We describe an enzyme-linked immunosorbent assay (ELISA) for urinary albumin. It requires only commercially available reagents, can detect as little as 16 micrograms of albumin per liter, and analytical recovery ranges from 92 to 116%. The assay is simple, rapid, and inexpensive. Albumin excretion was 6.2 (SD 4.1) mg/24 h in healthy subjects (n = 40), 14.7 (SD 7.2) mg/24 h in albumin-test-strip-negative Type I diabetics (n = 11), and 19.7 (SD 16.2) mg/24 h in patients with essential hypertension (n = 12)

Jesse, U.

Krämer, Bernhard K.

Ress, K. M.

Risler, T.

Schmülling, R. M.

English

University of Regensburg Publication Server

CLIN. CHEM. 33/4, 609-611 (1987)CLINICAL CHEMISTRY, Vol. 33, No. 4, 1987 609Enzyme-Linked Immunosorbent Assay for Unnary Albumin at Low ConcentrationsBernhard K. Kramer,1 Ute Jesse,1 Klaudla M. Ress,’ Reinhold-Michael SchmUiling,2 and Teut Risler’We describe an enzyme-linked immunosorbent assay (ELIsA)for urinary albumin. It requires only commercially availablereagents, can detect as little as 16 zg of albumin per liter,and analytical recovery ranges from 92 to 116%. The assayis simple, rapid, and inexpensive. Albumin excretion was 6.2(SD 4.1) mg/24 h in healthy subjects (n = 40), 14.7 (SD 7.2)mg/24 h in albumin-test-strip-negative Type I diabetics (n =11), and 19.7 (SD 16.2) mg/24 h in patients with essentialhypertension (n = 12).Additional Keyphrases: diabetes hypertension referenceintervalSlight (“micro”) albuminuria is thought to predict diabeticnephropathy in patients whose urines are albumin-negativeby Albustix test strips (1-3), but at a stage that can bereversed by sufficient metabolic control (4-6). Once albu-minuria is detectable by the Albustix test, the developmentof diabetic nephropathy usually can no longer be prevented.It is therefore important to have a sensitive, reliable, rapid,and inexpensive test available for urinary albumin. Exactestimation of microalbuminuria is also of special interest incontrol of hypertension and of patients with renal allografts(7,8).Various techniques have been used for measuring mi-croalbuminuria: radial immunodiffusion (9, 10), immuno-electrophoresis (11), radioimmunoassays (12-16), and en-zyme immunoassays (16-21). The main disadvantages ofradioimmunoassays are isotope-related health and safetyhazards and the short shelf-life of reagents.Our aim in this study was to develop a sensitive, simple,and rapid immunoassay for measuring urinary albuminwith use of only commercially available reagents.Materials and MethodsMaterialsReagents. Human serum albumin was obtained fromBehringwerke AG, Marburg, F.R.G. Bovine serum albuminwas obtained from Sigma, Taufkirchen, F.R.G.; casein,gelatin, creatinine, 250 mLIL sulfuric acid, hydrogen perox-ide (300 g/L), and all reagents for buffer solutions weresupplied by Merck, Darmstadt, F.R.G. Tween 20polyoxyethylene (20) sorbitan monolaurate, uric acid, urea,globulins, and the enzyme substrate o-phenylenediaminewere from Serva, Heidelberg, F.R.G.Antisera. Rabbit anti-human albumin antiserum (liquid;cat. no. ORCBO4/05) was obtained from Berhringwerke AG,and peroxidase-labeled goat anti-human albumin antise-rum (lyophilized; cat. no. 3201-034) was from Cooper Bio-medical, Frankfurt, F.R.G.Assay diluent, pH 9.6. Antigen and antibodies are dilutedin 0.2 mol/L carbonate/bicarbonate buffer, prepared by dis-‘Department of Internal Medicine III and’ Department of Inter-nal Medicine 1V,University ofTubingen, Otfried-Muller-Str. 10, D-7400 Tubingen, F.R.G.ReceivedDecember 16, 1986; accepted January 29, 1987.solving 16.72 g of NaHCO3 in 1 L of distilled water, andadjusting to pH 9.6 with a solution of 21.1 g of Na2CO3 in 1L of distilled water.Washing solution. Dissolve 42.71 g of Na2HPO4 and 53.06g of NaCl in 4 L of distilled water, adjust to pH 7.4 with amixture of 5.72 g of KH2PO4 and 6.14 g of NaC1 in 0.7 L ofdistilled water. To 0.5 L of this phosphate-buffered salineadd 2.5 g of casein and 300 L of Tween 20.Substrate solution. To 30 mg of o-phenylenediamine firstadd 100 mL of freshly prepared 0.1 mol/L citrate buffer, pH5.0(20.95 g of trisodium citrate dissolved in 0.7 L of distilledwater, adjusted to pH 5.0 with 6.3 g of citric acid dissolved in0.3 L of distilled water), then add 30 pL of the concentratedhydrogen peroxide solution. Prepare the substrate solutionin, and dispense it from, a brown bottle. Fresh preparation ofsubstrate solution before each run is recommended.Standards. We prepared a stock 1 mg/L solution bydissolving human serum albumin in the assay buffer; westored aliquots at -20 #{176}Cin tubes coated with the above-described washing solution. From this stock solution weprepared standards containing 0.03 to 8 mg per liter, inassay buffer.Equipment. Polystyrene microtiter plates were obtainedfrom Nunc, Wiesbaden, F.R.G. We measured absorbancewith a nliCrO-EuSA autoreader (MR 580; Dynatech, Denken-dorf F.R.G.).SamplesWe collected 24-h urine specimens in polyethylene con-miners that had