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Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening

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Abstract

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen–antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17–470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

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Acknowledgments

TR thanks the BBSRC for an Industrial Studentship.

Conflict of interest

Prof. Shaw is a Director of Attomarker Ltd. that holds the intellectual property for the array reader technology.

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Authors and Affiliations

  1. Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK

    Thomas Read, Rouslan V. Olkhov & Andrew M. Shaw

  2. Biomedical Sciences Department, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK

    E. Diane Williamson

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  1. Thomas Read
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  2. Rouslan V. Olkhov
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  4. Andrew M. Shaw
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Correspondence to Andrew M. Shaw.

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Read, T., Olkhov, R.V., Williamson, E.D. et al. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening. Anal Bioanal Chem 407, 7349–7357 (2015). https://doi.org/10.1007/s00216-015-8897-6

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  • DOI: https://doi.org/10.1007/s00216-015-8897-6

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