Clinical Chemistry 43: 2052-2057, 1997;
(Clinical Chemistry. 1997;43:2052-2057.)
© 1997 American Association for Clinical Chemistry, Inc.
Direct comparison of performance characteristics of two immunoassays for bone isoform of alkaline phosphatase in serum
Christopher P. Pricea,
Thomas P. Milligan and
Claude Darte
Department of Clinical Biochemistry, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Turner St., London E1 2AD, UK.
a Author for correspondence. Fax 44-171-377-1544; e-mail C.P.Price{at}mds.qmw.ac.uk
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Abstract
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A clinical need exists for a sensitive and specific assay for the
quantitation of the bone isoform of alkaline phosphatase in serum. The
majority of methods do not meet this requirement; however, the recent
development of immunoassays for this isoform may provide a solution. In
a detailed evaluation of two immunoassays, we found a degree of
imprecision that enables the discrimination of changes within the
reference range. The cross-reactivity of the liver isoform was found to
be between 7.1% and 12.7% when two different methods of assessment
were used. The comparison of results with an electrophoretic procedure
showed that the immunocapture method recovered less of the bone isoform
in samples from children than in samples from patients with Paget
disease; no such difference was found with the immunometric method.
This suggests that the immunocapture antibody may discriminate between
different bone isoforms in children whereas the immunometric assay does
not.
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Introduction
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The bone isoform of alkaline phosphatase (ALP) is a member of
the so-called tissue-nonspecific isoenzyme family, which also comprises
the liver and kidney isoforms. These three main isoforms are thought to
differ primarily in the degree of posttranslational glycosylation
(1)(2). Additional isoforms demonstrated in
vesicular structures are thought to be a consequence of shedding of the
enzyme attached to its membrane anchor region (3).
Many things may cause increases of ALP activity in serum, the most
common being obstructive liver disease and metabolic bone disease. An
increase of the liver or particularly the bone isoform in serum can
provide valuable diagnostic information. It is rare that the
kidney-derived isoform appears in the circulation, and whereas an
increase of the intestinal, placental, or germ cell isoenzymes is more
common, this is of limited diagnostic value except in some patients
with malignant disease.
The analytical challenge therefore is to achieve specific quantitation
of the liver and bone isoforms. This has been attempted with several
techniques, including heat inactivation (4), wheat germ
lectin precipitation (5), electrophoresis
(6), isoelectric focusing (7), HPLC
(8)(9), and immunoassay
(10)(11). The nonimmunological techniques have
in some instances failed to provide the required sensitivity or
specificity, whereas some of the separation techniques have identified
multiple fractions (bands) that have confused interpretation. In this
respect, the primary clinical application is quantitation of the bone
isoform, and although several immunoassays for this isoform have been
described, only two have been validated in any detail
(11)(12)(13)(14)(15)(16)(17)(18)(19).
The two immunoassays that have received the most attention are based on
immunometric (12) and immunocapture (11)
principles. The former detects bound isoform with a labeled second
monoclonal antibody, whereas the latter measures the activity captured,
in both cases, to a solid-phase-bound monoclonal antibody. Enzyme
derived from a human osteosarcoma cell line is needed as a calibrator
in both methods. However, the published validations of these methods
have resulted from a range of comparison methods to assess accuracy
and, in particular, the cross-reactivity with the liver isoform. The
results reported have thus been very variable. In an earlier study
(19), we found a significant difference in the correlation
of results by the immunocapture method and by an electrophoretic
technique, when studying samples from children and from patients with
Paget disease. We did not observe such a difference in patient groups
when assessing the immunometric assay (14). We have
therefore set out to validate both immunoassay methods with the use of
the same samples and identical comparison procedures. Particular
attention was paid to comparison with a nonimmunological method that
achieved complete discrimination between liver and bone isoforms, and
to assessment of apparent cross-reactivity of the liver isoform.
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Materials and Methods
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measurement of total alp activity
The total ALP activity was measured with the use of a centrifugal
analyzer (Monarch®, Instrumentation Laboratory).
