Preparation and identification of a single domain antibody specific for adenovirus vectors and its application to the immunoaffinity purification of adenoviruses

Abstract

Abstract Adenovirus belongs to the family of Adenoviridae. As a vaccine carrier, it has high safety and stimulates the body to produce cellular immunity and humoral immunity. This study prepared an adenoviral vector-specific single-domain antibody for use in adenovirus identification and purification. We successfully constructed a single domain antibody phage display library with a capacity of 1.8 × 109 by immunizing and cloning the VHH gene from Bactrian camel. After the second round of biopanning, clones specific for adenovirus were screened using phage ELISA. Twenty-two positive clones were obtained, and two clones with the highest binding affinity from ELISA were selected and named sdAb 5 and sdAb 31 for further application. The recombinant single-domain antibody was solublely expressed in E. coli and specifically bound to adenoviruses rAd26, ChAd63 and HAd5 in ELISA and live cell immunofluorescence assays. We established an effective method for immunoaffinity purification of adenovirus by immobilizing the single domain antibody to Sepharose beads, and it may be used to selectively capture adenoviruses from cell culture medium. The preparation of the adenovirus-specific single-domain antibody lays a foundation for the one-step immunoaffinity purification and identification of adenoviruses.

Keywords

• Adenovirus
• Single domain antibody
• Phage display library
• Immunofluorescence
• Immunoaffinity purification