Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide ‘maps’ prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.

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