Isolation, Characterization, and Molecular Modeling of a Rheumatoid Factor from a Hepatitis C Virus Infected Patient with Sjögren's Syndrome

Lee, Yu-Ching; Tsai, Keng-Chang; Leu, Sy-Jye; Wang, Tuan-Jen; Liu, Chia-Yu; Yang, Yi-Yuan

Human Fc. All the clones containing light chain of Fab were further tested for binding activity and specificity. GG3 and GG48 clones with high binding specificity to Fc [24] and LPSL and BL12 clones with anti-LPS and anti-red-blood surface Ag were applied as positive and negative controls, respectively. These selected Fab fragments showed significant binding activity to Fc but not to BSA (Figure 1(c) ). Moreover, their recognition of human Fc fragment immobilized on Western blot was tested. The results showed both serum of HCV-infected patient with SS disease (Figure 2(b) ) and the recombinant Fab molecule as represented by RFL11 (Figure 2(c) ) could clearly recognize Fc protein. No Fcbinding activity was observed when an irreverent Fab molecule was applied (Figure 2(d) ). In order to test the binding specificity, unrelated antigens including collagen, KLH, LPS, calf thymus ssDNA, and thyroglobulin were used in ELISA test. As shown in Figure 3 , the representative RFL11 Fab antibody as well as the positive GG3 clone showed specific binding activity to Fc fragment but minimal binding to the panel of unrelated antigens. The binding specificity and affinity of RFL11 was further determined by competitive inhibition assay. As demonstrated in Figure 4 , 89% inhibitory effect was found on the binding reactivity of RFL11 Fab against coated human Fc fragment in the presence of a concentration of 40 ug/mL of free Fc fragment. When performed in parallel, close to 50% of inhibitory effects were seen using positive GG3 Fab antibody and none were detected using negative BL12 Fab molecule. The value for RFL11 antibody was 6 × 10 −8 M calculated using the Klotz plot method [25] .