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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution.

Sean A Gray1, Kris M Weigel, Keith D Miller

  • 1Seattle Biomedical Research Institute, 307 Westlake Ave. N., Suite 500, Seattle, Washington 98109, USA. sean.gray@sbri.org

Biotechnology and Bioengineering
|December 3, 2009
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New methods enable the use of yeast-displayed single chain fragment variable (scFv) antibodies for diagnostics. These assays overcome challenges in converting yeast-displayed scFv into soluble forms, facilitating their application in immunoassays and reagent development.

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Area of Science:

  • Biotechnology
  • Immunology
  • Molecular Biology

Background:

  • Yeast-display libraries are a source of single chain fragment variable (scFv) antibodies.
  • Converting yeast-displayed scFv into soluble, active forms for immunoassays is challenging.
  • Limited scFv solubility and activity hinder their use as diagnostic reagents.

Purpose of the Study:

  • To develop novel methods for evaluating and utilizing scFv antibodies from yeast-display libraries.
  • To overcome limitations in soluble scFv conversion and application.
  • To facilitate the development of scFv-based diagnostic reagents.

Main Methods:

  • Developed competitive inhibition flow cytometry (CIFC) assays for detecting secreted scFv.
  • Utilized an epitope binning assay to identify scFv binding to non-overlapping epitopes.
  • Created a CIFC assay that uses yeast-displayed scFv to detect unlabeled antigen, bypassing soluble scFv expression.

Main Results:

  • Established assays employing both yeast-displayed and secreted scFv.
  • Identified scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei.
  • Demonstrated the ability to identify sandwich assay pairs and active soluble scFv in crude extracts.

Conclusions:

  • The developed methods facilitate the practical implementation of scFv antibodies from yeast-display platforms.
  • These assays address key challenges in scFv solubility and activity.
  • The findings will advance the use of scFv in diagnostic and research applications.