Problems in the Immunolocalization of Type IX Collagen in Fetal Calf Cartilage Using a Monoclonal Antibody

Monoclonal antibodies were prepared against the pepsin-resistant fragments (X1-X3) of bovine type IX collagen. One of the five hybridomas that gave a positive reaction in an enzyme-linked immunosorbent assay was selected (Hla) for structural analysis and immunolocalization of type IX collagen. The location of the epitope for Hla was deduced from immunoblots and electron microscopic observations after rotary shadowing. The Hla antibody binds to one end of the longest X2, X3, X4 molecules, and preferentially 40-55 nm from one end of Xl molecules thus, on or near the noncollagenous domain, NC2. Different immunolocalizations of type IX collagen in the superficial, middle and deep zones of fetal calf epiphyseal cartilage were observed depending on the thickness of the section and on hyaluronidase digestion conditions. In the middle and deep zones, staining with Hla throughout the matrix was obtained only with thin sections (5μm) and digestion for 1 h at 37°C. With thick sections (15μm) or with digestion for 1 h at 24°C, staining was restricted to the pericellular regions. Staining throughout the matrix was obtained in the superficial zone under all experimental conditions. Without hyaluronidase treatment, no immunofluorescent staining was seen with either Hla or polyclonal antibody to type II collagen, indicating that type IX collagen is present throughout the matrix in the different zones of fetal calf cartilage. This result is in good accordance with the recent demonstration of common cross-links between type II and type IX collagen in chicken and bovine cartilage. However, the preferential unmasking of type IX collagen antigenic sites in the pericellular regions of middle and deep zones of fetal calf cartilage does not preclude the presence in that region of a special pericellular organization of the collagenous network.