University of California, Berkeley - UCSF Graduate Program in Bioengineering, Berkeley, CA, 94720, USA.
Labcyte Inc., 1190 Borregas Ave, Sunnyvale, CA, 94089, USA.
Sci Rep. 2016 Dec 23;6:39774. doi: 10.1038/srep39774.
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) of each recombinant antibody and the target antigen. To characterize the K 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 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.
高质量的免疫试剂可提高免疫分析的性能和重现性，进而提高生物和临床测量的质量。高质量的重组免疫试剂是使用抗体噬菌体展示技术生成的。抗体质量的一个衡量标准 - 结合亲和力 - 通过每个重组抗体和目标抗原的解离常数（K）来定量。为了表征重组抗体和目标抗原的 K，我们引入了亲和电泳迁移率变动分析（EMSA），其采用适用于小体积样品的高通量格式。由独立式聚丙烯酰胺凝胶（fsPAG）分离槽组成的微流控卡支持使用单个电源在 30 秒内同时进行 384 次 EMSA。通过声控液滴喷射（ADE）将样品分配到微流控 EMSA 卡上，与使用手动或针工具进行样品分配相比，可降低 EMSA 的变异性。与传统的异质分析相比，使用约 25 倍少的样品质量和 5 倍少的时间报告了六成员片段抗原结合片段文库的每个 K。鉴于这种微流控和介观流控工作流程的形式因素和性能，我们已经开发出了一种节省样品、高通量、溶液相的生物分子亲和力表征的替代方法。