been washed three successive times withthe washing solution before use. We stored 2-mL aliquots ofurine in tubes coated as described above at -20 #{176}Cfor notlonger than two months before assay. Urine samples werecentrifuged at 3700 x g and diluted 10-fold in assay bufferbefore assay.PatientsUrines from all patients and apparently healthy controlsubjects were negative for albumin by #{176}Combur9-Test”(Boehringer Mannheim GmbH, Mannheim, F.R.G.; detec-tion limit for albumin 60 mgfL), and for leukocytes andnitrite. Serum creatinine concentration was within thenormal reference interval in all subjects, and results ofroutine clinical investigations were also within normallimits. Mean (±SD) age was 39.4 ± 11.2 years (range 23-55,n = 40) for the control subjects, 34.4 ± 10.4 years (range 22-48, n = 11) for Type I diabetics, and 55.0 ±7.6 years (45-66,n = 12) for patients with essential hypertension. For statis-tical evaluation of data we used Student’s t-test.ELISA ProcedureThe ELISA was a solid-phase-binding double-antibody as-say as described by Voller et al. (22). The procedure:1. To sensitize the wells, incubate each well with 150 p.Lof rabbit anti-human albumin (diluted 5000-fold in assaybuffer), keeping the plates covered for 4 h at 4#{176}C.2. After emptying the wells, wash them three times withthe washing solution, 150 pL per well. After the third wash0 standard curveudilution eaperiment002 003 004 007 01 0.20.3 050007 1 45 10mg1LoIbumn(0) and dilution41-50Females (n = 5)6.6(2.0-14.5)Males (n = 5)51-60AlbumInexcretIon, mg/24h, mean(andrange)6.6 5.9 8.9(2.0-21.0) (2.3-9.8) (3.1-11.7)6.3 4.5 7.5 3.5(3.5-10.3) (0.7-9.3) (5.4-9.9) (0.6-9.0)610 CLINICALCHEMISTRY, Vol. 33, No. 4, 1987incubate the plate for 30 mm at 20#{176}Cand then empty it,tapping it while it is inverted on paper towelling. Afterdrying, it is ready to receive urine samples.3. Pipette 100 L of standard solution, sample, or assaybuffer (as blank) into the wells in duplicate and incubate for1.5 h at 20 #{176}C.4. Wash the wells as described in step 2, but without theincubation step.5. To each well add 150 pL of peroxidase-labeled goatanti-human albumin (diluted 5000-fold in assay buffer) andincubate for 1 h at 20#{176}C.6. Wash the wells again as described in step 4.7. Add 150 ML of enzyme-substrate solution to each welland incubate for 20 mm at 20#{176}C.Stop the reaction byadding 50 L of sulfuric acid to each well.8. Measure the absorbance of each well at 450 nm.ResultsStandard curve. The limit of detection (mean ±2 SD ofreadings for 21 albumin-free wells) was 16 g/L, correspond-ing to a sensitivity of 1.6 ng of human serum albumin perwell. The standard curve was linear over the range of 0.06 to8.0 mg/L (Figure 1). A minimum of five standards isrecommended, and standards have to be run with eachplate.Precision. Within-run precision was measured by assay-ing three urine samples eight times in one assay (Table 1).Between-run precision was estimated by assaying foururine samples on 10 consecutive days (Table 1).Accuracy. To check the specificity of the ELISA system, weadded urea, uric acid, creatinine, and a-, -, and y.globulinsto the assay buffer in their greatest physiological concentra-tions and assayed. No cross reactivity was detected.Analytical recovery. Recovery of low, middle, and high10 ObsOrbOnce1 1450nm)05 -08-07-06-05-04 -03-02-0.1 -0-Fig. 1. Standard curve for human albuminin urinecurve (#{149})TabIAlbumIn cone 1. PrecisIon of the Methoden, mg/LSD CV,%MeanWithin-run4.19 0.386 9.21.05 0.053 5.10.10 0.008 7.6Between-run1.01 0.17 16.22.10 0.31 14.93.57 0.34 9.6concentrations of human serum albumin was between 92.3and 116.5% (Table 2). To demonstrate the absence of inter-fering substances we diluted, with assay buffer, urineshaving high, middle, and low concentrations of albumin(38.7, 5.0, 1.6 mgJL). The results of the dilution experimentare superimposable on the standard curve (Figure 1).Albumin in 24-h urines. Using this ELISA, we measuredalbumin excretion in albumin-test-strip-negative Type Idiabetics, in albumin-test-strip-negative patients with es-sential hypertension, and in control subjects (Table 3). Thisexcretion was significantly (p <0.05) higher in the twogroups of patients than in the control subjects. We coulddemonstrate no correlation between urinary albumin excre-tion and age in either the control subjects (Table 4) or thepatients.DiscussionThis enzyme iinmunoassay (double-antibody “sandwich”technique) is simple and rapid to perform; all the reagentsare both commercially available and inexpensive. Inter- andintra-assay variations and analytical recovery were satisfac-tory, although further improvements would probably bepossible if semi-automated techniques were used. The limitof detection, 16 g/L, corresponds to 1.6 ng of albumin perwell, and is sufficiently low to detect above-normal albuminconcentrations (5.0 ng per well in control subjects). ELISA-based urine-albumin assay techniques reported by severalTable 