Fig. 2.

Schematic overview to generate immune, naïve, and synthetic Nb libraries. Blood in camelids contains two kinds of B cells that secrete either homodimeric HCAbs or classical heterotetrameric antibodies. The mRNA of these B cells is converted into cDNA and a first PCR with primers annealing to the leader signal and conserved CH2 region (LS‐BACK and CH2‐FOR, respectively) on this cDNA will amplify a fragment of 600–650 bp with VHH‐hinge‐CH2 coding sequences of HCAbs and a fragment of 900 bp with VH‐CH1‐hinge‐CH2 sequences from the heavy chain of classical antibodies. The former amplicon is purified after agarose gel electrophoresis and amplified in a second, nested PCR with primers annealing at framework region 1 and 4 (FR1‐BACK and FR4‐FOR, respectively). This PCR amplification product is ligated into the, for example pMECS‐GG phagemid vector (procedure “1”) to generate pMECS‐Nb phagemids that are transformed in Escherichia coli. An immune Nb library (typically 10⁶–10⁸ clones) is obtained when starting from a 50 to 100 mL blood sample of an immunised camelid, whereas a naïve Nb library (typically 10⁹–10¹¹ clones) is generated when starting from much larger amounts of blood (~ 10 L) taken from several healthy camels, dromedaries, llama's or alpaca's that were not intentionally immunised. To construct a synthetic Nb library (procedure “2”), a single or a few Nbs are expressed form the previous libraries and selected for favourable (biochemical) properties (stability, expression level, solubility, unique paratope architecture). The codons for amino acids located in the Ag‐binding loops (yellow, orange and red for CDR1, 2 and 3) of these selected Nb scaffolds are then randomised and the sequences amplified by PCR. These PCR products are ligated, for example in phage display vector pMECS‐GG to obtain a synthetic pMECS‐Nb library after transforming E. coli cells.