Briefly, 3 µL of serum was mixed with 160 µL of
p-nitrophenyl phosphate in a 2-amino-2-methyl-1-propane
buffer at pH 10.5. The reaction was monitored at 405 nm for a total of
3 min after an incubation temperature of 37 °C was reached, and the
activity was calculated with reference to the absorptivity of the
product.
electrophoresis with neuraminidase pretreatment
An aliquot of sample (50 µL) was incubated with 10 µL of
neuraminidase (2000 U/L) at 20 °C for 10 min, and 5 µL of the
mixture was applied to a preprepared gel (Isopal Plus, Beckman
Instruments); an aliquot of untreated sample was also analyzed. Current
was applied at a constant 150 V for 25 min. The gel was then incubated
with freshly prepared substrate for 15 min at 37 °C. The gel was
then rinsed in water and acetic acid and dried. Quantitation of the
isoforms was performed by densitometry (Model GS-60 Imaging
Densitometer, Bio-Rad Labs.); the so-called
high-Mr liver isoform was quantitated from
densitometry of the untreated sample separation.
immunometric assay
Serum or calibrator (100 µL), together with 100 µL of
125I-labeled anti-bone ALP (mouse) monoclonal antibody
was added to a tube containing a bead coated with a second anti-bone
ALP (mouse) monoclonal antibody. All tubes were incubated for 19
± 2 h at 4 °C with the tubes partially immersed in a tray of
water. The beads were then rinsed three times with a detergent
solution, and the total and bound radioactivity was counted in an NE
1600 counter (Nuclear Enterprises). All calibrator and sample
measurements were performed in duplicate, and results were expressed in
µg/L. The calibrator was prepared from the SAOS-2 human osteosarcoma
cell line and supplied in a liquid form. The reagents were supplied by
Hybritech.
immunocapture assay
An aliquot (125 µL) of reaction buffer followed by 20 µL of
sample or calibrator was added to wells of a microtiter plate coated
with an anti-bone ALP (mouse) monoclonal antibody. The microtiter plate
was covered and incubated at room temperature for 3 h, followed by
removal of the contents and washing of the wells with a buffered
detergent solution. To each well was then added 150 µL of buffered
p-nitrophenyl phosphate, pH 10.4, and the contents were
incubated at room temperature for 30 min. The reaction was stopped by
the addition of 100 µL of 1 mol/L sodium hydroxide, and the
absorbance was read at 405 nm with an automated microtiter plate reader
(Molecular Devices). The assay was calibrated with the use of bone ALP
obtained from the SAOS-2 human osteosarcoma cell line, and the results
were reported as U/L at 37 °C (conversion from results at 25 °C
was established in a separate experiment (19)). The
reagents were supplied by Metra Biosystems.
samples
Serum samples were collected from children with no evidence of
bone disease and admitted to the hospital for another reason, patients
with Paget disease, and patients with obstructive liver disease. All
samples were stored at -20 °C until analysis, and all analyses on a
sample were performed within 7 h of its thawing.
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Results
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imprecision
Two serum pools were prepared containing different concentrations
of bone ALP, and the pools were aliquotted and stored at -20 °C.
One aliquot of each pool was thawed and analyzed in duplicate on each
day when another experiment was performed. The between-batch
imprecision for the two immunoassay methods is shown in Table 1
.
comparison of results
The bone ALP isoform concentration was determined by the two
immunoassay methods and the electrophoretic method for samples from
children and from patients with Paget disease. The data are shown in
Figs. 1–3
.
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Figure 1. Comparison of results for the measurement of bone ALP in
sera from children ( ) and patients with Paget disease (•) by the
immunometric and electrophoretic methods.
The regression statistics (Deming procedure (21)) are as
follows: children, y = 0.385x - 0.907,
r = 0.986; Paget, y = 0.407x
+ 20.03, r = 0.986.
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Figure 2. Comparison of results for the measurement of bone ALP in
sera from children and patients with Paget disease (•) by the
immunocapture and electrophoretic methods.
The regression statistics (Deming procedure (21)) are as
follows: children, y = 0.611x + 24.65,
r = 0.991; Paget, y =
0.906x - 5.10, r = 0.981.
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Figure 3. Comparison of results for the measurement of bone ALP in
sera from children () and patients with Paget disease (•) by the
immunocapture and immunometric methods.
The regression statistics (Deming procedure (21)) are as
follows: children, y = 0.632x - 16.61,
r = 0.974; Paget, y = 0.436x
+ 23.41, r = 0.941.
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cross-reactivity of liver isoform
The assessment of cross-reactivity of the liver isoform was
undertaken by two approaches: heat inactivation, and addition of liver
ALP.
Heat inactivation
. A total of 27 serum samples known to
contain various proportions of the bone and liver isoforms (from
electrophoretic evaluation) were used. For each sample, a 400-µL
aliquot of sample was placed in each of 6 thin-walled glass tubes. The
tubes were placed in a water bath at 56 °C and removed after 7.5,
15, 20, 25, or 30 min; each tube was then placed in a tray of crushed
ice. The total ALP and the bone ALP isoform was quantitated in the
latter by all three methods described. The ratio of apparent bone ALP
to total ALP was calculated at each of the incubation points, and the
data for the two immunoassays are shown in Table 2
. The electrophoretic method confirmed that no bone ALP activity
remained after heating of the sample for 25 min at 56 °C.
