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

Publication ,  Journal Article
Yu, X; Pegram, CN; Bigner, DD; Chandramohan, V
Published in: J Immunol Methods
March 2017

Monoclonal antibodies have become essential tools for diagnostic and therapeutic purposes. Antibody affinity is one of the critical factors influencing the therapeutic success of tumor-targeting antibodies. Therefore, developing an accurate and reliable method for determining antibody affinity is crucial. In this study, we describe a fluorescent cell-based immunosorbent assay that can accurately measure antibody affinity (KD) in the nanomolar range. This method involves the addition of fluorescently labeled antibodies to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The KD values obtained by this method were comparable with values obtained by the flow cytometry and radioactive (I125) scatchard assay. Our results demonstrate that this modified cell-based fluorescent method allows for a convenient and efficient identification of therapeutically relevant leads.

Duke Scholars

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Published In

J Immunol Methods

DOI

EISSN

1872-7905

Publication Date

March 2017

Volume

442

Start / End Page

49 / 53

Location

Netherlands

Related Subject Headings

  • Tumor Cells, Cultured
  • Transfection
  • Swiss 3T3 Cells
  • Spectrometry, Fluorescence
  • Reproducibility of Results
  • Protein Binding
  • Mutation
  • Mice
  • Membrane Proteins
  • Membrane Glycoproteins
 

Citation

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Yu, X., Pegram, C. N., Bigner, D. D., & Chandramohan, V. (2017). Development and validation of a cell-based fluorescent method for measuring antibody affinity. J Immunol Methods, 442, 49–53. https://doi.org/10.1016/j.jim.2016.12.004
Yu, Xin, Charles N. Pegram, Darell D. Bigner, and Vidyalakshmi Chandramohan. “Development and validation of a cell-based fluorescent method for measuring antibody affinity.J Immunol Methods 442 (March 2017): 49–53. https://doi.org/10.1016/j.jim.2016.12.004.
Yu X, Pegram CN, Bigner DD, Chandramohan V. Development and validation of a cell-based fluorescent method for measuring antibody affinity. J Immunol Methods. 2017 Mar;442:49–53.
Yu, Xin, et al. “Development and validation of a cell-based fluorescent method for measuring antibody affinity.J Immunol Methods, vol. 442, Mar. 2017, pp. 49–53. Pubmed, doi:10.1016/j.jim.2016.12.004.
Yu X, Pegram CN, Bigner DD, Chandramohan V. Development and validation of a cell-based fluorescent method for measuring antibody affinity. J Immunol Methods. 2017 Mar;442:49–53.
Journal cover image

Published In

J Immunol Methods

DOI

EISSN

1872-7905

Publication Date

March 2017

Volume

442

Start / End Page

49 / 53

Location

Netherlands

Related Subject Headings

  • Tumor Cells, Cultured
  • Transfection
  • Swiss 3T3 Cells
  • Spectrometry, Fluorescence
  • Reproducibility of Results
  • Protein Binding
  • Mutation
  • Mice
  • Membrane Proteins
  • Membrane Glycoproteins