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 (K_D) of each recombinant antibody and the target antigen. To characterize the K_D 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 K_D 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.

Figure 1. Principle and optimization of K_D determination by EMSA.

(A) Schematics of EMSA separation of recombinant Fab fragment (Fab) and eGFP. By EMSA, the eGFP-Fab immunocomplex has a larger molecular mass and therefore lower electrophoretic mobility than unbound (free) eGFP. Fluorescence intensity profiles for EMSA-based analysis of eGFP and Fab reaction and a negative control (no Fab fragment) are shown (elapsed t = 30 s, E = 50 V/cm; [Fab] = 5 nM; [eGFP] = 10 nM). (B) Time evolution of inverted grayscale fluorescence micrographs show the eGFP-Fab immunocomplex peak resolved from free eGFP peak by EMSA. R.F.U. Relative fluorescence units.

Figure 2. Schematic design of sample dispensing schemes and EMSA card for high-throughput K_D determination.

(A) Side-view schematics of sample dispensing via manual pipetting or acoustic droplet ejection (ADE) to one of the 384 sample reservoirs on the EMSA card. (B) Top-view schematics of the 384-plex EMSA card design.

Figure 3. Technical variation in EMSA performance is reduced by ADE sample dispensing as compared to manual dispensing.

(A) Fluorescence micrograph of PAGE of well-characterized protein ladder establishes quantitative estimates of technical noise in AUC and target mobility. Inset micrograph reveals the separation of protein ladder, including a BSA dimer (BSA2). Elapsed separation time 75 s; E = 50 V/cm. (B) Unit-to-unit variation in the AUC for the model BSA protein peak from the fsPAG assays displayed in (A). Left: A plot of BSA AUC value of both manual and ADE dispensing for fsPAG. Right: Box plot of BSA AUC value for both dispensing methods and their variation. (C) CV of the model BSA protein peak as a function of sample dispensing conditions (source-to-card distance). PAGE conditions are the same as in (A).

Figure 4. Screening-mode EMSAs of a six-member Fab library.

(A) Fluorescence micrograph shows EMSA device with replicate units highlighted. Six Fabs (rAB 1003, rAB 1004, rAB 1005, rAB 1006, rAB 1007 and rAB 1008) were assayed simultaneously. Fabs were arranged column-wise with yellow boxed regions indicating EMSA card location of each of the six library members. (B) Fluorescence micrographs and intensity profiles across the six Fabs ([eGFP] = 0.3 nM, [Fab] = 0–500 nM, E = 50 V/cm, separation time = 28 s). R.F.U.: Relative fluorescence units.

Figure 5. EMSA card reports the K_D of a six-member recombinant Fab library in 1 hour.

(A) Dose-response curves acquired during EMSAs over a titration range of [Fab] with [eGFP] = 0.3 nM. Determination of the K_D values uses least squares fitting to 8-point concentration response with 8 replicates each. (B) Empirically determined K_D values for the six-member library (n = 3 for rAB 1003 for benchmark study), including comparison to the biolayer interferometry gold-standard (Octet) for rAB 1003. Sample and EMSAs conditions as in Fig. 4.