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Table 2. Ratio of apparent bone ALP present to total ALP activity
remaining after incubation for different periods at
56 °C.
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The cross-reactivity of the liver isoform in the immunometric assay was
calculated from the ratio of apparent bone ALP to total ALP for the
liver isoform remaining after heating at 56 °C for 25 min divided by
the same ratio for the bone isoform in serum from patients with Paget
disease (i.e., the slope of the regression line in Figs. 1
and 2
). This
gave a figure for cross-reactivity (mean ± SD) of 7.1% ± 2.32%
(i.e., 2.88 ÷ 0.407 x 100 = 7.1%) for the
immunometric assay. The comparable calculation for the immunocapture
assay gave a figure of cross-reactivity of 7.9% ± 1.41% (i.e.,
7.17 ÷ 0.906 x 100 = 7.91%).
Addition of liver ALP
. A series of samples containing either
predominantly liver or bone ALP were obtained. A sample containing
predominantly liver isoform was added (to 10%, 20%, 30%, 40%, and
50% of the final volume) to a constant amount of serum from a patient
with Paget disease (50% of the final volume) with the difference made
up with human serum albumin (40 g/L in 0.9 g/L NaCl). All mixtures were
assayed by both immunoassays, whereas the original materials were also
assayed by the electrophoretic method to confirm the predominance
(>95%) of one of the isoforms. The total ALP of the samples
containing the liver isoform was also determined. The apparent bone ALP
increase resulting from added liver ALP was then plotted against the
ALP activity and added, and the data are shown in Figs. 4
and
5. The figure for cross-reactivity is obtained from the ratio of
apparent bone ALP to total ALP for the sera from patients with Paget
disease (i.e., the slopes in Figs. 1
and 2
). The calculated figures for
cross-reactivity of the liver isoform in the immunometric and
immunocapture assay are 12.7% and 8.7%, respectively (0.052 ÷
0.407 x 100 and 0.079 ÷ 0.906 x 100).
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Figure 4. Data on the apparent bone ALP mass measured in the
immunometric assay plotted against the amount of liver ALP activity
added (•).
The regression line a represents the correlation between ALP
mass and activity in Paget disease from Fig. 1
; line b is
the regression line relating the apparent bone ALP mass related to
liver ALP activity added, y = 0.052x +
6.624, r = 0.804.
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Discussion
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This study produced results for imprecision comparable with those
reported in earlier evaluations (11)(12)(13)(14)(15)(16)(17)(18)(19). Most
importantly, it confirmed some of the results obtained in previous
method comparisons, but not others
(14)(16)(19); thus whereas the
immunometric assay showed concordance in the relation between mass and
activity in samples from children and patients with Paget disease, the
same was not true in the case of the immunocapture assay. The
cross-reactivity of the liver isoform is very similar for both
immunoassay methods and uses two different experimental approaches.
There is no ideal approach to the assessment of cross-reactivity,
especially when it has been shown that analysis of a purified fraction
of the liver isoform gave 100% cross-reactivity (14). The
use of heat-inactivation studies assumes that all specimens have an
identical inactivation profile, which is known not to be the case
(4); furthermore, the technique assumes that catalytic
activity and immunoactivity are equally sensitive to elevated
temperatures. By use of this approach with the immunocapture assay,
Gomez et al. (11) studied five samples from patients with
liver disease and found the cross-reactivity to be within the range of
3% to 8%; our data showed a mean of 7.9% with a range of 5.8% to
10.0% when we studied 27 samples. A similar experiment in which the
immunometric assay was used with the same samples and the apparent bone
ALP:total ALP ratio of the heat-inactivated sample was compared with
the ratio in sera from patients with Paget disease indicated a mean
cross-reactivity of 7.0% with a range of 3.2% to 10.6%. This is the
first instance where this approach has been used with the immunometric
method. Hata et al. (15) used the heat-inactivation
approach with the immunocapture assay and found a cross-reactivity of
8.7% with the liver isoform.
The addition of liver isoform in serum from patients with obstructive
liver disease to a serum containing bone isoform and measurement of the
apparent increase in bone ALP indicated a cross-reactivity of 8.6% in
the immunocapture assay and 12.7% in the immunometric assay. This
result is based on the correlation data shown in Table 2
. These data
compare with similar figures in previous evaluations
(14)(19).
