Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

PloS One
Christine BeeArvind 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 (K(D) 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 K(D) 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 recombina...Continue Reading

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Citations

Apr 11, 2013·International Journal of Endocrinology·Min WangHou-De Zhou
Feb 26, 2013·Immunobiology·Sean Carroll, George Georgiou
Oct 30, 2012·Biochemical and Biophysical Research Communications·Yasuhiro FujinoTakuya Ueda
Oct 21, 2015·Analytical Chemistry·Farzad Jalali-YazdiRichard W Roberts
Nov 20, 2016·Nature Communications·Yik Andy YeungJavier Chaparro-Riggers
Jan 15, 2020·The American Journal of Tropical Medicine and Hygiene·Anizah RahumatullahRahmah Noordin
Jul 17, 2015·Journal of Biomolecular Screening·Daniela Della DucataBodo Brocks
Aug 28, 2020·Molecular Cancer Therapeutics·Amir A Al-KhamiShahram Salek-Ardakani
Jul 15, 2021·Computational and Structural Biotechnology Journal·Sonia BudroniOretta Finco

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Methods Mentioned

BETA
biosensor
antigen assay
antibody assay
transfection
ELISA
chip
biosensors
antigen
chips
PCR

Software Mentioned

Biacore T200
Biacore
Scrubber
KinExA Pro
KinExA

Related Concepts

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