title: Continuous microfluidic assortment of interactive ligands (CMAIL).
abstract: Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 10(5) CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 10(9) individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.
authors: Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan.|Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan.
authors: Yi-Hsing
authors: YH
authors: Hsiao
authors: Development Center for Biotechnology, New Taipei City 22180, Taiwan.
authors: Chao-Yang
authors: CY
authors: Huang
authors: Development Center for Biotechnology, New Taipei City 22180, Taiwan.
authors: Chih-Yung
authors: CY
authors: Hu
authors: Development Center for Biotechnology, New Taipei City 22180, Taiwan.
authors: Yen-Yu
authors: YY
authors: Wu
authors: Development Center for Biotechnology, New Taipei City 22180, Taiwan.
authors: Chung-Hsiun
authors: CH
authors: Wu
authors: Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan.|Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan.
authors: Chia-Hsien
authors: CH
authors: Hsu
authors: Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan.|Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.
authors: Chihchen
authors: C
authors: Chen
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D002999:Clone Cells; D004560:Electricity; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D020100:Hydrogels; D056656:Lab-On-A-Chip Devices; D008024:Ligands; D019151:Peptide Library; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry; D055672:Static Electricity; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D020100:Hydrogels; D008024:Ligands; D019151:Peptide Library; D057127:Single-Chain Antibodies; C053409:mammary serum antigen, human; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2