Relative level and kinetics of the antibody response to each truncated cartilage oligomeric matrix protein (COMP) fragment. (A) Graphical representation of the domain composition of the recombinant mouse COMP proteins used in this study. pCOMP: pentameric COMP; mCOMP: monomeric COMP; EGF: epidermal growth factor; TIII: thrombospondin type 3; CG: C-terminal globular domain. (B) Medium was collected from each transfected cell line and purified using Ni-NTA affinity resin followed by a concentration step using ion-exchange chromatography as described in Materials and methods, and 10 μl of the purified proteins were subjected to 4 to 10% gradient SDS-PAGE under reducing conditions. (C) Relative level of antibody response to different recombinant COMP fragments. Full length COMP (pCOMP) and COMP fragments (10 μg/ml in 100 μl) were tested by ELISA for COMP autoantibody response. Serum samples from COMP+/+Ncf1*/*mice were taken at day 50 after immunization with rat COMP and diluted 1:1000 for the assay. Background levels were determined for each test using antigen, coated, but without primary antibodies in the wells. Results are expressed as absorbance reading at 405 nm and represent the mean and SEM of 13 mice. The lower binding to the TIII1-CG fragment compared to pCOMP, mCOMP or EGF1-TIII8 was confirmed to be statistically significant (P ≤ 0.05). (D) Kinetics of antibody response to rat COMP, full length mouse COMP (pCOMP) and mouse COMP fragments. Serum samples from COMP+/+Ncf1*/* mice were taken at days 15, 30, 50 and 85 after immunization with rat COMP and were subjected to ELISA. Sera were diluted 1:1000 for the assays. Data represent the mean values from 13 mice.
Geng et al. Arthritis Research & Therapy 2012 14:R191 doi:10.1186/ar4022