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Quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence

Dafeng Jiang1, Lei Wang, Wei Jiang

  • 1School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.

Analytica Chimica Acta
|January 22, 2009
PubMed

Insights

This study introduces a sensitive single-molecule counting method using total internal reflection fluorescence microscopy and quantum dots for antibody quantification. The technique enables precise measurement of antibody concentrations with a wide linear range.

Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Immunology

Background:

  • Accurate antibody quantification is crucial for various biological and medical applications.
  • Traditional immunoassay methods can suffer from limited sensitivity and dynamic range.

Purpose of the Study:

  • To develop a highly sensitive method for quantifying antibodies.
  • To utilize single-molecule counting principles for precise antibody measurement.

Main Methods:

  • Immobilization of biotinylated monoclonal anti-human IgG on a silanized glass substrate.
  • Labeling of target antibodies with streptavidin-coated quantum dots as fluorescent probes.
  • Detection and counting of single fluorescent spots using total internal reflection fluorescence microscopy and an electron multiplying charge-coupled device.

Main Results:

  • Successful implementation of single-molecule counting for antibody quantification.
  • Achieved a linear range from 8.0 x 10(-14) to 5.0 x 10(-12) mol/L for antibody concentration.
  • Demonstrated high sensitivity in detecting and quantifying target antibody molecules.

Conclusions:

  • The developed method offers a sensitive and precise approach for antibody quantification.
  • Single-molecule counting via TIRF microscopy provides a robust platform for biomarker detection.
  • This technique has potential applications in diagnostics and fundamental biological research.

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