Rapid Production of Cyclic Citrullinated Peptide Monoclonal Antibody in Nicotiana benthamiana for the Early Detection and Diagnosis of Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting 0.5% to 1% of the population. It could ultimately result in joint destruction, functional decline, work disability, and enhanced mortality. Cyclic citrullinated peptide antibodies (CCP Abs) are useful biomarkers for the early detection and diagnosis of RA. In this study, we used plant viral-based expression vectors that produce rapidly large quantities of CCP-specific monoclonal antibodies. Heavy and light chain genes of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant viral-based expression vectors, TMV and PVX. A cyclic citrullinated peptide monoclonal antibody was produced in Nicotiana benthamiana through an Agrobacterium-mediated transient expression system. The expression of CCP mAb in tobacco plants was confirmed by dot blot, western blot analysis, and enzyme-linked immunosorbent assays (ELISA). It was shown that tobacco plants could accumulate CCP mAbs up to 0.35% of total soluble protein. Accumulated CCP mAb from infiltrated leaves was purified by protein G affinity chromatography. Immunoblot assays and ELISA showed plant-produced CCP mAbs successfully bound to a synthetic CCP peptide antigen. This system provides a fast strategy for the production of pharmaceutical CCP mAbs in tobacco plants.