Characterization of monoclonal antibodies recognizing different fragments of cartilage oligomeric matrix protein in human body fluids.
Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.
Vilim
V
V
Institute of Rheumatology, Praha, Czech Republic. vilim@mbox.cesnet.cz
Lenz
M E
ME
Vytasek
R
R
Masuda
K
K
Pavelka
K
K
Kuettner
K E
KE
Thonar
E J
EJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D002356:Cartilage / Q000276:immunology; D064236:Cartilage Oligomeric Matrix Protein; D003720:Densitometry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D064235:Matrilin Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D013582:Synovial Fluid / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D064236:Cartilage Oligomeric Matrix Protein; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins; D010446:Peptide Fragments; C577553:TSP5 protein, human; D014357:Trypsin