Rapid and sensitive immunoassay for albumin determination in urine

269

Clinica Chimica Acta, 149 (1985) 269-274 Elsevier

CCA 03205

Brief technical

note

Rapid and sensitive immunoassay for albumin determination in urine Aimo Harmoinen,

Ilpo Ala-Houhala

and Pauli Vuorinen

Tampere University Central Hospital, SF- 33520 Tampere (Finland) (Received

November

22nd, 1984; revision March 4th, 1985)

Key words: Albuminuria; Diabetic nephropathy; Kidney function; Nephelometry; Non-isotopic immunoassay

Introduction

Slightly elevated urinary excretion of albumin has been shown to be predictive of clinical nephropathy in insulin-dependent diabetics [l]. Such a slight increase in albumin excretion is not usually measurable by the tests ordinarily used to detect proteinuria, and it has been called microalbuminuria [1,2]. Because this increase seems to be reversible [3], the need of a simple, but a method sensitive enough for albumin determination in urine is obvious. The most current method for sensitive albumin measurement is radioimmunoassays [4-71 involving ‘251-labelled albumin. The disadvantages of using this reagent include its short shelf life, cumbersome labelling procedure and long incubation time. Non-isotopic immunoassays for albumin determination have also been developed [8-131, but some of them are not sensitive enough to detect low albumin concentrations [8,9], and some of the others need complicated reagents [ll-131. Here we describe a simple and rapid nephelometric method for albumin determination sensitive enough for measurements of albumin concentration in normal urine. We have also determined reference values for albumin excretion and examined if urine collection could be replaced by the albumin/creatinine ratio of a single sample. Materials and methods

Analytical methods LC-Partigen@-albumin immunodiffusion plates (Behringwerke AG, Marburg, FRG) and Pharmacia Albumin RIA (Pharmacia, Uppsala, Sweden) were used according to the manufacturers’ recommendations. PEG-buffer, containing polyethylene glycol (PEG 6000, Merck, Serva, FRG) 50 g/l, NaCl 0.15 mol/l, phosphate 0.01 mol/l and NaN, 0.015 mol/l, pH 7.4. Standard, pooled human serum, the concentration of which was determined against Seronorm protein (Nyegaard & Co., Oslo, Norway, albumin concentration 45 g/l) and Protein Standard Set (Orion

0009-8981/85/$03.30

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

270

ALBUMIN

Fig. 1. A typical

standard

(mg;l

)

curve. The assay range from 0.75-90

mg/l.

Both axes on a log scale.

Diagnostica, Helsinki, Finland). For nephelometric determination, the standard was diluted 1: 400; 1: 800; 1: 1600; 1: 6400; 1: 25 600; and 1: 51200. Antiserum, swine antihuman albumin serum (Orion Diagnostica, Helsinki, Finland). For nephelometric determination the antiserum was prediluted with PEG-buffer 1 : 5. With this dilution the antigen excess will not become apparent until the Albustix@-reaction (Ames Division, Miles Labs., Elkhart, IN, USA) is strongly positive. The creatinine determinations by the Jaffe reaction were carried out on an Auto-Analyzer (Technicon Comp, Terrytown, NY, USA). Nephelometer, Transcon 102 FN fluoronephelometer (Orion Analytica, Espoo, Finland) is a microprocessor-controlled semi-automated discrete batch analyzer, which makes rapid nephelometric and fluorometric measurements possible. The functions of the instrument in measurement and calculation of results are controlled by a microprocessor. The settings of the instrument for albumin determination: wavelength 610 nm, anode voltage 650 V, aperture 4. The nephelometric albumin determination was as follows: the urine samples were centrifuged and prechecked with Albustix and, if necessary, diluted to correspond to the assay range (see Fig. 1). One hundred microliters of the standards and the samples in duplicates, and 800 ~1 of PEG-buffer were pipetted with an automated dilutor (Dilutrend@, Clinicon Mannheim, Mannheim, FRG) into cuvettes and the blank values were measured. Then 100 ~1 of the diluted antiserum were added rapidly with Eppendorf Multipipette (Eppendorf, Hamburg, FRG), the cuvettes were mixed gently and after an incubation time of 15 min, the cuvettes were measured again. The nephelometer subtracted the blank values and calculated the results automatically. Patients and reference material

Forty-one insulin dependent diabetic patients with intermittent proteinuria (Albustix) were studied. Twenty of them were female, aged 17-46 yr, and 21 were male, aged 19-34 yr. The mean duration of their diabetes was 18.1 yr f 5.9 (SD) in females and 17.7 yr f 6.0 (SD) in males. Five females and six males had retinopathy. The control group was composed of 42 healthy volunteers, 23 females, aged 25-63 yr and 19 males, aged 26-43 yr, who were devoid of renal diseases.

