase-conjugated ELISA antigen detection [6,7].…”

“…The presence of the PCR product, through gel electrophoresis or threshold cycle analysis by real-time PCR, reveals the existence of the complexes and thus is used to calculate the presence of the antigen [6]. One advantage of the immuno-PCR method is that it is approximately 10 5 -fold more sensitive than alkaline phosphatase-conjugated ELISA antigen detection [6,7]. Unfortunately, temperature changes during the PCR cycles are detrimental to the stability of proteins in the system, and denatured proteins can greatly interrupt the PCR reaction thus reducing the reliability of this approach.…”