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Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

ABSTRACT: Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

SUBMITTER: Hunt JS

PROVIDER: S-EPMC1144927 | biostudies-other | 1985 May

REPOSITORIES: biostudies-other

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Effects of freezing on the estimated amounts of Tamm--Horsfall glycoprotein in urine, as determined by radioimmunoassay.

Project description:Freeze-drying or freezing of salt-free solutions of human Tamm--Horsfall glycoprotein appeared to lead to changes in the structure of the latter, changes that increased its ability to bind with antibody raised, in rabbits, against it. This alteration in avidity of the glycoprotein was observed irrespective of whether antiserum was raised against freeze-dried or non-frozen antigen. The implications of this finding for the radioimmunoassay of the glycoprotein in urine samples were studied. Appropriate treatment for urine samples, before assay, was devised. The amount of Tamm--Horsfall glycoprotein excreted was shown to range from 30 to 138 mg in normal males and 43 to 126 mg in normal females per 24 h.

| S-EPMC1162033 | biostudies-other

The development of a radioimmunoassay for Tamm--Horsfall glycoprotein in serum.

Project description:The Tamm-Horsfall glycoprotein prepared by salt precipitation from urine was found to comprise a heterogeneous collection of aggregates. These could be disaggregated with 8m-urea, following which chromatography on a column of Bio-Gel A.15m yielded a homogeneous glycoprotein of mol.wt. 73000 together with several unidentified impurities. Gel filtration of normal plasma showed the glycoprotein to exist predominantly in a form that is eluted identically with the purified preparation. In one case, material of higher molecular weight was also detected. The purified glycoprotein was used to develop a rapid specific radioimmunoassay for its measurement in human serum or plasma by the use of the Tamm-Horsfall glycoprotein, labelled with (125)I by the chloramine-t method as the tracer, an antiserum raised in rabbits, and separation of the bound and free fractions by a second antibody covalently linked to magnetizable particles. Parallelism was demonstrated between the standard preparation and samples. Recovery of added standard to serum varied between 99 and 109%. Total assay time was less than 4h with an intra-assay and inter-assay coefficient of variation of less than 10%. There were no significant differences in the ranges covered with regard to either age or sex, and no circadian rhythm was observed in normal subjects. A physiological range of 70-540ng/ml was established based on serum samples from 95 subjects with normal renal function, as defined by their serum creatinine and urea concentrations. No Tamm-Horsfall glycoprotein was detected in the serum of six anephric patients.

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Subunits of Tamm-Horsfall glycoprotein.

Project description:1. The effect of 6m-guanidine hydrochloride on the urinary glycoprotein described by Tamm & Horsfall (1952) is to produce a homogeneous subunit of molecular weight approx. 100000. 2. Complete reduction of the disulphide bonds of this subunit does not decrease the molecular weight, suggesting that all disulphide bonds are intrachain. 3. Comparison of sedimentation and viscometric behaviour of unreduced and reduced material in 6m-guanidine hydrochloride is consistent with reduction causing an opening-up of intrachain disulphide bonds to give a more asymmetric molecule.

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An improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein. Investigation and resolution of factors affecting its quantification.

Project description:A rapid, specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. A dilution of 1 in 100 was chosen for routine assay. Whole urine was centrifuged and the dissolved precipitate and supernatant assayed to quantify the proportion of TH glycoprotein of TH glycoprotein initially present in highly aggregated form. This correlated positively and significantly with increasing osmolality, decreasing pH and increasing TH glycoprotein concentration. When the urine was diluted 1 in 100 in water, no TH glycoprotein was precipitated by centrifugation and the measured concentrations were unaffected by alterations of urine pH or calcium concentration or by addition of sodium dodecyl sulphate. Parallelism was demonstrated between the diluted samples and the disaggregated standard preparation. Recovery of added standard to diluted urine varied between 96 and 114%. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4 degrees C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22--56 mg/24 h, based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance.

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Tamm-Horsfall urinary glycoprotein. The subunit structure.

