title: Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.
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 KDof 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 KDfor 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.
authors.affiliation: University of California, Berkeley - UCSF Graduate Program in Bioengineering, Berkeley, CA, 94720, USA.
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Labcyte Inc., 1190 Borregas Ave, Sunnyvale, CA, 94089, USA.
authors.forename: Eric K
authors.initials: EK
authors.lastname: Sackmann
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158 USA.
authors.forename: Karolina
authors.initials: K
authors.lastname: Wypisniak
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158 USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Hornsby
authors.affiliation: Labcyte Inc., 1190 Borregas Ave, Sunnyvale, CA, 94089, USA.
authors.forename: Sammy S
authors.initials: SS
authors.lastname: Datwani
authors.affiliation: University of California, Berkeley - UCSF Graduate Program in Bioengineering, Berkeley, CA, 94720, USA.|Department of Bioengineering, University of California, Berkeley, CA, 94720, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins