Scand J Clin Lab Invest 1985; 45: 367-371

(S2-microglobulin in urine and serum determined by ELISA technique

L A R S H E M M I N G S E N * & P O U L S K A A R U P t

*Department of Clinical Chemistry, Central Hospital, N y k ~ b i n g F. and ?Department of Urology, Herlev University Hospital, Denmark

Hemmingsen L , Skaarup P. f32-microglobulin in urine and serum determined by ELISA technique. Scand J Clin Lab Invest 1985; 45: 367-371.

A two-site solid-phase enzyme immunoassay for the determination of p2- microglobulin in serum and urine is established using polystyrene tubes as solid phase. Horseradish peroxidase-conjugated antihuman P2-microglobulin was used as labelled antibody. The technique is based on a general ELISA procedure and can be established without any specific equipment.

Key words: clearance of fi2-microglobulin, reference interval

P. Skaarup, Department of Urology, Herlev University Hospital, 2730 Herlev, Denmark

Evidence for the importance of p2- microglobulin in clinical medicine has accumu- lated considerably, since the protein was iso- lated by BerggArd & Bearn [l].

In urine, P2-microglobulin can be used as a marker of renal tubular dysfunction in a variety of conditions, including Fanconi’s syndrome, Wilson’s disease, Balkan nephropathy [2, 31, treatment with several tubulotoxic agents such as aminoglycosides [4], CIS-platinium [S], or other cancer chemotherapeutic drugs [6], and exposure to cadmium [3]. The urinary concen- tration of P2-microglobulin has also been em- ployed to trace the extent of urinary tract infection [7].

In serum, the ~2-microglobulin concentration is a valuable indicator following reha1 trans- plantation, especially for estimation of the viability of an apparently non-functioning graft in the anuric period [2]. It has also proved helpful in the detection of rejection episodes [8]. Patients with various neoplastic diseases., have increased serum concentration of P2- microglobulin [9], which may be of prognostic value in myelomatosis [lo].

Determination of urinary and serum concen- trations of ~2-microglobulin is almost entirely performed by radioimmunoassay methods as in the studies referred to above. Although other methods have been described (11, 121, the radioimmunoassay methods are still preferred.

The aim of the present study has been to evaluate the enzyme-linked immunosorbent assay (ELISA) to avoid the disadvantages of radioisotope methods, (i.e. short life, cost, environmental problems, assay time) and to make the analysis of ~2-microglobulin possible, when radioisotopes and laboratory facilities are not available. In the meantime an ELISA method has been made available commercially. However, we find it useful to publish our experiments, which show that an ELISA tech- nique can be established without any specific equipment.

Since impaired renal function results in high serum concentration of b2-microglobulin [ 131 with subsequent overflow proteinuria 114) it is mandatory in several clinical situations to deter- mine both the urinary and serum concentration of the P2-microglobulin and express its urinary

367

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368 L. Hemmingsen & P. Skuurup

excretion as protein clearance related to glomerular filtration rate.

M A T E R I A L A N D M E T H O D S

We investigated 45 apparently healthy subjects; 28 were women and 17 men, aged 24-69 yr. They all fulfilled the criteria for a reference material [15]. They had normal renal function, i.e. creatinine clearance of 68 mlimin-213 ml/min, according to age, sex, and body sur- face. None of them had a history of renal disease, hypertension, febrile diseases within the last 3 months or any previous history of inflammatory diseases at all. Urinary examina- tion for erythrocytes, leucocytes, bacteria and casts were entirely normal. The pH of urine from all subjects was above 5.5.

Serum and 24 h urine samples were collected and flz-microglobulin and creatinine (Auto- Analyzer, Technicon Corp.) were determined within a day or two.

The relative clearance of P2-microglobulin was calculated by the formula:

where UP,, and S,,, denote the urinary and serum concentrations of P2-microglobulin, Ucr and SCr the urinary and serum concentrations of creatinine, and V the volume of urine per unit time.

ELISA-technique

A two-site solid-phase enzyme immunoassay was applied for quantification of fi2- microglobulin. The ELISA technique was based on the general ELISA procedure as published by Dakopatts, (February 1984).

Reagents. Solid phase was polystyrene tubes 75 x 12 mm (immunotest-maxisorp tube Cat. no. 44202. NUNC A/S, Denmark).

Coating antibody was rabbit antihuman (32-

microglobulin (code no. A 072, Dakopatts, Denmark).

Labelled antibody was horseradish peroxidase-(HRP-)conjugated rabbit antihu- man fi2-microglobulin (code no. P 174, Dako- patts, Denmark).

