bioj294 at emoryu1.cc.emory.edu
Thu Jan 6 17:00:15 EST 1994
I have gotten several replies from my immuno-PCR post...mostly
asking what it is....i shall now post the abstract (some
paraphrasing) and a new reference as supplied to me recently.
The objective of this technique is to have a greater sensitivity in
the detection of a particular antigen, using the amplification of
Science 2 october 1992 158:120-122.
"Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific
An antigen detection system, termed immuno-PCR was developed in
which a specific DNA molecule is used as the marker. A
stepavidin-protein A chimera that pocesses tight and specific
binding affinity both for biotin and IgG was used to attach a
biotinylated DNA specifically to antigen-MAb complexes that have
been immobilized on microtiter plate wells. Then, a segment of the
attached DNA was amplified by PCR. Analysis of the PCR products by
gel after staining with EtBr allowed as few as 580 antigen
molecules (9.6 x 10 to the -22 moles) to be readily and
reproducibly detected. Direct comparison with enzyme linked
immunosorbant assay with the use of a chimera-alkaline phosphatase
conjugate demonstrates that enhancement (approx. x 10 to the fifth)
in detection sensitivity was obtained with immuno-PCR.
also look at Orlando and Paro Cell 75: 1187-1198
Here they immunoprecipitate putative Ab-protein-DNA complexes
then they make sticky ends on the unknown DNA and ligate to
sticky primers (this is a quick synopsis)...then they amp.
DNA...they then use DNA to screen southerns etc etc..
More information about the Methods