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Related Experiment Videos

Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution.

H T Zhang1, J E Kacharmina, K Miyashiro

  • 1Department of Pathology, Abramson Institute for Cancer Research, University of Pennsylvania, Philadelphia, PA 19104-6082, USA.

Proceedings of the National Academy of Sciences of the United States of America
|April 26, 2001
PubMed
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We developed immuno-detection amplified by T7 RNA polymerase (IDAT), a highly sensitive method for single-cell protein analysis. This technique significantly surpasses ELISA sensitivity, enabling precise detection of biomarkers like p185(her2/neu).

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Proteomics

Background:

  • Current methods for single-cell analysis of proteins, lipids, and metabolites have limitations in sensitivity and scope.
  • There is a need for highly sensitive techniques to detect and quantify biomolecules and their modifications at the single-cell level.

Purpose of the Study:

  • To develop an extremely sensitive technique for monitoring proteins, lipids, and metabolites at the single-cell level.
  • To improve upon existing immunoassay methods, such as ELISA and immuno-PCR, in terms of sensitivity and applicability.

Main Methods:

  • Developed immuno-detection amplified by T7 RNA polymerase (IDAT), conjugating a T7 promoter-containing oligonucleotide to an antibody (Ab).
  • Utilized T7 RNA polymerase to amplify RNA from the oligonucleotide-Ab complex within an Ab-antigen complex for detection.

Related Experiment Videos

  • Demonstrated detection of p185(her2/neu) receptor in cell lysates using IDAT, with potential use of single-chain Fv fragments or complementarity determining region peptides.

    • Main Results:

    • Achieved extremely high sensitivity, detecting p185(her2/neu) at a 10(-13) dilution from crude cell lysate.
    • IDAT demonstrated 10(9)-fold greater sensitivity compared to conventional ELISA methods.
    • Modified protocols allow for a universal detector species by coupling oligonucleotides to a common epitope antibody.

    Conclusions:

    • IDAT offers a significant advancement in sensitivity over immuno-PCR due to the linear amplification capability of T7 RNA polymerase.
    • The technique is versatile, capable of monitoring proteins, lipids, metabolites, and their modifications at the single-cell level.
    • IDAT holds potential for development into a robotic platform for high-throughput proteomics and biomarker discovery.