Clinica Chimica Acta, 155 (1986) 69-16 69

Elsevier

CCA 03402

Measurement of beta-2-microglobulin in serum

and plasma by an enzyme-linked immunosorbent
assay (ELBA)
Ole W. Bjerrum and Henrik S. Birgens
Department of Medicine and Haematologv C, Gentofte Hospital, Niels Andersensvej 65,
University of Copenhagen, DK-2900 Hellerup (Denmark)

(Received June 18th, 1985; revision September 18th, 1985)

Key words: beta-2-Microglobulin; Blood; ELISA

Summary

A simple technique for the measurement of beta-Zmicroglobulin (P2M) in serum

was developed. The method was designed as a sandwich technique using rabbit
anti-human antibodies, employing commercially availabe reagents in an enzyme
linked immunosorbent assay (ELISA). The assay was of high specificity, sensitivity,
accuracy and reproducibility. P2M in serum was strongly correlated with age
( p < O.OOS),but independent of sex. Values in heparin and citrate plasma were
significantly lower than in serum (p < O.OOl),whereas values in serum and EDTA
plasma were similar. Release of P2M from normal blood cells was not observed in
vitro before the test procedure. An excellent correlation between the results obtained
in the ELISA and a RIA was demonstrated (rs = 0.99, p < 0.0001).

Introduction

beta-2-Microglobulin (P2M) is a single polypeptide chain of 100 amino acids

(mol wt = 11.815), first isolated from urine by Berggard and Bear-n [l]. The molecule
consists of the light chain of the histocompatibility antigens, present on the plasma
membrane of most human cells [2,3]. As a result of the continuous turnover of
HLA-ABC antigens, /32M is released from the cell surfaces into the extracellular
compartment [4], and is detectable in various biological fluids and serum, where 98%
can be recovered as the monomer form [5].
fi2M is eliminated by glomerular filtration and subsequently reabsorbed almost
completely in the proximal tubule cells, where it is catabolized [6]. Serum levels of
/?2M (S-fl2M) rise as glomerular filtration rate falls. Correlation formulas between

0009-8981/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

70

S-/32M and S-creatinine have been presented, enabling the calculation of the
expected S-P2M for the corresponding creatinine concentration [7].
The clinical role of P2M measurements in serum and urine has been shown in
nephrology [6,8,9] and in a wide variety of benign and malignant disorders [lo-151.
Raised serum /32M concentrations may occur without a fall in glomerular filtration
rate. Clinical interest in S-/32M has mainly focused on myelomatosis, in which
S-P2M may have important prognostic significance, being correlated with stage and
reflecting response to chemotherapy [16]. Recently, detection of elevated S-/32M in
conjunction with other laboratory tests has been suggested as a useful marker for
early stages of aquired immune deficiency syndrome [17,18].
The vast majority of all previous reports have measured S-P2M by radioim-
munoassay (RIA) [19,20]. An enzyme immunoassay have been described [21], based
on reagents, which implies techniques to purify P2M and to develop antisera.
We now describe a rapid enzyme linked immunosorbent assay (ELISA) in which
all reagents are commercially available for routine determinations of S-,/?2M with
high specificity and sensitivity.

Materials and methods

Blood samples
Serum or plasma were isolated from 5 ml whole blood, using Na-heparinate (0.1
ml 2% w/v), Na-citrate (0.5 ml 3.8% w/v) or lyophilized EDTA (from 0.5 ml 0.17%
w/v) as anticoagulants respectively. The serum or plasma were removed at room
temperature (RT), 18-22C after centrifugation at 1000 X g for 6 min. The time for
removal never exceeded 5 h from sampling. The samples were stored immediately at
- 20C.

Reference interval
Blood samples from 29 males and 38 females, all subjectively healthy, 18-86 yr
old, were obtained and considered to represent a normal material.

Buffered solutions
The following solutions were used. A. Carbonate/hydrogencarbonate buffer, 0.05
mol/l, pH 9.6. B. Phosphate buffer, 0.008 mol/l, pH 7.2, containing 0.5 mol/l
NaCl, 0.003 mol/l KC1 and 1% (v/v) Triton X-100. C. Buffer B, added 1% (w/v)
bovine albumine (Sigma Chemicals, St. Louis, MO, USA). D. Phosphate/citrate
buffer, 0.1 mol/l, pH 5.0. E. Substrate solution: buffer D, added 0.04% (w/v)
l.Zphenyldiamine (Sigma) and 0.006% (v/v) H,Oz. The buffered solutions A and C
were coloured by 20 mg phenol red/l.

Antisera
Rabbit anti-human P2M and rabbit anti-human P2M conjugated with horsera-
dish peroxidase were purchased from Dakopatts (Copenhagen, Denmark).
 71

/32M standard
beta-2 Micro Control Low (Pharmacia Diagnostics, Uppsala, Sweden) containing
1.4 mg P2M/l redistilled water was used in two-fold dilutions from 1 : 25 in buffer
C.

