Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution.
We have developed an extremely sensitive technique, termed immuno-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring proteins, lipids, and metabolites and their modifications at the single-cell level. A double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the Ab in the Ab-antigen complex. By using this technique, we are able to detect the p185(her2/neu) receptor from the crude lysate of T6-17 cells at 10(-13) dilution, which is 10(9)-fold more sensitive than the conventional ELISA method. Single-chain Fv fragments or complementarity determining region peptides of the Ab also can be substituted for the Ab in IDAT. In a modified protocol, the oligonucleotide has been coupled to an Ab against a common epitope to create a universal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in terms of sensitivity and has the potential to provide a robotic platform for proteomics.
Zhang
H T
HT
Department of Pathology, Abramson Institute for Cancer Research, University of Pennsylvania, Philadelphia, PA 19104-6082, USA.
Kacharmina
J E
JE
Miyashiro
K
K
Greene
M I
MI
Eberwine
J
J
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D051379:Mice; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D020543:Proteome; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011506:Proteins; D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases