Characterization Of Two Monoclonal Antibodies Specific Against Cartilage Oligomeric Matrix Protein

Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease.The main characterisyics are articular cartilage inflammation damage and synovial tissue hyperplasia.The fundamental reason of RA is autoantigen induced immune responses.RA have been considered to be related to some cartilage-specific antigens such as cartilage oligomeric matrix protein(COMP),proteoglycans,collagen types II,IX,and XI,fibrinogen,aggrecan,a-enolase,vimentin and GPI.These antigens active cross-linking of effector cell bound IgG,B and T cells.COMP belongs to the thrombospondin(TSP)protein family,which we also named TSP-5.It is an important element of the cartilage extracellular matrix,which is closely related to the metabolism of cartilage tissue and cartilage formation.It also play an important role in adjusting the formation of fiber structure of extracellular matrix and maintaining the integrity of the collagen network.What is more,recent study found that autoimmune response to COMP antigen is important to RA.In earlier studies,we produced 20 monoclonal antibodies specific to COMP.In this research,we studied characterization of two monoclonal antibodies by phage displaying?peptide synthesis and bioinformatics analysis.The main research results are as follows:1.The characterization of mAb 1D10:A filamentous phage library displaying random linear dodecapeptides was used to identify the epitope of mAb 1D10.After repeated screenings using bio-panning method,five phages were isolated.Two related peptide-presenting phages(HSFKWLDSPRLR-phage and HSFQWLDSPRLR-phage)were identified,which interacted specifically with 1D10 mAb.Homology search and structure analysis with Pymol identified that the peptide sequence is indeed corresponding to 680-689 residues of COMP,which adopts a stable ?-sheet structure.Competitive ELISA demonstrated that the binding of 1D10 antibody could be inhibited by the synthetic peptide derived from aa 680-689 of COMP,selected phages as well as full-length COMP molecule.Preferential reactivity of the mAb with EDTA-treated COMP and western blot analysis suggest recognition of an open conformational structure of the epitope that is normally present inside the COMP molecule.This study demonstrates that a pathogenic autoantibody recognizes a well-defined structure on native COMP.However,this region is embedded inside the molecule and thus the binding of the antibody to cartilage in vivo is more likely due to structural changes caused by mechanical stress/inflammation or the presence of a variant of the COMP molecule in the joint cartilage.2.The characterization of mAb 15A11:Another monoclonal antibody named 15A11 was used to select the random phage 12 peptide library.After repeated screenings using bio-panning method,clone 1 and clone 2 were identified,which interacted specifically with mAb 15A11 After repeated screenings using bio-panning method,2 clones were identified,which interacted specifically with mAb 15A11.Homology search did not find succession consensus sequence within COMP molecular,which indicated that the epitope is not linear.PyMol Structure analysis identified the rationality of conformational epitope.Western blot analysis and ELISA of EDTA-treated COMP further prove an conformational structure of the epitope recognized by mAb 15A11.ELISA analysis of COMP-derived peptides demonstrated both disulfide bonds between 229C-243C and 237C-253C and every epitope amino acid of 232G?238H?240H?241 A?244V?247R and 251R are essential to the binding of mAb 15A11 with COMP.In this study,the potential B cell antigentic epitopes of mAb 15A11 was identified by phage display library.The epitope amino acids sequence and characterization were also recognized.It may have important theoretical value for the study of reaction mechanism of COMP antibody and antigen and may also show application significance in the detection of rheumatoid arthritis.