DETERMINATION OF CHONDROITIN-6-SULFATE BY A COMPETITIVE ENZYME-IMMUNOASSAY USING A BIOTINYLATED ANTIGEN

A competitive enzyme immunoassay was developed to determine chondroiti n-6-sulphate in body fluids and cell cultures. The assay uses a monocl onal anti-chondroitin-6-sulphate antibody, immobilised to microtitre p lates, and it involves a competitive binding reaction between chondroi tin-6-sulphate in the samples and the biotinylated antigen. This assay enables the quantification of chondroitin-6-sulphate in the low conce ntration range of 16-120 mu g/l. The intra-assay and inter-assay coeff icients of variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/l ph osphate-buffered saline, an albumin solution (40 g/l in phosphate-buff ered saline) and cell culture medium (containing 100 ml/l foetal calf serum). Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal range was 5 5-169 mu g/l. In men the mean value was estimated at 102.2 +/- 37.1 mu g/l and in women at 98.7 +/- 26.4 mu g/l. No age or sex dependence wa s observed. The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 +/- 21.1 mg/kg creatinine (mean +/- standard deviation) a nd in females (n = 10) 53.5 +/- 21.3 mg/kg creatinine. The clearance r ate in men was 0.41 +/- 0.22 ml x min(-1) and in women 0.38 +/- 0.15 m l x min(-1). No sex dependence was found. Furthermore, the enzyme immu noassay was modified to measure the specific incorporation of a radioa ctively labelled precursor (C-14galactosamine) into chondroitin-6-su lphate. This modification rapidly gives information on the cellular gl ycosaminoglycan synthesis in cell culture. Using this method our exper iments with cultivated human chondrocytes showed that the synthesis of chondroitin-6-sulphate decreased in the presence of interleukin-1 alp ha (60.0% less), tumour necrosis factor alpha (64.4%), gamma-interfero n (21.6%) and lipopolysaccharide (53.4%).