Proteoglycans from bovine nasal cartilage. Immunochemical studies of link protein

Antisera have been prepared against purified proteoglycan monomer and link protein from bovine cartilages. The immunological properties of these species have been studied by immunodiffusion, Laurell rocket immunoelectrophoresis, crossed immunoelectrophoresis, hemagglutination, and immunofluorescence. Antisera raised against either adult bovine articular or foetal bovine epiphyseal proteoglycan monomer were monospecific. They did not react with link protein, type II collagen, nor with any other molecular species isolated from bovine articular and epiphyseal cartilages. Antisera to link protein, purified from bovine nasal cartilage by dissociative density gradient centrifugation followed by chromatography under dissociative conditions on Sephacryl S-200, showed no reaction with proteoglycan monomer by immunodiffusion. However, antisera to link protein showed a weak reaction with native monomer on Laurell rocket immunoelectrophoresis (not detectable when chondroitinase ABC-treated monomer was used), and with chondroitinase ABC-treated monomer by hemagglutination assay. These traces of antibody to proteoglycan monomer were inactivated by immunoreaction with proteoglycan monomer in solution to provide a monospecific antiserum to link protein. Purified antibodies specific for proteoglycan monomer, some of which did not react with the hyaluronic acid binding region of bovine nasal monomer, were prepared from anti-link sera by affinity chromatography using chondroitinase ABC-treated bovine nasal proteoglycan monomer. Purified link protein, which contains 48,000- and 44,000-dalton subunits, contains two immunologically distinguishable species. An immunoassay for purified link protein was developed using Laurell rocket electrophoresis. A micro-method, using crossed-immunoelectrophoresis, for studying the binding of link protein to proteoglycan monomer and to hyaluronate is described. The complex of link protein bound to hyaluronate was dissociated on reaction of link protein with antibody.