Human uromucoid

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.

mentioning

confidence: 99%

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

Hunt¹,

McGiven²,

Groufsky³

et al. 1985

“…A small study has shown that urinary TH glycoprotein output is decreased in chronic renal failure but detailed assessment of the value of its measurement has been hampered by the laborious and relatively insensitive immunoassays that have been used to date. Such methods have included varying combinations of sample treatment before assay, e.g., dialysis, gel filtration, ultrafiltration, freezing, incubation with detergents and adjustment of pH (Bichler et al, 1973;Mazzuchi et al, 1974;Wieslander et al, 1977;Akesson et al, 1978;Samuell, 1978;Goodall & Marshall, 1980) in attempts to achieve a uniform state of disaggregation and, by implication, a degree of immunoreactivity dependent solely on concentration.…”

mentioning

confidence: 99%

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

Dawnay¹,

Thornley²,

Cattell³

1982

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.

“…However, Keutel (1965) gave its location as the proximal tubule, and Tsantoulas et al (1974) located a material in liver cell membranes with similar immunological activity. Various methods including radial immunodiffusion (Bichler et al, 1973;Mazzuchi et al, 1974), electroimmunoassay (Bichler et al, 1973;Wieslander et al, 1977;Samuell, 1978) and radioimmunoassay (Grant & Neuberger, 1973) have been used to measure TH glycoprotein excretion in urine, and normal values have been reported as lying between 39 and 78 mg/24 h. However, all encountered technical difficulties in trying to obtain the glycoprotein in a uniform state of disaggregation. 0306-3275/80/030679-09 $1.50/1…”

mentioning

confidence: 99%

The development of a radioimmunoassay for Tamm–Horsfall glycoprotein in serum

Dawnay

McLean

Cattell

1980

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.