Coating buffer was 50 mmol/l carbonatel hydrogen carbonate buffer p H 9.6.

Washing solution was 20 mmoVl phosphate buffer, p H 7.2 (KH2P04, 0.2 g/l; Na2HP04,

2Hz0, 1.15 g/l) 150 mmol/l sodium chloride, 0.2 g/l Tween-20.

Dilution buffer was 0.5'i: bovine albumin in washing solution.

Substrate buffer (pH 5.0) was citric acid, H 2 0 7.3 g/I and Na2HP04, 2H20 11.81 g/l.

OPD-Substrate was 8 mg Ortho-Phenyl- Diamine, HC1 and 5 ~1 30% H 2 0 2 in 15 ml

Stundurd substunce was Phadebas-reference serum (Pharmacia, Sweden), 1.6 mg/l.

Coated tubes were prepared by adding 200 yl of a 1 in 300 dilution of antihumane p2- microglobulin in coating buffer. After over- night incubation at room temperature the tubes were decanted and washed three times with washing solution 4 ml per tube.

H20.

/3*-microglobulin assay

Urine samples were prediluted 1 in 50, serum samples 1 in 400 and standards from 1 in 100 to 1 in 800 in dilution buffer. Two hundred yl of sample/standard were incubated in coated tubes for 30 min. After decanting, the tubes were washed three times with 4 ml wash solution per tube. Two hundred yl of HRP-conjugated antihumane fi2-microglobulin prediluted 1 in 200 with dilution buffer were added to the tubes, and incubated for 30 min. After decant- ing, the tubes were washed three times with 4 ml wash solution per tube. Three hundred p1 OPD-substrate was added to the tubes, and after 20 min incubation in dark, the reaction was stopped by adding 400 yl 1 mol/l H2SO4.

The colour development was read on a LKB-calculating absorptiometer and calculated by means of a power function (y=axb) based on a two-point calibration.

R E S U L T S A N D D I S C U S S I O N

Standard curve

The standard curve is shown in Fig. 1. As can be seen, the dose-response curve is approxi- mately linear' in a double logarithmic co- ordinate system below 16 &I. The standard co-ordinates used for the two-point calibration are 16 ys/l and 2 &I. Below 2 &I the standard curve is extrapolated until the detection limit (0.25 &I) is reached.

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P2-rnicroglobulin by ELlSA technique 369

FIG.

1.0

0.50

g 0.20

e :: 0.10

C 0

17 Q

0.05

0.02

&- microglobulin ( p g / L )

1. Dose-response curve of \3,-mieroglobulin. The standard curve is demonstrated in a double logarithmic coordinate system

Although the standard co-ordinates cover only the upper part of the dose-response curve, readings below the lower standard were calcu- lated by extrapolation of the two-point calibra- tion curve.

Precision

The assay variation was calculated at two levels, the serum level and the urine level. The intra-assay variation was determined by adding 1.3 mg/l of a fi2-microglobulin substance (Cal- biochem Behring Cat. no. 475823) to a serum pool and 0.18 mg/l to a urine pool. The intra-assay variation of serum and urine (n=20) was 7.1% and 3.8% respectively.

The inter-assay variation of /32-microglobulin in serum and urine (n=31) was.8.2% and 4.7% respectively.

Sensitivity

The detection limit was 0.25 pg/1 as c 9 be seen from the dose-response curve. This corres- ponds to 0.01 mg/l related to urine samples and 0.10 mg/l related to serum samples.

Non-specific binding of HRP-conjugated antibody demonstrated an average (n=12) on absorbance of 0.200 f 0.010 (+2 SD).

Recovery

Serum. 1.3 mg/l of fiz-microglobulin was added to ten different serum samples in the interval 0.89-2.30 mgll. The recovery was 1.35k0.20 (SD) mg/l corresponding to 102%k15'X.

Urine. 0.26 mg/l of fiz-microglobulin was added to ten different urine samples in the interval 0.04-0.55 mg/l. The recovery 0.25+0.03 (SD) mg/l corresponding 96% + 14%.

Comparison with radioimmunoassay

For comparison purposes we used

was to

the Phadebas@ fi2-microtest, radioimmunoassay (Pharmacia, Sweden).

There was a linear correlation between the ELISA technique and the RIA technique. The regression coefficients (a and b) and the cor- relation coefficients (r) are illustrated in Fig. 2.

