Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

TitleDetermination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.
Publication TypeJournal Article
Year of Publication2016
AuthorsPan, Yuchen, Sackmann Eric K., Wypisniak Karolina, Hornsby Michael, Datwani Sammy S., and Herr Amy E.
JournalSci Rep
Volume6
Pagination39774
Date Published2016 Dec 23
ISSN2045-2322
Abstract

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

DOI10.1038/srep39774
Alternate JournalSci Rep
PubMed ID28008969
PubMed Central IDPMC5180089
Grant ListP41 CA196276 / CA / NCI NIH HHS / United States
R01 CA203018 / CA / NCI NIH HHS / United States
U54 HG006436 / HG / NHGRI NIH HHS / United States

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