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

Abstract

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.

Theranostics in immuno-oncology using nanobody derivatives

“…Recently, efforts has been made to develop in vitro selection of nanobodies against a target protein by for example cDNA display methods with synthetic or semi-synthetic libraries 22-25. Although some studies reported this method for selection of nanobodies against their target of interest, it must be mentioned that affinities were usually lower, in the range of 10 -7 to 10 -8 M, and that this approach demands large library sizes and sophisticated selection procedures, which currently is prohibitive for widespread implementation 26-28.…”

Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells

“…Detailed information on primer design, amplicon lengths and restriction enzymes used are presented in Supplementary Data Sheet 2 . Digestion of amplified PCR products, ligation into a pQE-30-mCherry-GFP plasmid (in-house modified vector pQE-30 UA, Qiagen Comor et al, 2017 , transformation and selection of clones were performed as described earlier Jimenez-Munguia et al, 2018 . Insertion of encoding genes was confirmed by sequencing with vector specific primers UA Insertom F and R (Supplementary Data Sheet 2 ).…”

Exploring cellular biochemistry with nanobodies

“…For library construction, B cells from naive or immunized camelids can serve as the point of departure, as can cultured camelid B cells exposed to antigens of interest (9). Purified proteins (10), cells or cell lysates containing antigens of interest (11,12), or recombinant DNA to induce antigen expression in the host (13,14) can serve as immunogens.…”