A new and rapid immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein.
Tamm-Horsfall glycoprotein was purified to apparent homogeneity from human urine by repeated precipitation with 0.58 mol/l NaCl and gel permeation chromatography under dissociating conditions on Bio-Gel A1.5M. The protein was found to consist of a single polypeptide chain of Mr 100,000 under non-reducing conditions and Mr 75,000 under reducing conditions. Antibodies to Tamm-Horsfall glycoprotein were raised in rabbits and subsequently purified by affinity chromatography using the glycoprotein linked to Sepharose 4B. The specificity of these antibodies was confirmed by Western blotting and by indirect immunofluorescence staining of human kidney tissue. The purified antibodies were labelled with 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methyl-9-acridinium carboxylate fluorosulphonate, an acridinium ester, to a specific activity of 6 X 10(5) photon counts/ng of protein, and used to establish a two-site immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein in serum and urine. The bound and the free fractions were separated by a second antibody to Tamm-Horsfall glycoprotein linked to paramagnetic particles. The bound antibodies were quantified by chemiluminescence. The assay had a sensitivity of detection of 2 ng/ml and a working range, as determined by inter-assay precision profiles, of 30-500 ng/ml. The range in serum samples from volunteers with normal renal function (n = 92) was 74-520 ng/ml and the mean 24-h excretion rate in healthy subjects (n = 32) was 70 +/- 26 mg.
Horton
J K
JK
Department of Renal Medicine, University of Wales College of Medicine, Cardiff, UK.
Davies
M
M
Woodhead
J S
JS
Weeks
I
I
D000818:Animals; D002846:Chromatography, Affinity; D005455:Fluorescent Antibody Technique; D006801:Humans; D007120:Immunochemistry; D008163:Luminescent Measurements; D009088:Mucoproteins / Q000097:blood / Q000652:urine*; D011817:Rabbits; D058949:Uromodulin
D009088:Mucoproteins; C548612:UMOD protein, human; D058949:Uromodulin