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

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.


K D measurements of Alexa Fluor ® 647 labeled eGFP -rAB 1003 binding on Octet Red384
To benchmark the K D measurements on fsPAGE, we performed binding analysis of Alexa Fluor ® 647 (AF647) labeled eGFP and rAB1003 in HEPES. The concentration of the reagents and experimental conditions used in measurements were detailed in the Methods section.
The results were shown in Figure S1A. The reaction, composed of both association and dissociation processes, was monitored in real-time through the thickness of bio-layer, representing the formation/decomposition of binding complex. k on and k off were extracted by fitting the 1:1 kinetic equation to the binding curves. K D values, then, were calculated by the ratio of k on and k off. The resulting K D is 3.53 ± 0.03 nM for the AF647 labeled eGFP -rAB1003.

2-step registration workflow for alignment of EMSA card to destination microplate
Registration proceeds in two steps: (i) printing of a target grid and (ii) assembly of a gel-gridplate stack. ( Figure S2A) • First, a target grid outlining the geometry of 384 well plate was printed with ADE on a transparent microplate sealing film attached on the top surface of the destination plate, using a red food dye spiked water solution.
• In this method, the target grid is precisely mapped out on the location of where the droplet lands. Next the grids were labeled with permanent marker pen to register the location information for the next step.
• Next, the EMSA card was aligned onto the sealing firm with sample well overlapping the target grid.
• Finally, the gel-plate structure was inserted to Echo for liquid transfer.
Further, we set out to quantify the performance of the alignment and assess the location accuracy of the droplet dispensing. To do that, we integrated the ADE transfer with the 384-plex EMSA card and measured center-to-center displacement ( Figure S2B) using a liquid sample doped with red dye. For quantitation of location accuracy, the bright-field image of a ADE loaded EMSA card was converted to grayscale and offset between the center of each droplet and the fluid well  8

ANOVA analysis for unit-to-unit variation
The area-under-curve (AUC) and migration distance of BSA for unit-to-unit variation are listed in Figure S3.  ANOVA test between interior group and row 1: The sum of squares (SS) of all units SS t = 1.14 mm 2 The sum of SS in both group: SS w =1.13 mm 2 The SS between groups: SS b = SS t -SS w =0.01 mm 2 All F value calculated between side perimeter groups and interior groups are below F critical = 4.
Therefore, these results indicated no significant variation between the EMSA performance in units in the chip interior and on the perimeter ( Figure S3), which also corroborated our analysis of low-density 96-plex fsPAG devices 36 .
Next to seek any spatial variation due to the sample differential evaporation during the sample delivery stage, we applied an ANOVA (analysis of variance) test to scrutinize differences in AUC and migration distance of BSA between column 1 (first filled column) and column 24 (last filled column).