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 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.

High glucose removes natural anti‑α‑galactoside and anti‑β‑glucoside antibody immune complexes adhering to surface O‑glycoproteins of normal platelets and enhances platelet aggregation

“…6 (lane 1). The marked jacalin reactivity of the strongly jacalin-reactive band, despite lectin-carbohydrate interactions being much weaker [association constant (K a ) ~1.67x10 5 M -1 for jacalin binding to fetuin] ( 19 ) than antigen-antibody interactions (Ka in the range 1x10 6 -10 8 M -1 ) ( 20 ), suggesting its high O-glycan content. The size of this dominant O-glycoprotein on the platelet surface was determined to be 116 kDa ( Fig.…”

A Novel Assay Format as an Alternative to ELISA: MINA Test for Biotin

“…By comparison, antigen binding fragments (Fab) typically possess K d values in the pM–nM range ,. Monoclonal anti‐biotin antibodies have a K d value of 900 nM .…”

In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage

“…The sensitivity and reliability of PLA are linked to the nature of the probes: The applications in situ are generally based on the use of two primary antibodies, characterized by binding to the target molecules with high specificity, and two species-specific, oligonucleotide-conjugated secondary antibodies (as probes). This two-step procedure guarantees the reduction in potential PLA binders cross-reactivity, which might be a serious concern especially when the target molecule is not abundant or when the antibody dissociation constant (Kd) for the intended molecule is slightly different from the one for the cross-reactive molecule at similar concentrations [46].…”