An ultrasensitive, nonisotopic immunoassay for hyaluronan using the streptavidin-biotin system.
A time-resolved fluoroimmunoassay for measuring hyaluronan concentrations in plasma and several biological fluids is described. The solid-phase immunoassay is based on the competition between aggregation of hyaluronan with the cartilage proteoglycan monomer, followed by binding of a monoclonal antibody to keratan sulfate of the proteoglycan and a biotinylated anti-mouse IgG. Fluorescence can be measured by a time-resolved fluorometer after binding of Eu(3+)-labelled streptavidin to the biotinylated IgG. The assay is precise and correlates well (r = 0.986) with the only established radioimmunoassay known. The results show that it is essential to perform a blank run without addition of proteoglycan, as endogenous proteoglycan disturbs the measurement and causes underestimation of plasma hyaluronan. The distinguishing feature of this assay is its extreme sensitivity (< 0.24 microgram/l of plasma). The mean analytical recovery after serial dilutions and addition was 100.3 and 101.3%, the within-assay and between-assay coefficients of variation were 3.67% and 7.02%, respectively.
Rössler
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Department of Physiology, School of Medicine, Karl-Franzens University, Graz, Austria. roessler@balu.kfunigraz.ac.at
D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001710:Biotin; D002356:Cartilage / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000378:metabolism; D015200:Fluoroimmunoassay; D006801:Humans; D006820:Hyaluronic Acid / Q000097:blood*; D007118:Immunoassay / Q000379:methods*; D007632:Keratan Sulfate / Q000378:metabolism; D008107:Liver Diseases / Q000097:blood; D011509:Proteoglycans / Q000097:blood; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D011509:Proteoglycans; D001710:Biotin; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D019809:Streptavidin; D007632:Keratan Sulfate