Yeast Surface Display in Combination with Fluorescence‐activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens

• Published in: Biotechnology journal 2019-04, Vol.14 (4), p.e1800466-n/a
• Main Authors: Grzeschik, Julius, Yanakieva, Desislava, Roth, Lukas, Krah, Simon, Hinz, Steffen C., Elter, Adrian, Zollmann, Tina, Schwall, Gerhard, Zielonka, Stefan, Kolmar, Harald
• Format: Article
• Language: English
• Subjects: Animals; Antibody Affinity; Antigens - chemistry; Antigens - immunology; chicken antibody; Chickens - immunology; Chorionic Gonadotropin - genetics; Chorionic Gonadotropin - immunology; EGFR; Epitopes - immunology; Flow Cytometry - methods; Genes, erbB-1 - genetics; Genes, erbB-1 - immunology; hCG; Humans; Immunization - methods; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - immunology; Immunoglobulin Fragments - isolation & purification; Peptide Library; Recombinant Proteins - genetics; Recombinant Proteins - immunology; RNA, Messenger - genetics; Saccharomyces cerevisiae - genetics; scFv; yeast surface display
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201800466

Yeast surface display emerged as a viable tool for the generation of human and murine monoclonal antibodies. This platform technology enables the careful definition of selection conditions, the potential for high‐throughput screening, as well as the isolation of antibodies recognizing predefined epitopes. In this study, the applicability of yeast surface display in combination with fluorescence‐activated cell sorting (FACS) for the isolation of antigen‐specific chicken‐derived antibodies is demonstrated. To this end, yeast‐displayed recombinant antibody libraries from splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction. A large number of antigen binding scFvs were readily isolated in a convenient screening process. Target‐specific scFv‐Fc molecules were produced as soluble proteins and more extensively characterized by confirming specificity, thermostability and high affinity. Essentially, we demonstrated the biotechnological applicability of binders directed against both antigens via specific cellular binding for EGFR and in the context of a lateral flow test by utilizing hCG‐binding scFvs as capturing antibodies for pregnancy detection. Altogether, the described strategy using yeast surface display expands the repertoire of display methods for the isolation of antibodies resulting from chicken immunization campaigns. Monoclonal antibodies from immunized chickens are useful tools for many biotechnological and diagnostic applications. In this study, the authors demonstrate for the first time that chicken‐derived antibodies can rapidly be isolated by transferring the antibody coding information from chickens to baker's yeast followed by high‐throughput screening using fluorescence‐activated cell sorting (FACS). Among binders to a tumor target, the authors isolated antibody fragments with high sensitivity for the detection of the pregnancy hormone HCG in a lateral flow test.