Development of a novel immunoassay for the measurement of type II collagen neoepitope generated by collagenase cleavage.

Background: To evaluate cartilage degeneration in arthritis, we developed a novel enzyme-linked immunosorbent assay (ELISA) with the capacity to determine urinary concentrations of type II collagen neoepitope (CIINE) generated by collagenase cleavage.

Methods: Two monoclonal antibodies, 20A10 and 6G4, were generated. Of these antibodies, 20A10 recognized CIINE regardless of hydroxylation of Pro⁷⁷¹, and 6G4 recognized the type II collagen-specific region adjacent to the neoepitope. A sandwich ELISA was constructed using these antibodies.

Results: The ELISA positively determined CIINE concentrations from human and dog urine samples, and from tissue culture supernatant of rat and bovine cartilage. Validation with human urine samples revealed that the ELISA had a detection limit of 100 pmol/l, with intra- and inter-assay coefficients of less than 15%. Recovery of extraneously added CIINE peptide to human urine samples was 83.1-104%. Measurement with the ELISA demonstrated that urine samples from OA patients contained CIINE at significantly higher concentrations compared with those from healthy controls (P<0.01).

Conclusions: The ELISA can determine the CIINE concentration in human urine sensitively and accurately. This assay may also be useful to determine the concentration of CIINE of various animal samples.