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Development and validation of a cell-based fluorescent method for measuring antibody affinity.

Xin Yu1, Charles N Pegram1, Darell D Bigner1

  • 1Department of Pathology, Duke University Medical Center, Durham, NC, USA.

Journal of Immunological Methods
|December 28, 2016
PubMed
Summary

A new fluorescent cell-based assay accurately measures antibody affinity (KD) for tumor-targeting therapies. This method provides a convenient way to identify promising therapeutic antibody leads.

Keywords:
Antibody affinityFlow cytometryFluorescent cell-based assayScatchard

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Area of Science:

  • Immunology
  • Biotechnology
  • Biochemistry

Background:

  • Monoclonal antibodies are vital for diagnostics and therapeutics.
  • Antibody affinity is critical for the efficacy of tumor-targeting antibodies.
  • Accurate antibody affinity measurement is essential for therapeutic development.

Purpose of the Study:

  • To develop and validate a fluorescent cell-based immunosorbent assay for measuring antibody affinity (KD).
  • To assess the assay's reliability and convenience for identifying therapeutically relevant antibody leads.

Main Methods:

  • A fluorescent cell-based immunosorbent assay was developed using antigen-positive and antigen-negative cell lines.
  • Fluorescently labeled antibodies were added to cells in 96-well plates.
  • Specific binding signals were determined by subtracting nonspecific binding to negative control cells.

Main Results:

  • The assay accurately measured antibody affinity (KD) in the nanomolar range.
  • Results were comparable to established methods like flow cytometry and radioactive Scatchard assay.
  • The method demonstrated convenience and efficiency in identifying therapeutic leads.

Conclusions:

  • The modified cell-based fluorescent assay is a reliable and efficient tool for determining antibody affinity.
  • This assay facilitates the identification of promising antibody candidates for cancer therapy.
  • The method offers a practical approach for antibody affinity characterization in drug discovery.