Generation of the Phage-Display VHH-Library

The anti-hTNC nanobody phage-display VHH-library was constructed as previously described with slight modifications (39, 49, 50). Briefly, 3 days after the last boost of antigen injection, 150 mL of anti-coagulated blood sample was collected from the jugular vein of the immunized dromedary as recently detailed (51). Peripheral blood mononuclear cells (PBMCs) were extracted by density gradient centrifugation using Lymphoprep (catalog number 17-829 LONZA, Basel, Switzerland). Subsequently, total RNA was extracted and purified. An amount of 40 μg of total RNA was reverse transcribed into cDNA with oligo-dT primer and the SuperScript II First-Strand Synthesis System for RT-PCR (catalog number 18064-014 Invitrogen, Carsbad, CA, USA). Thereafter, cDNA fragments were used as template to amplify heavy-chain IgG encoding variable domains using specific primers [CALL001 (5′-GTCCTGGCTGCTCTTCTACAAGG-3′) and CALL002 (5′-GGTACGTGCTGTTGAACTGTTCC-3′)]. The 700 bp PCR fragment (VHH-CH2 without CH1 exon, corresponding to heavy-chain antibodies) was purified from a 1% agarose gel using the Qiaquick gel extraction kit (catalog number 28704 Qiagen, Hilden, Germany). Subsequently, these sequences were used as template in a nested PCR to amplify VHH-only variable domains with nested-PCR primers [SM017 (5′-CCAGCCGGCCATGGCTGCATGGTGCAGCTGGTGGAGTCTGG-3′) and PMCF (5′-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3′)], annealing at the Framework 1 and Framework 4 regions, including NcoI and NotI restriction sites, respectively (catalog numbers R0193T and R3189M New England Biolabs, UK, respectively). The PCR product was ligated into the pMECS phagemid vector (T4 DNA Ligase, catalog number 15224-041 Invitrogen, Carsbad, CA, USA) using a molar ratio 1:3 in favor of the inserts. Freshly prepared electro-competent E. coli TG1 cells were transformed by the ligated product and plated overnight (O/N) on selective Luria-Bertani Miller (LB) media supplemented with (100 μg/mL) ampicillin (catalog number 271896 Sigma Aldrich, MO, USA) and glucose 2% (catalog number G8270 Sigma Aldrich, MO, USA). Colonies were recovered from the overnight-incubated plates at 37°C. Library size was estimated by serial dilutions.