2. Analytical Recovery oAlbumIn, mg/Lf AlbuminRecovery,%Added Measured0.5 0.58 1161.0 1.01 1012.0 1.94 973.0 2.75 92Table 3. Albumin Excretion (mg/24 h) by ControlSubjects and PatientsMean± SD (and range)Controls Type I diabetIcs EssentialhyportenslvesFemales6.6 ± 5.1 14.2 ± 4.8 23.3 ± 17.2(0.7 - 21.0) (9.0 - 20.6) (4.6 - 50.4)n=20 n= 5 n= 8Males5.4 ± 3.1 15.0 ± 9.2 12.6 ± 13.3(0.6 - 10.3) (3.3 - 30.9) (1.7 - 30.6)n=20 n= 6 n= 4Total6.2 ± 4.1 14.7 ± 7.2 19.7 ± 16.2 -(0.6 - 21.0) (3.3 - 30.9) (1.7 - 50.4)n=40 n=11 n=12Table 4. Relation between Age and Albumin Excretionin Control SubjectsAge, y 20-30 31-40CLINICAL CHEMISTRY, Vol. 33, No. 4, 1987 611investigators (16-21) also give satisfactory results, but mostare either relatively time consuming and complicated orrequire reagents that are not commerically available. Usingour immunoassay in clinical studies, the values we found foralbumin in 24-h urine specimens from normal controlsaccorded well with results of other investigators (13, 14,20,23). Above-normal excretions of albumin were demonstrat-ed in patients with essential hypertension and Type Idiabetes, although routine laboratory methods (albumin-test-strip, serum-creatinine) had shown normal results forthese subjects.Others have reported conflicting results regarding age-dependence of albumin excretion in healthy children (24,25); we found no such correlation in adults (Table 4).References1. Mathieson E, Oxenb#{248}llB, Johansen K, SvendsenPA, Deckert T.Incipient nephropathy in Type I (insulin-dependent) diabetes.Dia-betologia 1984;26:406-10.2. MogensenCE, Christensen CK. Predictingdiabeticnephropathyin insulin-dependentpatients. N Engl J Med 1984;311:89-93.3. Viberti (IC, Hill RD. Jarrett M, ArgyropoulosA, Mahmud U,Keen H. Microalbummuria as a predictor ofclinicalnephropathyininsulin-dependent diabetes mellitus. Lancet 1982;ii:1430-2.4. Viberti GC, PickupJC, Jarrett RJ, Keen H. Effectof control ofbloodglucoseonurinary excretionofalbumin and -microglobulinin insulin-dependentdiabetes.N Engl J Med 1979;300:638-41.5. Mohamed A, Wilkin T, Leatherdale BA, Rowe DJF. Responseofurinary albumin to submaximal exercise in newly diagnosednon-insulin dependentdiabetes. Br Med J 1984,288:1342-3.6. MogensenCE. Renal function changesin diabetics. Diabetes1976:25:872-9.7. De Venuto G, Andreotti C, Mattarei M, Pegoretti G. Long-termcaptopril therapy at low dosesreduces albumin excretion in pa-tients with essentialhypertension andnosign ofrenal impairment.J Hypertension 1985;3(Suppl2):S143-5.8. Woo J, Floyd W, Cannon DC. Albumin and -microglobinradioimmunoassays applied to monitoring of renal-allograft func-tion and in differentiating glomerular and tubular diseases.ClinChem 1981;27:709-13.9. Mancini G, Carbonara AO, Heremans JF. Inununochemicalquantitation of antigens by single radial immunodiffusion. Immu-nochemistry 1965;2:235-9.10. Hobbs JR. Simplified radial immunodiffusion. Am CollegePhysicians Broadsheet 1970;68:1-8.11. Laurell CB. Quantitative estimation of proteins by electropho-resis in agarose gel containing antibodies. Anal Biochem1969;15:45-9.12. Keen H, Chlouverakis C. An immunoassay method for urinaryalbumin at low concentrations. Lancet 1963;ii:913-4.13. Miles DW, Morgensen CE, Gundersen HJG. Radioim-munoassay for urinary albumin using a single antibody. Scand JClin Lab Invest 1970;26:5-11.14. WooJ, Floyd M, Cannon DC, Kahan B. Radioimmunoassay forurinary albwnin. Clin Chem 197824:1464-7.15. Berglund AB, Carlsson LA, Dahlquist GO. Solid-phaseRIA-asimple technique for the early detection of albuminuria in diabetes.Diabetic Nephropathy 1984;3:89-91.16. Silver A, Dawnay A, London J, Cattell WR. Immunoassays forlowconcentrations ofalbumin in urine. Cliii Chem 1986;32:1303-6.17. Fielding BA, PriceDA, Houlton CA. Enzyme immunoassay forurinary albumin. Clin Chem 198329:355-7.18. Mohamed A, Wilkin T, Leatherdale B, Davies R A microen-zyme-linked immunosorbent assay for urinary albumin and itscomparison with radioimmunoassay. J Immunol Methods1984;74:17-22.19. Feldt-Rasmussen B, Dinesen B, Deckert M. Enzyme iznmuno-assay:an improveddetermination of urinary albumin in diabeticswith incipientnephropathy. ScandJ Clin Lab Invest 1985;45:539-44.20. Chesham J, Anderton SW, Kingdon CFM. Rapid, competitiveenzyme immunoassay for albumin in urine. Clin Chem1986;32:669-71.21. Townsend JC. A competitive immunoenzymometric assay foralbumin in urine. Clin Chem 1986;32:1372-4.22. Voller A, Bidwell DE, Bartlett A. Enzyme immunoassays indiagnostic medicine. Bull WHO 1976;53:55-65.23. Berggard I, Risinger C. Quantitative immunochemical deter-mination of albumin in normal human urine. Acta Soc Med Ups1961;66:217-29.24. Davies AG, PostlethwaiteRJ, PriceDA, Burn JL, Houlton CA,Fielding BA. Urinary albumin excretion in school children. Arch[ha Child 1984;54:625-30.25. Rowe DJF, Hayward M, Bagga H, Bibbe P. Effect of glycaemiccontrol and duration of diseaseon overnight albumin excretion indiabetic children. Br Med J 1984;289:957-9.

