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Sci Rep ; 10(1): 14103, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839506

RESUMO

"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 105 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32-63-fold improved Ka values (> 1010 M-1), compared with the wild-type scFv (Ka = 3.8 × 108 M-1), none of which could be recovered via conventional panning procedures operated for the entire library.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Bacteriófagos/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Afinidade de Anticorpos/imunologia , Bacteriófagos/genética , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/genética
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