OBTENTION OF SPECIFIC CAMELID SINGLE DOMAIN ANTIBODIES, VHH OR NANOBODIES, AGAINST MURINE IgG1 FOR DEVELOPMENT OF A NEW METHOD FOR PURIFICATION OF MONOCLONAL ANTIBODIES

MARCHESE F; BOZZO J; MATTION N; GRIPPO V

Mar del Plata

Congreso; REUNION CONJUNTA SAIC SAI SAFIS 2018; 2018

SAIC SAI SAFIS

Last 30 years, monoclonal antibodies (mAbs) industry has grown exponentially, due to their wide use in research, diagnosis and therapy. Currently, most used method for mAb purification is affinity chromatography with protein A (ProtA). However, it presents some disadvantages as acidic elution and high cost. On the other hand, biotechnological application of camelid single domain antibodies (VHH or nanobodies) has notably increased in the last years. These nanobodies present several advantages as small size, high solubility and physicochemical stability. For this reason, our goal was to obtain VHH against murine IgG1 (mIgG1) to be used as ligand in a new resin for mAb purification.Firstly, two llamas were immunized with a mix of murine IgG (total and subtype 1). After immunization, both llamas presented a high humoral response against mIgG1 measured by ELISA. Two immune VHH libraries were constructed starting from llama peripheral blood and library quality controls were performed. Both libraries presented high size (more than 5x108 CFU/ml) and 100% of full-length insert clones. Phages expressing specific VHH against mIgG1 were obtained after two rounds of selection. Reactivity of total phage libraries and eluted phages was tested by polyclonal Phage-ELISA. For 88 selected clones, specific reactivity was confirmed in VHH-ELISA. The 24 most reactive nanobodies were analyzed by fingerprinting, in order to evaluate VHH diversity. As a result, 14 different digestion patterns were obtained and 9 of them were confirmed as unique by sequencing.In conclusion, two immune VHH libraries were constructed and specific nanobodies against mIgG1 were obtained by Phage Display methodology. Even more, 14 out of the 24 most reactive nanobodies presented a unique fingerprinting pattern confirming VHH diversity. Further characterization of these nanobodies will allow us to develop a new method based in VHH as ligand, with potential wider use and lower cost for mAb purification.