A competitiveimmunoenzymometric assay for albumin in urine.

A microenzyme-linked immunosorbent assay for urinary albumin and its comparison with radioimmunoassay.

Albumin and -microglobin radioimmunoassays appliedto monitoring of renal-allograft function and in differentiating glomerular and tubular diseases.Clin Chem

An immunoassay method forurinary albumin at low concentrations.

competitive enzyme immunoassay for albumin in urine.

Dahlquist GO.Solid-phaseRIA-a simple technique fortheearly detection ofalbuminuria in diabetes.

Effect ofglycaemic control and duration ofdiseaseonovernight albumin excretion in diabetic children.

Effectof control of bloodglucose onurinary excretionofalbumin and -microglobulin in insulin-dependent diabetes.N

Enzyme immunoassay for urinary albumin.

Enzyme immunoassays in diagnostic medicine.

Enzyme iznmunoassay:an improveddetermination of urinary albumin in diabetics with incipientnephropathy.

Immunoassays for lowconcentrations ofalbumin in urine.

Incipient nephropathy in Type I (insulin-dependent)

Inununochemical quantitation of antigens by single radial immunodiffusion.

Long-term captopril therapy at low dosesreduces albumin excretion in patientswith essentialhypertension andnosign ofrenal impairment.

Microalbummuria asa predictor ofclinicalnephropathyin insulin-dependent diabetes mellitus.

Predictingdiabeticnephropathy in insulin-dependentpatients.

Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies.

Quantitative immunochemical determination of albumin in normal human urine. Acta SocMed Ups

Radioimmunoassay for urinary albumin using a single antibody.

Radioimmunoassay for urinary albwnin.

Renal function changesin diabetics.

Response of urinary albumin to submaximal exercise in newly diagnosed noninsulindependentdiabetes.

Simplified radial immunodiffusion.

Urinary albumin excretion in school children.

Enzyme-linked immunosorbent assay for urinary albumin at low concentrations

Enzyme-linked immunosorbent assay for urinary albumin at low concentrations

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