Specimen preparation for assay of albumin in urine.
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Albumins↗
Biomedical subjects
A Dawnay
Publications and source records attributed to A Dawnay.
Immunoassays for low concentrations of albumin in urine.
We have developed and validated simple, rapid immunoassays to measure concentrations of albumin in urine ranging from 5 to 200 mg/L. We use antiserum to human albumin, raised in sheep, and we separate the antibody-bound and free fractions of albumin by using a second antiserum, produced in donkeys, against the Fc fragment of sheep IgG. These two antisera can be mixed and added to assay tubes as a single reagent without inhibiting antigen binding. Samples or standards are incubated for at least 30 min with fluorescein-labeled albumin and the premixed antiserum reagent. After brief centrifugation, the supernate is discarded and the precipitate containing the bound fraction is dissolved and its fluorescence measured. Alternatively, 125I-labeled albumin can be used as tracer with all other reagents unchanged; in this case one simply counts the radioactivity of the bound fraction. A reference interval of less than 0.2-2.8 mg/mmol of creatinine was obtained for untimed, daytime, midstream urine specimens from 80 healthy subjects.
Adolescent↗
Human Tamm-Horsfall glycoprotein: urinary and plasma levels in normal subjects and patients with renal disease determined by a fully validated radioimmunoassay.
A recently developed, simple and sensitive radioimmunoassay has been used to examine 24 h excretion and plasma levels of Tamm-Horsfall glycoprotein (THG) in normal subjects, stone formers and patients with stable chronic renal disease. In normal subjects THG excretion ranged from 22 to 66 mg/24 h, with no sex difference and no correlation with creatinine clearance or body surface area. There was no correlation between 24 h THG excretion and urine volume, pH or osmolality, excretion of Na+, K+ or Ca2+ or free-water clearance. There was a small significant correlation between plasma THG concentration and urinary THG excretion. A good correlation was obtained between the THG/creatinine ratio in 24 h and random samples. This made possible the use of random samples to establish a reference range for THG excretion of 0.15-0.50 micrograms/ml of creatinine clearance which did not depend on sex or age. The excretion rate of THG in stone formers was generally within the reference range. It was not significantly different in those who were hypercalciuric or in those taking thiazides. In patients with chronic renal disease there was a good correlation between 24 h THG excretion, plasma THG concentration and creatinine clearance. The range of excretion of THG per ml of creatinine clearance was greater than in normal subjects, independent of the type of renal disease and unrelated to proteinuria. In patients with glomerulonephritis the excretion of THG per ml of creatinine clearance was significantly higher in those with well-preserved tubules compared with those with tubular atrophy.
Adolescent↗
The development of a fluoroimmunoassay for beta-2-microglobulin in serum.
A liquid-phase fluoroimmunoassay has been developed for the measurement of beta-2-microglobulin in undiluted serum or plasma. Protein labelled with fluorescein-isothiocyanate at a molar ratio of 1:2 respectively is employed as a tracer together with an antiserum to beta-2-microglobulin raised in a sheep. Separation is achieved by means of rabbit anti-sheep immunoglobulin G and, following precipitation of the bound fraction by centrifugation, the free fraction together with any potentially interfering factors is removed in the supernatant. Finally the precipitate is dissolved and the fluorescence measured. The assay is simple and rapid in that all reactants, including the second antibody, can be added at the start of the assay and equilibrium is attained within 60 min. The assay is reproducible and sufficiently sensitive to allow measurement of normal serum levels and has a wide range avoiding the need for sample dilution. Results correlated well with those of an established radioimmunoassay.
Fluorescein-5-isothiocyanate↗
A radioimmunoassay for retinol-binding protein in serum and urine.
In this radioimmunoassay for human retinol-binding protein (RBP), sample (serum, diluted as much as 3600-fold; or urine, diluted or undiluted) or calibrant (purified RBP in phosphate buffer containing bovine serum albumin) is incubated with 125I-labeled RBP and rabbit anti-human RBP antiserum for 24 h before separation with second antibody/polyethylene glycol. The assay's sensitivity is 5 micrograms/L, its useful working range 10 to 200 micrograms/L. The between-batch CV is 12.5% at 12 micrograms/L, 7% at 120 micrograms/L. Results correlate well (r = 0.99) with those by radial immunodiffusion. The reference interval for RBP in serum of men is 39-75 mg/L (mean 57), significantly (p less than 0.01) broader than for women (29-66 mg/L; mean 48). The 2.5-97.5 centile interval for RBP in urine is from less than 0.5 to 12.2 micrograms per millimole of creatinine (median 5.8). RBP in urine is unstable below pH 5.0 at 37 degrees C.
Animals↗
A two-site immunoradiometric assay with magnetizable particles: human liver ferritin as a model.
Using human liver ferritin as a model, we have developed a two-site immunoradiometric assay. Sample (or standard) is incubated with excess rabbit antiserum and 125I-labeled specific sheep antibodies directed against different antigenic determinants until equilibrium is reached. Then excess sheep anti-rabbit immunoglobulin G serum, covalently linked to magnetizable particles, is added. The radioactivity bound to these particles is directly proportional to the amount of ferritin present. The assay is reproducible and precise throughout the clinically important range for ferritin in serum. Late addition of second antibodies coupled to magnetizable particles is a novel, simple, rapid, and universally-applicable separation technique for two-site immunoradiometric assays. By this approach, the first immunological reaction can occur in liquid phase, which shortens the reaction time, maximizes sensitivity, and avoids the need for continuous mixing, as compared with methods in which one of the first antibodies is immobilized on a solid phase.
Adult↗
A genetic polymorphism of a human urinary mucin.
We report here a novel genetically determined polymorphism of a human urinary mucin which is demonstrable by the separation technique of SDS polyacrylamide gel electrophoresis, followed by detection with radio-iodinated lectins. The mucins are demonstrable using various lectins but the polymorphism is most easily recognized using peanut agglutinin and we therefore propose to designate this new genetic locus PUM (peanut-reactive urinary mucin). Four common alleles have been identified and an autosomal codominant mode of inheritance has been found in the families studied so far.
Alleles↗
A novel immunoradiometric assay for human liver ferritin.
Rivanol, the cationic salt of an acridine base, has been used as a novel separation procedure in an immunoradiometric assay for human liver ferritin. The separation step is based on the differences in charge and molecular weight between the labelled antibody-ferritin complex and free labelled immunoglobulins. The resultant assay is simple, reproducible and sufficiently sensitive to determine serum concentrations of ferritin.
Antigen-Antibody Complex↗
In vitro segregation of the metanephric nephron.
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Animals↗
The development of a radioimmunoassay for Tamm--Horsfall glycoprotein in serum.
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.
Amino Acids↗
Evaluation of therapy for cast nephropathy: failure of colchicine to alter urinary Tamm Horsfall glycoprotein excretion in normal subjects.
Tamm Horsfall glycoprotein (THG) is a major constituent of renal tubular casts including the light chain casts of myeloma. Animal studies suggest that the anti-inflammatory agent colchicine reduces urinary THG excretion and prevents light chain cast formation. Six normal male subjects were given therapeutic doses of colchicine (0.5 mg twice daily for 6 days) and excretion of THG, albumin, creatinine and N-acetyl glucosaminidase (NAG) was determined. Colchicine therapy had no effect on the urinary excretion of THG, albumin or NAG or on renal function as assessed by creatinine clearance. This suggests that colchicine will not be a useful therapeutic adjunct to the treatment of light-chain nephropathy.
Acetylglucosaminidase↗