Project description:1. Subunit molecular weights of 76000-82000 were obtained for native and alkylated Tamm-Horsfall glycoprotein by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. 2. A further estimate of the subunit molecular weight of 79000+/-4000 was obtained by disc gel electrophoresis in sodium dodecyl sulphate. 3. A minimum value of the chemical molecular weight of 79000+/-6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 4. Similar values were obtained for the subunit molecular weight of Tamm-Horsfall glycoprotein from patients with cystic fibrosis. 5. On ultracentrifugation both in 1.0% sodium dodecyl sulphate and in 70% formic acid, Tamm-Horsfall glycoprotein sedimented as a single component, slightly faster than serum albumin. 6. On reduction of the disulphide bonds the same subunit molecular weight was obtained, which suggested that these bonds are intrachain.

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Tamm-Horsfall urinary glycoprotein. The chemical composition.

Project description:1. A revised amino acid and carbohydrate composition of human Tamm-Horsfall glycoprotein is presented. 2. No significant differences were obtained in the amino acid composition of Tamm-Horsfall glycoprotein isolated from patients with cystic fibrosis. 3. The glycoprotein was shown to possess a high half-cystine content of 1 per 11-12 amino acid residues, which has been confirmed by performic acid oxidation and S-alkylation with iodoacetate and iodoacetamide. No thiol groups were detected in the glycoprotein. 4. Treatment of the glycoprotein with 0.5m-sodium hydroxide at 4 degrees C for 2 days did not release heterosaccharide material, which suggests that the predominant carbohydrate-protein linkages present are not of the O-glycosidic type. 5. No N-terminal amino acid was detected in the glycoprotein.

| S-EPMC1179613 | biostudies-other

Properties of a glycopeptide isolated from human Tamm-Horsfall glycoprotein. Interaction with leucoagglutinin and anti-(human Tamm-Horsfall glycoprotein) antibodies.

Project description:A sialylated glycopeptide isolated after Pronase digestion of human Tamm-Horsfall glycoprotein behaves as a powerful monovalent hapten in the precipitin reaction between human Tamm-Horsfall glycoprotein and leucoagglutinin, but fails to inhibit the interaction of the glycoprotein with rabbit anti-(human Tamm-Horsfall glycoprotein) antibodies. The glycopeptide is much less active than the intact glycoprotein as an inhibitor of lymphocyte transformation induced by leucoagglutinin.

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Tamm-Horsfall glycoprotein engages human Siglec-9 to modulate neutrophil activation in the urinary tract.

Project description:Urinary tract infections are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic Escherichia coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared with wild-type mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

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Tamm-Horsfall glycoprotein interacts with renal outer medullary potassium channel ROMK2 and regulates its function.

Project description:Tamm-Horsfall glycoprotein (THGP) or Uromodulin is a membrane protein exclusively expressed along the thick ascending limb (TAL) and early distal convoluted tubule (DCT) of the nephron. Mutations in the THGP encoding gene result in Familial Juvenile Hyperuricemic Nephropathy (FJHN), Medullary Cystic Kidney Disease type 2 (MCKD-2), and Glomerulocystic Kidney Disease (GCKD). The physicochemical and biological properties of THGP have been studied extensively, but its physiological function in the TAL remains obscure. We performed yeast two-hybrid screening employing a human kidney cDNA library and identified THGP as a potential interaction partner of the renal outer medullary potassium channel (ROMK2), a key player in the process of salt reabsorption along the TAL. Functional analysis by electrophysiological techniques in Xenopus oocytes showed a strong increase in ROMK current amplitudes when co-expressed with THGP. The effect of THGP was specific for ROMK2 and did not influence current amplitudes upon co-expression with Kir2.x, inward rectifier potassium channels related to ROMK. Single channel conductance and open probability of ROMK2 were not altered by co-expression of THGP, which instead increased surface expression of ROMK2 as determined by patch clamp analysis and luminometric surface quantification, respectively. Despite preserved interaction with ROMK2, disease-causing THGP mutants failed to increase its current amplitude and surface expression. THGP(-/-) mice exhibited increased ROMK accumulation in intracellular vesicular compartments when compared with WT animals. Therefore, THGP modulation of ROMK function confers a new role of THGP on renal ion transport and may contribute to salt wasting observed in FJHN/MCKD-2/GCKD patients.

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Tamm-Horsfall protein and urinary exosome isolation.

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