Characterization of rabbit polyclonal antibody against camel recombinant nanobodies

Abstract

Nanobodies (Nbs) are recombinant single-domain fragments derived from camelids’ heavy-chain antibodies (HCAbs). Nanobodies are increasingly used in numerous biotechnological and medical applications because of their high stability, solubility, and yield. However, one major obstacle prohibiting Nb expansion is the affordability of specific detector antibodies for their final revelation. In this work, the production of a specific anti-Nb antibody as a general detector for camel antibodies, conventional cIgG, and HCAb, and their derived Nbs was sought. Thus, a T7 promoter plasmid was constructed and used to highly express six different Nbs that were used in a successful rabbit immunization. Affinity-purified rabbit anti-Nb rIgG was able to detect immobilized or antigen-bound Nbs via enzyme-linked immunosorbent assay, and its performance was comparable to that of a commercial anti-6× His antibody. Its capacities in dosing impure Nbs, detecting Nbs displayed on M13 phages, and revealing denatured Nbs in immune blotting were all proven. As expected, and because of shared epitopes, rabbit anti-Nb cross-reacted with cIgG, HCAbs, and 6× His-tagged proteins, and the percentage of each fraction within anti-Nb rIgG was determined. Anti-Nb is a promising tool for the checkpoints throughout the recombinant Nb technology.

Keywords

• nanobody
• camel
• polyclonal antibody
• heavy-chain antibody
• phage display