Withold et al. (16) and Garnero and Delmas
(13) studied the cross-reactivity of the immunocapture and
immunometric assays, respectively, by comparing the ratio of apparent
bone ALP to total ALP in sera from patients with liver disease and
found mean cross-reactivities of 20% and 16%, respectively. This
approach assumes that no bone isoform is present in the sera of
patients with liver disease, which is unlikely to be the case,
especially in chronic liver disease. Consequently, these figures are
probably an overestimate of cross-reactivity. Panigrahi et al.
(12) studied the cross-reactivity of the liver isoform in
the immunometric assay by enriching heat-inactivated serum with a
series of dilutions of sera from patients with predominantly bone or
liver ALP present. The ratio of apparent bone ALP mass to total ALP
activity was calculated for the bone and liver dilutions; the ratio of
the liver dilutions was 14.7% of the bone dilutions, indicating the
amount of cross-reactivity. Again, on the basis that the ratio of mass
to activity did not change in the majority of samples, the authors
concluded that each sample contained only one isoform; this
figure may therefore reflect an overestimate of the cross-reactivity.
The immunometric assay showed a relation similar to that found with a
catalytic method as that found by Garnero and Delmas (13)
and Panigrahi et al. (12). All of these studies used serum
samples from patients with a range of metabolic bone disorders.
None of the previous studies has independently assessed the comparison
of results by different methods with samples from children and from
patients with Paget disease; our studies have shown a difference in the
relation between activity captured by the monoclonal antibody and that
found by the electrophoretic method. No difference was found with the
immunometric assay in this or in a previous study (14).
The observations made with the immunocapture assay may be due to
(a) a different specific activity of the bone isoform
present in children compared with patients with Paget disease;
(b) differences in the posttranslational modification, which
alters the epitope that is recognized in the immunocapture assay; or
(c) the presence of different isoforms of bone origin, the
proportions of which differ in the subject groups studied. It is
unlikely that a difference in specific activity accounts for the
observation; otherwise, differences would have been noted in the
immunometric assay. The possibility of variations in posttranslational
modification influencing either the conformation of the epitope or the
production of a subfamily of isoforms is possible. The broad
distribution of the bone isoform after electrophoresis points to a
heterogeneity of sialylation—treatment with neuraminidase producing a
more discrete band. Furthermore, both isoelectric focusing and HPLC
separations have identified more than one bone isoform, although the
predominance of individual fractions in particular diseases has not
been reported (7)(9)(21). Miura et
al. (22) studied the sugar moieties with the use of two
neuraminidases and O-glycanase and concluded that the
structural epitope differences between liver and bone ALP may depend on
the nearby O-linked sugar moieties as well as sialic acid
residues. The authors did not, however, link the variations to any
particular type of bone disease. Schoneau et al. (23),
using anion-exchange chromatography, found two bone isoforms in sera of
children, and Parviainen et al. (9) found that one isoform
predominated in sera from patients with osteoporosis, osteomalacia,
bone metastases, and Paget disease. Onica et al. (24)
found two bone fractions on electrophoresis, and Anderson et al.
(25) also found evidence of change in the glycosylation of
bone ALP in patients with bone disease. Langlois et al.
(26) compared the immunometric assay with the
electrophoretic technique used in this study and found that the ratio
of activity to mass was substantially lower in patients with
hyperthyroidism than in controls and patients with osteoporosis; this
was considered to reflect a difference in posttranslational
modification in different pathological conditions.
The literature shows that different isoforms of bone ALP exist; the
immunocapture method appears to recognize an isoform that is different
from that recognized by the immunometric assay. At this stage, the
clinical significance of this observation is not clear, albeit there is
an apparent reduction in the detection limit compared with the
electrophoretic method. The application of a sensitive assay for bone
ALP in children in clinical practice is not apparent to date, in part
because of the large biological variation in this analyte in children;
however, it might be important in children with metabolic bone disease
in that the relative increase will be diminished.
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Figure 5. Data on the apparent bone ALP activity captured in the
immunocapture assay plotted against the amount of liver ALP activity
added (•).
The regression line a represents the correlation between
bone ALP captured and activity in Paget disease from Fig. 2
; line
b is the regression line relating the apparent bone ALP to liver
ALP activity added, y - 0.079x + 1.55,
r = 0.814.
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Acknowledgments
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We gratefully acknowledge the financial support of Hybritech Europe
for the purchase of all reagents used in this study.
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Top
Abstract
Introduction