Plates
Nunc I~u~oplat~ (Roskilde, Denmark), containing 96 wells per polystyrene
plate, were used.

ELI&$ technique
All steps were performed at RT and included incubations of 100-1.11portions for
60 min. The plates were washed 3 times for 3 min with buffer B between each step,
unless otherwise stated. Initially, the plates were coated with ~ti-hums &2M
diluted 1: 2000 in buffer A and subsequently washed once. Then followed the
successive binding of (a) buffer C in order to block non-specific binding of protein
to the plates, (b) the standard or test samples diluted in buffer C, (c) the peroxidase-
conjugated anti-human 82M, diluted 1: 4000 in buffer C, and (d) buffer E. Between
the last two steps the wash was repeated four times, followed by an equilibration of
the plates by a single wash with buffer D. The enzymatic reaction was stopped after
45 min in darkness with 100 ~1 2 mol/l H,SO,. The optical density (OD) of the
wells were determined in a spectrometer (Biotek EIA-reader, model 307) at 490 nm.
After subtraction of the non-specific background density, obtained by o~tting /32M
during incubations (usually < O.OZS),the /32M concentrations in the test samples
were calculated from the standard curve by a computer program, employing a
quadratic regression analysis by the least square method.

Samples (3 ml) of pooled serum or EDTA plasma from 7 he~thy subjects were
filtrated on a Sephadex G-200 column (Pharmacia), 2 x 95 cm, at 4*C, with a flow
rate of 10 ml/h and fraction volumes of 2 ml. The column was equilibrated with
0.05 mol/l Tris-HCl buffer, pH 8.0, containing 0.1 mol/l NaCl and 0.01 mol/l
EDTA. The column was calibrated with cytochrome e (Sigma). The elution position
of cytochrome c was determined by reading the OD at 280 nm. The position of
activity in the fractions was determined using the ELISA method.

RIA
Sera from 38 patients with myelomatosis or other malignant blood disorders were
measured by RIA (Pharmacia /32micro RIA), and compared to results of measure-
ment by the ELISA method.

Results

The standard curve produced with pure /32M is shown in Fig. 1, which also
demonstrates almost complete parallelism between the standard curve and serum
dilutions, indicating lack of interference from other serum proteins in the assay.
72

0b0 nm

i 5 lb S-B2rrixlO.3 m9/1

Fig. 1. The standard curve and serial dilutions of sera. Squares represent the standard curve, diluted from
1: 100 to 1: 3 200. Open circles represent a normal serum sample, diluted from 1: 200 to 1: 1600. Closed
circles represent a normal serum sample, diluted from 1: 100 to 1: 1600. The triangles represent a serum
sample from a patient with myelomatosis, diluted from 1: 500 to 1: 4000. Test samples were diluted from
1: 300 routinely.

Identical results were obtained with EDTA plasma (data not shown). Dilutions of
test samples by a factor 300 or more were applied routinely, whereby measurements
were performed on the lineary part of the standard curve (Fig. 1). Furthermore,
supporting the specificity of the assay, was the finding that specific rabbit anti-hu-
man /32M added in excess to serum samples 1 h before incubated on the plates,
completely abolished the activity. Finally, ELISA analysis of the eluents obtained by
gelfiltration showed that the activity in serum and EDTA plasma was restricted to a
low molecular weight fraction, slightly less than that of cytochrome c (mol wt
13 000).
The SD for the OD of the standard curve, determined in 25 consecutive standard
curves, was 7% for the dilution 1 : 1000, increasing to 19% for the dilution 1 : 3 200.
The detection limit, defined as the lowest concentration of fi2M that can be
distinguished from zero, was 440 rig/l (Fig. l), since the difference in OD for this
standard concentration and the non-specific background was significant (Students t
test, p < 0.001, n = 27).
The accuracy of the assay was examined by addition of 40 ng pure j32M to 20 ~1
normal serum or EDTA plasma. In these experiments the recovery was 94% in serum
(mean of 5 tests: 84, 92, 96, 97, 101%) and 95% in EDTA plasma (3 tests: 94, 94,
97%). Likewise, addition of 100, 200 or 500 ng pure P2M to 20 ~1 serum from
patients with myelomatosis, who had S-j32M concentrations of 7, 11 and 23 mg/l,
respectively, showed the recovery of 94% (mean of 3 tests: 96, 90, 96%).
The reproducibility was determined in both normal serum and EDTA plasma.
The interday-variation coefficient was 8 and lo%, respectively (serum: 1.98 k 0.16
mg/l; EDTA plasma: 1.63 f 0.16 mg/l, n = 10 in both cases). The intra-day
variation coefficient was 3% in serum (2.00 k 0.06 mg/l, n = 16) and 2% in EDTA
plasma (1.54 + 0.03 mg/l, n = 16).
The duration of storage of whole blood at RT before separation of serum or
plasma from cells did not influence the concentration of j32M for up to 24 h.
 13

