A competitive immunoenzymometric assay for albumin in urine

I describe a competitive immunoenzymometric assay for urinary albumin, with use of an immobilized albumin-thyroglobulin conjugate and an affinity-purified antibody. The assay is rapid, easy to perform, has a range of 0.5 to 78 mg/L, and is ideally suited for screening purposes. Higher concentrations may be assayed by further dilution of the urine sample. Results compare favorably with those of a commercially available radioimmunoassay kit.     

Rapid, robust method for measuring low concentrations of albumin in urine

We describe a rapid particle-enhanced turbidimetric immunoassay for albumin in urine. Intra- and interassay CVs were less than 5% and less than 10%, respectively, the detection limit is 2 mg/L, and the working range extends to 200 mg/L. Mean analytical recovery of albumin added to centrifuged urines was 100% (SD 10.6%), and, when results were compared with those by the Pharmacia RIA, the correlation coefficient was 0.99. The working reagents are stable for at least six months; thus this assay is suited for both batch and urgent analysis.     

An albumin dimer in urine

An albumin dimer (approximately 134 000 Da) was present, along with monomeric albumin, in freshly voided urine but not in serum from a 53-year-old man with alcoholic liver disease and chronic renal failure. The dimerization, evidently via disulfide bonds, resulted in a loss of [125I]thyroxin-binding capacity. This suggests that the S--S bridging is at a site different from that previously reported. The dimer was unstable at all storage temperatures studied.     

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.     

Solid-phase enzymoimmunoassay of estrone in saliva

In this solid-phase enzymoimmunoassay for estrone in saliva, microtiter plates are used after extraction of the sample with diethyl ether. No chromatographic step is involved. The detection limit of the assay is 450 fg per well (33 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in saliva were respectively 4.2, 12.7; 5.2, 8.7; and 2.7, 5.8%. Using this assay, we found a highly significant correlation (P less than 0.001) between estrone concentrations in time-matched serum and saliva samples. The analytical procedure is rapid and relatively simple. One person can assay 50-60 saliva samples during a normal working day. We conclude that the assay is very suitable, even in small laboratories, for saliva estrone measurements, which, in facilitating serial sampling, enables dynamic observations of estrone concentrations and ovarian activity to be more easily made.     

Purification of hapten-enzyme conjugates for enzymoimmunoassay

Chromatography on hydroxyapatite, including a potassium phosphate lineargradient elution, was applied to the purification of hapten-enzyme conjugates to be used as immunoenzymatic tracers (namely, progesterone, phenobarbital, diphenylhydantoin coupled to glucose-6-phosphate dehydrogenase). The method effectively removed unreacted enzyme and separated the conjugate classes according to the number of substitutions between enzyme linking groups and hapten derivatives. The adequacy of the Chromatographie procedures in improving the analytical performance of the enzymatic tracer appears to depend on direct interdependence between the degree of substitution and the immunological properties of the conjugates.     

High-sensitivity dye binding assay for albumin in urine.

We developed a dye-binding method for albumin in urine based on bis (3',3"-diiodo4'4"-dihydroxy-5'5"-dinitrophenyl)-3,4,5,6-tetrabr omosulfonphthalein (DIDNTB), a dye that has a higher chemical sensitivity and specificity for albumin when compared to two other commonly used dyes. We prepared urine dipsticks with DIDNTB and certain other compounds to prevent "nonspecific" binding to the dipstick matrix. The detection limit for albumin with DIDNTB as the dye is about 10 mg/L. The extent of dye binding to proteins and other compounds was studied using ultracentrifugation and a selectively permeable membrane that permitted the passage of free but not bound dye; we believe this method is superior to photometric titration. The affinity of the dyes for albumin was found to be pH dependent with stronger binding at pH 1.8 than at pH 7.0. At pH 1.8, DIDNTB had a ca.10-fold greater binding coefficient to albumin when compared to the widely used dyes, tetrabromophenol blue (CI 4430-25-5) or bromophenol blue (CI 115-39-9). We developed a system that minimized nonspecific binding by the dye through the use of polymethyl vinyl ethers and bis-(heptapropylene glycol) carbonate. DIDNTB showed a greater chemical specificity for albumin when compared to most other proteins. The new albumin dipsticks are resistant to many potential interferences at substantial concentrations, making the dipsticks suitable to screen for albuminuria.     

Solid-phase enzymoimmunoassay of estrone in serum

This rapid solid-phase enzymoimmunoassay for estrone in serum or plasma is done on microtiter plates after the serum is extracted with diethyl ether. No chromatographic or centrifugation steps are involved. The detection limit of the assay is 380 fg per well (28 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in plasma were respectively 4.4, 9.3; 2.3, 9.1; and 2.0, 6.3 percent. There was a good correlation (correlation coefficient 0.95) between estrone concentrations measured with this assay and with a commercial radioimmunoassay. The analytical procedure is simple, and one person can assay 80 serum samples per working day. We conclude that the assay is very suitable for serum estrone measurements and is more convenient than published radioimmunoassays.     

Rapid, specific assay for plasma cortisol by competitive protein binding.

We describe a modified competitive protein-binding method for assay of plasma cortisol. Plasma samples are deproteinized by dilution with an ethanol/phosphate buffer, followed by heating at 100 degrees C for 2 min. Horse serum is used as the source of transcortin. Free radioactivity is separated from the protein-bound component by partition into liquid scintillation counting within 60 min. The assay has better specificity and precision than a competitive protein-binding assay in which ethanol extraction and Florisil adsorbent are used, and results correlate well with those of a specific radioimmunoassay method.     

重视尿白蛋白检测的临床应用

尿白蛋白检测的临床应用日趋广泛.选取合适的尿白蛋白检测标本的留取方式和判断值是面临的重要问题.本文就这两个问题进行讨论.同时指出,应进行更多的临床应用研究以获得循证医学的支持.     

Direct solid-phase enzymoimmunoassay of testosterone in saliva

This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.     

重视尿白蛋白检测的临床应用

尿白蛋白检测的临床应用日趋广泛.选取合适的尿白蛋白检测标本的留取方式和判断值是面临的重要问题.本文就这两个问题进行讨论.同时指出,应进行更多的临床应用研究以获得循证医学的支持.