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370 L. Hemmingsen & P. Skaarup

Urine Serum

0.4

0.3

0.2

0.1

a -0.015 b = 1.198 r = 0.991

0.1 0.2 0.3 0.4

6

-.I w E 2.01 - o =-0.013

b - 1.149 t r = 0.943

I . , . ,

2.0 3.0 4.0

R I A ( r n g / l ) R I A ( rng / l )

FIG. 2. fl,-microglobulin in urine and serum determined with ELISA-technique and RIA-technique. The coefficients of the linear regression line (y=a+bx) and the correlation coefficients (r) are indicated in the figure.

Reference interval Plasma and urinary concentration, 24 h

urinary excretion, clearance, and relative clear- ance of (&-microglobulin appear in Table I . The plasma concentration of &-microglobulin was significantly higher in individuals older than 40 yr compared to those younger than 40 yr, whereas no differences were encountered for the other parameters (Mann-Whitney test).

Although the p H of all urine samples was above 5.5 the rather high variation in urinary excretion of P2-microglobulin may be explained by a partial breakdown of the protein by urine samples with p H values between 5.5 and 6.0

[16, 171. This phenomenon, already taking place in the collecting tubules and in the bladder, can only be avoided by supplying patients and controls with sufficient bicarbon- ate to produce urine with p H values above 6.0.

We recommend the ELISA technique for quantitative determination of fi2-microglobulin in serum and urine. The assay is easy to perform and the sensitivity is high. As demon- strated in Fig. 2, the correlation to RIA is almost identical for urine and serum samples. For clinical purposes it is essential to determine Pz-microglobulin in urine and serum simul- taneously.

TABLE I. Concentration in plasma and urine, urinary excretion, clearance, and relative clearance of fi2- microglobulin in 4.5 apparently healthy subjects determined by an ELISA method.

t 4 0 years (n=29) >40 years (n=16) Total (n=4S) Median Median Median

(obs. range) (obs. range) (obs. range)

Plasma concentration (mg/l) 1.32* (0.74-2. 50)

Urinary concentration (rndl) 0.05

Urinary excretion (mg/24h) 0.07

Clearance (pVmin) 41

(0.01-0.10)

(0.01-0.19)

(6103) Q:93

(0.060.75) Relative clearance ( I X lo-’)

*pt0.001

1.92* (1.28-2.44)

0.05 (0.01-0.19)

0.08 (0.03-0.15)

30 ( 1666)

0.29 (0.20-0.83)

1.44 (0 .742 .SO)

0.05 (0.01-0.19)

0.08 (0.0 1-0.19)

33 (6103)

0.32 (0.0&0.83)

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Pz-rnicroglobulin by ELISA technique 371

R E F E R E N C E S

1 Berggird I, Bearn AG. Isolation and properties of a low molecular weight P,-microglobulin occur- ing in human biological fluids. J Biol Chem 1968; 243: 4095-103.

2 Karlsson FA, Wibell L, Evrin PE. /3?- microglobulin in clinical medicine. Scand J Clin Lab Invest 1980; 40. Suppl 154: 27-37.

3 Morgan DB. Assessment of renal tubular function and damage and their clinical significance. Ann Clin Biochem 1982; 19: 307-13.

4 Schentag JJ, Plaut ME. Patterns of urinary P,-microglobulin excretion by patients treated with aminoglycosides. Kidney Int 1980; 17: 654- 61.

5 Cohen AJ, Harberg J , Citrin DL. Measurement of urinary P,-microglobulin in the detection of cis-platin nephrotoxicity. Cancer Treat Rep 1981 ; 65: 1083-5.

6 Fleming JJ, Parapia L, Morgan DB, Child JA. Increased urinary P,-microglobulin after cancer chemotherapy. Cancer Treat Rep 1980; 64: 581-7.

7 Schardijn G, Statius Van Eps LW, Swaak AJG, Kager JCGM, Persijn JP. Urinary P2- microglobulin in upper and lower urinary tract infections. Lancet 1979; i: 805-7.

8 Light JA, Biggers JA, Alijani MR, Smith M, Oddendino K . Serum beta-2-microglobulin: an adjunctive monitoring test in renal transplanta- tion. Proc Clin Dial Transplant Forum 1980; 10: 67-73.

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Bernard AM, Moreau D, Lauwerys R. Compari- son of retinol-binding protein and p2- microglobulin determination in urine for the early detection of tubular proteinuria. Clin Chim Acta 1982; 126: 1-7. Bastable MD. P,-microglobulin in urine: not suitable for assessing renal tubular function. Clin Chem 1983; 29: 9967.

1975; 35, SUPPI 144: 1-44.

9 Teasdale C, Mander AM. Fifield R, Keyser JW, Received 26 July 1984 Newcombe RG, Hughes LE. Serum /3,- Accepted 20 December 1984

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