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

We have developed monoclonal antibody 5109 against a unique highly acidic s equence in type II collagen. When paired with previously reported monoclona l 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. T he assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyPr oX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is pro line or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid o f the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epito pe with a K=1.2x10(-8) M independently of hydroxylation of X-759. When X-77 1 is proline, the sequence is 90X more sensitively detected by this ELISA t han when it is hydroxyproline. Type II collagen of human articular cartilag e was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% h ydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine o f control individuals of 50-70 years of age, we failed to detect the presen ce of the collagen fragment in a majority (8/10) of specimens. The two cont rols with measurable levels averaged 123 pM. In a similar age cohort of ost eoarthritic patients, the majority (9/10) showed measurable values of urina ry collagen fragments averaging 312 pM. This assay can be used for monitori ng type II collagen metabolism in patients with osteoarthritis. (C) 2001 El sevier Science B.V. All rights reserved.