Author:
Zhang Hong-Tao,Kacharmina Janet E.,Miyashiro Kevin,Greene Mark I.,Eberwine James
Abstract
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 p185her2/neu
receptor from the crude lysate of T6–17 cells at 10−13
dilution, which is 109-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.
Publisher
Proceedings of the National Academy of Sciences
Cited by
67 articles.
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