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Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

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  • Christine Bee
  • Yasmina N Abdiche
  • Donna M Stone
  • Sierra Collier
  • Kevin C Lindquist
  • Alanna C Pinkerton
  • Jaume Pons
  • Arvind Rajpal

Abstract

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

Suggested Citation

  • Christine Bee & Yasmina N Abdiche & Donna M Stone & Sierra Collier & Kevin C Lindquist & Alanna C Pinkerton & Jaume Pons & Arvind Rajpal, 2012. "Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions," PLOS ONE, Public Library of Science, vol. 7(4), pages 1-13, April.
    • Handle: RePEc:plo:pone00:0036261
    DOI: 10.1371/journal.pone.0036261
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    1. Anders Nykjaer & Ramee Lee & Kenneth K. Teng & Pernille Jansen & Peder Madsen & Morten S. Nielsen & Christian Jacobsen & Marco Kliemannel & Elisabeth Schwarz & Thomas E. Willnow & Barbara L. Hempstead, 2004. "Sortilin is essential for proNGF-induced neuronal cell death," Nature, Nature, vol. 427(6977), pages 843-848, February.
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    1. Christine Bee & Yasmina N Abdiche & Jaume Pons & Arvind Rajpal, 2013. "Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum," PLOS ONE, Public Library of Science, vol. 8(11), pages 1-1, November.

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