211 TABLE

I

Precision

of the proposed

method SD

Mean

Method

n

(w/l)

Within-day 2.64 15.23 12.39

0.05 0.20 1.32

1.82 1.30 1.83

15 15 15

2.41 14.40 69.20

0.10 0.30 1.49

4.12 2.07 2.16

8 8 8

Day-to-day

Two 12-h collections of urine, during the day and during the night, were obtained from all subjects. Results

The reproducibility of the method proposed was tested on 15 duplicate determinations of albumin in three urine samples in one run (within-run precision), and on 8 duplicate determinations in the same urine samples during 2 wk (betweenrun precision). The CV ranged from 1.3 to 4.1% (Table I). This nephelometric method was also compared with the radial immunodiffusion method (Fig. 2.) and with the radioimmunoassay (Fig. 3). 100, y= 0.965x

=c F

.2.06

r=0.994 n-51

y =0.965x

50-

*1.59

r-0.991 -

n=40

G t; I:

101

s !i ii z

5-

0 0

i,

RID

,

,

, , , ,,,, 5 10

lmg/l)

I S"', 50 100 RIA

Fig. 2. A comparison of the method proposed with the radial immunodiffusion method. limit of the radial immunodiffusion method is about 25 mg/l. Both axes on log scale. Fig. 3. A comparison

of the proposed

method

with the radioimmunoassay.

ImQ/l)

The sensitivity

Both axes on a log scale.

212 TABLE Reference

II values for urinary

12-h collection,

and albumin/creatinine

ratio

Mean

Ref. values

Range

(mg/mmol)

2.82 0.45

1.12-7.08 0.18-1.12

0.60-7.20 0.13-1.01

(mg/mmol)

3.41 0.53

1.38-8.71 0.15-1.82

1 .oO-9.20 0.40-l .46

6.60

2.60-16.60

2.30-16.70

night

Albumin (mg) Albumin/creatinine 12-h collection,

albumin

day

Albumin (mg) Albumin/creatinine 24-h collection albumin

(mg)

Mean, logarithmic reference material.

mean;

reference

values, mean f 2

SD

range:

range, lowest and highest

values found in

Table II shows the reference values of albumin excretion and albumin/creatinine ratio in 12- and 24-h collections. The excretions of the men and the women did not differ statistically from each other. We had to delete two collections in the control men’s group and two collections in the control women’s group because they did not fulfill the population criteria for Student’s t test (2% discrimination limit). In our diabetic group, the urinary albumin excretion was elevated in 51% of the patients during the day and in 53% of the patients during the night.

The most characteristic feature of the excretion of plasma proteins in urine is the great dispersion of the values and the uneven distribution [14]. A logarithmic distribution seems to be characteristic of the albumin excretion, and, therefore, we have used a logarithmic transformation for the calculation of the reference values. Our values are in good accordance with those derived from other methods [5,6,14-161. Some investigators have suggested that urine collections could be replaced by determining the albumin/creatinine ratio of a single voided urine sample [17,18]. Although we also found a fairly good correlation between this ratio and albumin excretion (r = 0.82-0.97), we cannot recommend the use of this ratio. Firstly, at low albumin excretions, the ratio gives misleading results, e.g. in the night urine collections of our diabetic group, the ratio was above the upper limit reference in eight cases (20%) when the albumin excretion was normal, and normal in two cases (5%) when the albumin excretion was elevated. On the other hand, to get some benefit from the use of this ratio, random urine samples should be used, and then the correlation is unacceptable [19]. At most, this ratio can be used as a semiquantitative screening test, if the collection has not been adequate for some reason.

