Determination of chondroitin-6-sulphate by a competitive enzyme immunoassay using a biotinylated antigen.
A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitin-6-sulphate in the samples and the biotinylated antigen. This assay enables the quantification of chondroitin-6-sulphate in the low concentration range of 16-120 micrograms/l. The intra-assay and inter-assay coefficients 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 phosphate-buffered saline, an albumin solution (40 g/l in phosphate-buffered 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 55-169 micrograms/l. In men the mean value was estimated at 102.2 +/- 37.1 micrograms/l and in women at 98.7 +/- 26.4 micrograms/l. No age or sex dependence was observed. The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 +/- 21.1 mg/kg creatinine (mean +/- standard deviation) and in females (n = 10) 53.5 +/- 21.3 mg/kg creatinine. The clearance rate in men was 0.41 +/- 0.22 ml x min-1 and in women 0.38 +/- 0.15 ml x min-1. No sex dependence was found. Furthermore, the enzyme immunoassay was modified to measure the specific incorporation of a radioactively labelled precursor ([14C]galactosamine) into chondroitin-6-sulphate.(ABSTRACT TRUNCATED AT 250 WORDS)
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
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H
Kähnert
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Brinkmann
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Gässler
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Kleesiek
D000911:Antibodies, Monoclonal / Q000378:metabolism; D000941:Antigens / Q000378:metabolism*; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism*; D002356:Cartilage / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000032:analysis* / Q000097:blood / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D003470:Culture Media / Q000737:chemistry; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008297:Male; D012016:Reference Values; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000941:Antigens; D003470:Culture Media; D001710:Biotin; D002809:Chondroitin Sulfates