Monoclonal Antibodies Reactive with Keratan Sulfate-Bearing Tryptic Fragments of Bovine Nasal Cartilage Proteoglycan

Abstract

Monoclonal antibody-producing cell lines were derived from BALB/c mice immunized with a testicular hyaluronidase digest of tryptic fragments of bovine nasal cartilage proteoglycan. Sera and hybridoma culture supernatants were screened by solid-phase immunoassay for reactivity against a chondroitinase ABC digest of the same proteoglycan fragment fraction. Antibody specificity was determined by competitive inhibition with purified proteoglycan fragment subfractions and their enzymatically modified derivatives. Two monoclonal antibodies were produced which reacted with keratan sulfate-rich fragments from bovine nasal and human articular cartilage proteoglycan. One, monoclonal LC8.13, is directed against keratan sulfate itself, but differs from 5-D-4, a previously described monoclonal antibody to keratan sulfate, in its lesser reactivity with keratanase-treated fragments. The second, monoclonal F1.2, appears to be directed against a conformation-dependent determinant on the core protein of this segment of the cartilage proteoglycan monomer. Monoclonal F1.2 does not react with the keratan sulfate species in human and fetal calf serum and can therefore detect the production of keratan sulfate-bearing proteoglycan by chondrocytes cultured in serumcontaining media.