213

Diabetic patients with normal urinary albumin excretion at rest may excrete increased amounts of albumin during exercise [20,21]. Hence, values obtained after exercise may be a more sensitive measure of abnormal renal handling of albumin [20]. This must, however, be examined in well-controlled circumstances [20-241, and, therefore, we recommend a 12-h urine collection in the night for routine use. Besides its simplicity, sensitivity and rapidity the method described above is also cheap, about 1.0 DM in duplicate, and thus it is very suitable for routine determinations always when microalbuminuria is examined. Acknowledgements

We thank Mrs. Tuulikki Harmoinen for her excellent technical assistance and all the staff members who took part in the urine collections. RIA determinations were done in Tutkimuslaitos Elmeri. The Pharmacia Albumin RIA kit was a kind gift from Oy Star Ab. References A, Mahmud U, Keen, H. Microalbuminuria as 1 Viberti GC, Hill RD, Jarret RJ, Argyropoulos predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet 1982; 1: 1430-1432. predicts clinical proteinuria and early mortality in maturity-onset 2 Mogensen CE. Microalbuminuria diabetes. N Eng J Med 1984; 310: 356-360. 3 Mogensen CE. Renal function changes in diabetes. Diabetes 1976; 25: 872-879. C. An immunoassay for urinary albumin at low concentrations. Lancet 1963; ii: 4 Keen H, Chlouverakis 913-916. HJG. Radioimmunoassay for urinary albumin using a single 5 Miles DW, Mogensen CE, Gundersen antibody. Stand J Clin Lab Invest 1970; 26: 5-11. for urinary albumin. Clin Chem 1978; 6 Woo J, Floyd M, Cannon DL, Kahan B. Radioimmunoassay 24: 1464-1467. New techniques for the study of proteinuria. 7 Zager RA. Lysozyme and albumin radioimmunoassays. Invest Urol 1980; 17: 526-528. AO, Heremans SJ. Immunochemical quantitation of antigens by single radial 8 Mancini G, Garbonara immunodiffusion. Immunochemistry 1965; 2: 235-254. of albumin and immunoglobulin G in urine. Clin Chem 1982; 28: 9 Teppo A-M. Immunoturbidometry 1359-1361. L, Skaarup P. Evaluation of quantitative methods for determination of protein 10 Borg N. Hemmingsen in urine. Clin Chim Acta 1975; 64: 247-252. 11 Harper JR, Mahmoudi N. Orengo A. Protein A-bearing Staphylococcus aweus as the solid phase in an enzyme immunoassays and its application to determination of urinary albumin. Clin Chem 1982; 28: 2378-2382. 12 Bernard A, Lauwerys R. Latex immunoassay of urinary albumin. J Clin Chem Clin Biochem 1983; 21: 25-30. 13 Fielding BA, Price DA, Houlton CA. Enzyme immunoassay for urinary albumin. Clin Chem 1983; 29: 355-357. 14 Hemmingsen L, Skaarup P. The 24-hour excretion of plasma proteins in the urine of apparently healthy subjects. Stand J Clin Lab Invest 1975; 35: 347-353. 15 Berggard I, Risinger C. Quantitative immunological determination of albumin in normal human urine. Acta Sot Med Uppsal 1961; 66: 217-221. 16 Poortsman J, Jeanloz RW. Quantitative immunological determination of 12 plasma proteins excreted in human urine collected before and after exercise. J Clin Invest 1968; 47: 386-393.

214 17 Ginsberg JM, Chang BSk, Matarese RA, Carella S. Use of single voided urine samples to estimate quantitative proteinuria. N Engl J Med 1983; 309: 1543-1556. 18 Shaw AB, Risdon P, Lewis-Jackson JD. Protein creatinine index and Albustix in assessment of proteinuria. Br Med J 1983; 287: 929-932. 19 Wilkin T, Mohamed A, Gatling W, Rowe D. Protein creatinine index and Albustix in assessment of proteinuria. Letter. Br Med J 1983; 287: 1883. 20 Mogensen CE, Vittinghus E. Urinary albumin excretion during exercise in juvenile diabetics. Stand J Clin Lab Invest 1975; 35: 295-300. 21 Viberti GC, Jarret RJ, McGartney M, Keen H. Increased glomerular permeability to albumin induced by exercise in diabetic subjects. Diabetologia 1978; 14: 293-300. 22 Viberti GC, Pickup JC, Bilous RW, Keen H, Macintosh D. Correction of exercise-induced microalbuminuria in insulin-dependent diabetics after 3 weeks of subcutaneous insulin infusion. Diabetes 1981; 30: 818-822. 23 Mohamed A, Wilkin T. Leatherdale BA, Rowe D. Response of urinary albumin to submaximal exercise in newly diagnosed non-insulin dependent diabetes. Br Med J 1984; 288: 1342-1343. 24 Mogensen CE. Urinary albumin excretion in early and long-term juvenile diabetes. Stand J Clin Lab Invest 1971: 28: 183-193.