Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA

• Published in: Journal of immunological methods 2001, Vol.247 (1), p.25-34
• Main Authors: Downs, James T, Lane, Caryl L, Nestor, Nestor B, McLellan, Thomas J, Kelly, Michele A, Karam, George A, Mezes, Peter S, Pelletier, Jean-Pierre, Otterness, Ivan G
• Format: Article
• Language: English
• Subjects: Aged; Amino Acid Sequence; Animals; Biological and medical sciences; Cartilage, Articular - chemistry; Cartilage, Articular - pathology; Collagen - analysis; Collagen - immunology; Collagenase-neoepitope ELISA; Collagenases - metabolism; Enzyme-Linked Immunosorbent Assay - methods; Epitopes, B-Lymphocyte - immunology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Human viral diseases; Humans; Hydroxyproline - metabolism; Infectious diseases; Medical sciences; Mice; Mice, Inbred BALB C; Microbiology; Middle Aged; Molecular immunology; Molecular Sequence Data; Osteoarthritis; Osteoarthritis - immunology; Osteoarthritis - urine; Proline - metabolism; Surface Plasmon Resonance - methods; Techniques; Tumor Cells, Cultured; Type II collagen fragments; Viral diseases; Viral diseases of the lymphoid tissue and the blood. Aids
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(00)00302-1

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGly GluX 759 GlyAspAspGlyProSer GlyAlaGlu GlyProX 771 GlyProGlnGly 775 where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly 775 corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2×10 −8 M independently of hydroxylation of X 759. When X 771 is proline, the sequence is 90× more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline 771 averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50–70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.