Abstract
Monoclonal antibodies were raised against proteoglycan core protein isolated after chondroitinase ABC digestion of human articular cartilage proteoglycan monomer. Characterization of one of the monoclonal antibodies (1/20/5-D-4) indicated that it specifically recognized an antigenic determinant in the polysaccharide structure of both corneal and skeletal keratan sulfate. Enzyme immunoassay analyses indicated that the mouse monoclonal IgG1 recognized keratan sulfate in native proteoglycan aggregate and proteoglycan monomer preparations isolated from hyaline cartilages of a wide variety of animal species (human, monkey, cow, sheep, chicken, and shark cartilage). The 1/20/5-D-4 monoclonal antibody did not recognize antigenic determinants on proteoglycan isolated from Swarm rat chondrosarcoma. This finding is consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue. A variety of substructures isolated after selective cleavage of bovine nasal cartilage proteoglycan (Heinegård, D., and Axelsson, J. (1977) J. Biol. Chem. 252, 1971-1979) were used as competing antigens in radioimmunoassays to characterize the specificity of the 1/20/5-D-4 immunoglobulin. Substructures derived from the keratan sulfate attachment region of the proteoglycan (keratan sulfate peptides) showed the strongest inhibition. Both corneal and skeletal keratan sulfate peptides as competing antigens in radioimmunoassays showed similar inhibition when compared on the basis of their glucosamine content. Therefore, the 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties. Chemical desulfation of the keratan sulfate reduced the antigenicity of the glycosaminoglycan. The antibody did not recognize determinants present in dermatan sulfate, heparin, heparin sulfate, or hyaluronic acid.
Highlights
From the $Biochemistry Department, West Virginia University Medical Center, Morgantown, West Virginia26506 and the §Institute of Dental Research, School of Medicine and Diabetes Research and Training Center, Universityof Alabama in Birmingham, Birmingham, Alabama35294
Proteoglycans glycan core protein isolated after chondroitinaseABC from bovine nasal cartilage anbdovine tracheal cartilage have digestion of human articular cartilage proteoglycan been the most thoroughly studied [2, 3] in recent monomer
Enzyme immunoassay analyses indicated that the mouse monoclonal IgGl recognized keratan sulfate innative proteoglycan aggregateand proteoglycan monomer preparations isolated from hyalinceartilages
Summary
X 10') cells and themyeloma cells were suspended in 10 ml of RPMI 1640medium, counted on a Coulter counter, and mixed at a lymphocyte to myeloma ratio of 2:l. After 10 days of tissue culture inhypoxanthine-aminopterin-thymidine medium, a 0.5-ml aliquot of each culture well was taken and tested in an EIA for the presence of mouse immunoglobulins directed against HAC-PG-Core(ABC). A MonoclonKaSelurAlafntaaottinebody culture supernatants (100-200 &w' ell) were added and incubated for 90 min at 37 "C.The plates were washed three times with PBS-azide. Enzyme-linked second antibody (alkaline phosphatase-conjugated goat anti-mouse K and X light chains) was added (200 pllwell) and incubated. HAC-PG-Core(ABC) was coated on the EIA plate, unreacted sites were blocked with EIA buffer, and the supernatants from the wells of positive clones were incubated as described above. Changes indicated above, and measured the ability of unlabeled BNC proteoglycan antigens to compete with '251-BNC-PG-Core(ABC)for a known dilution of the 1/20/5-D-4 culture medium. 1/20/5-D-4monoclonal IgG1. '251-RC-PG-Core(ABC)3was not recognized by the 1/20/5-D-4 IgGl, showing similar resultstothe EIA analyses which used intact proteoglycan preparations as antigens
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