TABLE I

S-fi2M in healthy, adult subjects

Age No. of Median Range

(yr) subjects S+2M
Low High
(male/female) (m8/1)
m8/1 m8/1
18-29 19 (9/10) 1.39 1.16 2.66
30-50 36 (17/19) 1.60 1.05 2.56
51-86 12 (3/9) 1.97 1.44 3.41

18-86 67 (29/38) 1.60 1.05 3.41

No differences were demonstrated when portions (n = 30) of the same serum or

plasma had been stored in plastic tubes or polypropylene or polystyrene versus glass
tubes.

fl2M in healthy persons

The results of measurement in sera from 67 healthy persons are given in Table I.
The 0.05 fractile/median/0.95 fractile of S-P2M for this population was
1.19/1.60/2.62 mg/l. The S-P2M was positively correlated with age (Spearman
rank correlation coefficient, r, = 0.35, p < 0.005). The serum concentrations of P2M
in 26 males and 29 females, all between 18 and 50 yr of age, were.not significantly
different (Mann-Whitney rank sum test, p > 0.1).
Simultaneous determination of the fi2M concentration in serum and plasma,
performed in 54 control persons, 18-63 yr old, yielded the results presented in Fig.
2. The concentration of P2M was significantly lower in heparin and citrate plasma

02m
3.00 mgll

e-_-_
Serum CilWh Hcpin Edla

Fig. 2. Distribution of concentrations of 82M obtained by simultaneous measurement in serum and

plasma. Samples from 54 healthy adults, 18-63 yr old. ~ indicates median value.
74
mg 82 m/i
, RIA
1
24-
*
20 -

16 - /

8-

4-

ELISA
,
t

4 8 12 16 20 24 mgf32mll

Fig. 3. Comparison of RIA and the ELISA method. Sera from 38 patients with haematologic disorders,
measured by a RIA and the presented ELISA technique. The linear regression line was y = 0.89x -0.11,
Ys= 0.99, p < o.ooo1.

compared to serum (Wilcoxon signed rank test, p -CO.OOl), whereas no difference

was found comparing serum and EDTA plasma.

Comparison with RIA

The range of /32M levels in the 38 test sera was from below 1 mg/l to above 20
mg/l. The results of measurement by the ELISA and the RIA are presented in Fig. 3
and showed a highly significant correlation between the two methods.

Discussion

This study describes an ELBA technique for j32M measmement in serum. It has
a high specificity and sensitivity, it is easily performed and all reagents are commer-
cially available. The assays reproducibility is within acceptable limits. All data
characterizing the ELBA are compatible with those of the RIA [19,20], and the
results of measurement by the ELISA correlates with those obtained by RIA (Fig.
3). In contrast to work with radioactive material, the ELISA imposes no special
demands for staff, laboratories and equipment.
Interference by other plasma proteins was not detected (Fig. 1). Likewise, no
immunological cross reactivity have been reported with known proteins.
The measured /32M concentration was independent of, whether it had been
stored in polypropylene, polystyrene or glass tubes. The lack of difference in binding
affinity to test tubes has not been reported before.
The results obtained for the S-P2M in normals confined a strong age-correlation
{Table I), independent of sex 17,221.The normal values for S-/32&f measured by the
 75

ELISA (Table I) are compatible with the normal concentrations measured by RIA
P21*
Our results for the P2M concentration in normal subjects revealed, although in
absolute values limited, significantly lower concentration in heparin and citrate
plasma compared with serum or EDTA plasma (Fig. 2). Hence, the difference is
probably not due to a simple coagulation phenomenon, which has been reported to
affect the measurement of for instance lactoferrin 1231.The divergence might reflect
a systemic, non-specific effect contributed by heparin and citrate on P2M. Both
EDTA plasma, which contains very small amounts of /32M complexed bound to
HLA-ABC antigens [24], and serum are applicable in the ELISA. Preliminary results
in our laboratory indicate that this ELISA also may be utilized for the measurement
of /32M in cerebrospinal fluid. In this context, promising results have been reported
for the detection and monitoring of patients with CNS involvement in malignant
lymphoma [25].
The time course from blood sampling to separation of cells from normal serum or
plasma did not influence the concentration of fi2M for up to 24 h, as reported for
other cellular proteins [23]. This detail for /32M measurement has not been reported
previously.

We want to thank Miss Eva Gaarsdal Holst for skillful technical assistance and E
Kjarsgaard, MA, for the design of the computer programme. The majority of
samples from normal persons were obtained by permission from the chief of the
Blood bank, Gentofte Hospital, R Jordal, M.D. The RIA-kit was kindly provided by
Pharmacia.

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