Construction of VHH library and expression of recombinant VHHs

An adult alpaca was immunized with intact unfractionated mouse splenocytes (5 × 10⁶ cells per injection) at 2–3-wk intervals for a total of seven injections. We assessed the host response by immunoblotting of alpaca serum against membrane and cytosolic fractions of murine splenocytes that were isolated by selective plasma membrane permeabilization. Mononuclear cells from peripheral blood were isolated from the immunized alpaca by Ficoll gradient separation, followed by an established VHH library–generation protocol (10). Total RNA was extracted (RNeasy RNA Purification kit; QIAGEN), and cDNA was prepared (Superscript III First Strand Synthesis Kit; Life Technologies). The diversity of the cDNA preparation was maximized by using three sets of complementary oligonucleotides for first-strand cDNA synthesis: oligo-dT, random hexamers, and Ig-specific primers. The DNA sequences of the signal sequence from conventional and H chain–only Ig genes are not distinguishable based on the use of specific primers. However, two unique and distinct hinge regions (short and long hinge) are generated between the VHH domain and the C region (CH2). Thus, we amplified the VHH repertoire from the immune animal using alpaca VHH specific–primers that target these particular hinge sequences. The purified VHH PCR products were pooled and ligated into a phagemid vector to construct a phage display library through in-frame fusion of the amplified VHH sequences with the pIII gene of the M13 phagemid. The diversity of the phagemid library was estimated by serial dilution. The resulting VHH library consisted of ~10⁸ independent clones. We performed several rounds of panning against mouse splenocytes, as well as DC and B cell lines. VHH clones that showed reactivity by ELISA were enriched and expressed in WK6 Escherichia coli and grown in Terrific Broth overnight at 30°C after isopropyl β-D-thiogalactopyranoside induction (1 mM) at an OD₆₀₀ of 0.6. VHHs were harvested from the periplasm by osmotic shock and purified by metal ion affinity using Ni-NTA agarose beads (QIAGEN) size-exclusion chromatography (SEC) on a Superdex 75 16/600 (GE Healthcare) in PBS or Tris HCl (50 mM [pH 7.5]).