Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

Microbial Cell Factories

Research

Saskia Dolinska¹  Irene Jiménez-Munguía¹  Lenka Potocnakova¹  Lucia Pulzova¹  Katarina Bhide¹  Zuzana Flachbartova¹  Evelina Kanova¹  Elena Bencurova¹  Lubos Comor¹  Mangesh Bhide² 

[1] Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia;Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia;Institute of Neuroimunnology, Slovak Academy of Sciences, Bratislava, Slovakia;

关键词: VHH;    In vitro immunization;    OspA;    Phage display;    Single domain antibodies;   

DOI  :  10.1186/s12934-017-0630-z

 received in 2016-10-01, accepted in 2017-01-17,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundCamelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens).ResultsIn this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry.ConclusionA proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

【 授权许可】

CC BY   
© The Author(s) 2017