title: Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis.
abstract: BACKGROUND
  Camelids 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).
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  A 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.
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Lubos
authors.initials: L
authors.lastname: Comor
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Saskia
authors.initials: S
authors.lastname: Dolinska
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Katarina
authors.initials: K
authors.lastname: Bhide
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Lucia
authors.initials: L
authors.lastname: Pulzova
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Irene
authors.initials: I
authors.lastname: Jiménez-Munguía
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Elena
authors.initials: E
authors.lastname: Bencurova
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Flachbartova
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Lenka
authors.initials: L
authors.lastname: Potocnakova
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia.
authors.forename: Evelina
authors.initials: E
authors.lastname: Kanova
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, 73, 04181, Kosice, Slovakia. bhidemangesh@gmail.com.|Institute of Neuroimunnology, Slovak Academy of Sciences, Bratislava, Slovakia. bhidemangesh@gmail.com.
authors.forename: Mangesh
authors.initials: M
authors.lastname: Bhide
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D002161:Camelids, New World / Q000276:immunology*; D061505:Cell Surface Display Techniques / Q000191:economics / Q000379:methods; D003362:Cost-Benefit Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D007114:Immunization / Q000191:economics / Q000379:methods; D007376:Interleukin-2 / Q000276:immunology; D015847:Interleukin-4 / Q000276:immunology; D008074:Lipoproteins / Q000276:immunology; D008213:Lymphocyte Activation; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D007376:Interleukin-2; D008074:Lipoproteins; C072045:OspA protein; D019151:Peptide Library; D061905:Single-Domain Antibodies; D015847:Interleukin-4
keywords: In vitro immunization; OspA; Phage display; Single domain antibodies; VHH