ELISA detection of glycosaminoglycan (GAG)-linked proteoglycans in gingival crevicular fluid

The purpose of this study was to develop an ELISA method to detect chondroitin sulfate isomer-linked proteoglycans in gingival crevicular fluid (GCF), and to elucidate the role played by the glycosaminoglycans (GAGs) in GCF during experimentally-induced periodontitis in dogs. Experimental periodontitis was induced by placement of a silk ligature below the gingival margin of the molar teeth in 3 mongrel dogs. GCF was collected using microcapillary tubes at 0, 7, 21 and 60 days after ligature placement. To compare with GAG in GCF, bovine nasal cartilage proteoglycan monomer, dog's serum and supernatant of homogenized gingival tissue were prepared. Combination of monoclonal antibodies, 3B3 and 9A2, and specific enzymatic digestion made possible the identification of chondroitin 4 sulfate (C4S), chondroitin 6 sulfate (C6S) and dermatan sulfate (DS). The ELISA method detected very small amount of chondroitin sulfate (CS) isomers (15-1000 ng/ml of bovine nasal cartilage proteoglycan). The ELISA value of CS isomers in GCF was lower than that of homogenized gingival tissue but higher than that of the serum. The ELISA value of C4S, C6S and DS, although fluctuating, increased in proportion to the severity of the inflammation.