Enzyme-linked immunosorbent assay analyses of the hyaluronate-binding region and the link protein of proteoglycan aggregate.
An enzyme-linked immunosorbent assay, in combination with an independent inhibition step, was established to quantitate two components of the proteoglycan aggregate, namely link protein and hyaluronate-binding region, at concentrations below 100 ng/ml. The presence of other components of the aggregate in the samples to be tested influenced quantitation in a specific manner. The apparent antigenicity of link protein increased 2-5 times when either purified proteoglycan monomer, purified hyaluronate-binding region, or purified hyaluronate (macromolecular or oligomers) were present in the link protein samples. These findings are interpreted as showing different states of conformation or degree of association of the link protein with other components of aggregate in solution. In separate experiments, a 2-4-fold increase in the apparent antigenicity of purified hyaluronate-binding region was observed when hyaluronate molecules with at least 20 disaccharides were present in the samples. Co-incubation of the hyaluronate-binding region or proteoglycan monomer with either purified link protein or with smaller hyaluronate oligomers did not change its antigenicity in the assay. However, when hyaluronate oligomers with 8 disaccharides were included in a mixture of macromolecular hyaluronate with hyaluronate-binding region, the increase in apparent antigenicity was blocked. The results illustrate the inherent difficulties in using the enzyme-linked immunosorbent assay for the quantitation of link protein or proteoglycan monomers in samples where these macromolecules can associate with themselves or other components of proteoglycan aggregates.
D000818:Animals; D002813:Chondrosarcoma / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid / Q000378:metabolism*; D007700:Kinetics; D011506:Proteins / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D012513:Sarcoma, Experimental / Q000378:metabolism; D000099249:Proteoglycan Link Protein
D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein