CHARACTERIZATION OF SPECIFIC CAMELID SINGLE DOMAIN ANTIBODIES AGAINST MURINE IgG1 TO BE USED AS LIGAND IN CONVENTIONAL MONOCLONAL ANTIBODY PURIFICATION

MARCHESE, FLORENCIA; GRIPPO, VANINA; LÓPEZ, NORA; BOZZO, JOAQUIN

Congreso; Reunión Anual de Sociedades de Biociencias; 2020

Sociedad Argentina de Investigación Clinica, Sociedad Argentina de Inmunología, Sociedad Argentina de Fisiología

In addition to classical antibodies (Abs), the adaptive immune system of camelids comprises heavy-chain only antibodies (HCAb), where antigen-binding is mediated exclusively by one variable domain. These single domains are the smallest functional Abs, known as VHH or nanobodies (Nbs). Due to their inherent favorable attributes, such as high affinity and specificity for their antigen, easy cloning and small size, VHH have great potential for diagnostic, therapeutic and biotechnological applications. Thus, Nbs are used as protein purification ligands because of their high stability, monomeric nature, and easy directional immobilization to solid supports. For this reason, our aim was to characterize VHH against murine IgG1 (mIgG1) to be used as ligands in a new affinity chromatography method to purify conventional monoclonal Abs (mAbs). To achieve our goal, two immune VHH libraries were previously constructed starting from llama blood, and specific Nbs against mIgG1 were selected by Phage Display methodology. Moreover, 9 out of the 24 most reactive selected VHH were confirmed as unique by sequencing. In this work, these Nbs were expressed as soluble protein in E. coli WK6 strain. Later, 4 of these VHH were purified from periplasmic extract by IMAC. Reactivity of these soluble-expressed Nbs against mIgG1 was confirmed by ELISA and Western Blot (WB). Also, the reactivity against IgGs from other species was evaluated by Dot Blot and WB. Moreover, all these VHH were able to successfully bind and immunoprecipitate IgG from mouse sera and ascites. In conclusion, we have characterized four specific VHH expressed as soluble protein, which showed high reactivity against mIgG1 in ELISA and WB as expected. All of them were able to recognize their target in a biological protein mixture. Even more, two of them were highly specific for only murine IgG. Further characterization of these Nbs will allow us to develop a new VHH-based method of mAb purification with potential advantages.