Clinical Chemistry 45: 1557-1561, 1999;
(Clinical Chemistry. 1999;45:1557-1561.)
© 1999 American Association for Clinical Chemistry, Inc.
Evaluation of a Particle-enhanced Turbidimetric Immunoassay for the Measurement of Immunoglobulin E in an ILab 900 Analyzer
Sandra Guaita,
Josep MA Simó,
Natàlia Ferré,
Jorge Joven and
Jordi Campsa
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Carrer Sant Joan s/n, 43201-Reus, Catalunya, Spain.
a Author for correspondence. Fax 34-77-31-25-69;
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Abstract
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Background: The measurement of immunoglobulin E (IgE) in serum is
widely used in the diagnosis of allergic reactions and parasitic
infections. We describe here a fully automated assay for human IgE
suitable for routine application in a general chemistry analyzer.
Methods: We used an ILab 900® analyzer. This
instrument automates a particle-enhanced immunoturbidimetric assay with
an analysis time of 9 min.
Results: The assay was linear in the range 4–1000 kIU/L
(r = 0.9998). The intra- and interassay CVs at 57,
235, and 434 kIU/L were <3.5% and <7.4%, respectively. The
detection limit was 4 kIU/L. Hemoglobin (
16 g/L), bilirubin (250
µmol/L), and myeloma paraproteins did not interfere with the assay.
The assay showed good correlation with a microparticle enzyme
immunoassay (r = 0.998) with a mean difference
between methods of -6 ± 26 kIU/L.
Conclusion: The new automated serum assay for IgE is an
attractive alternative that avoids the need for dedicated
instrumentation.
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Introduction
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Immunoglobulin
(Ig)1
E is an antibody involved in the reactions of allergic disease
and parasitic infections (1). Several methods for measuring
total serum IgE have been reported. These include competitive binding
assays (2), ELISA (3), microparticle
enzyme immunoassay (MEIA) (4), and time-resolved
fluoroimmunoassays (5). These methods fulfill the rigorous
requirements for sensitivity and specificity that permit the detection
of microgram per liter concentrations of IgE even in the presence of
gram per liter concentrations of IgG, IgM, and IgA. However, they are
often time-consuming and need special, often dedicated, instruments.
Conversely, turbidimetric immunoassay is a well-established technique
for the rapid quantification of analytes at gram per liter
concentrations (6) and is widely used for the measurement of
immunoglobulins other than IgE. The concentration range for
turbidimetric assays may be extended by using the light-scattering
properties of the immunoaggregates obtained by attaching the antibody
to latex particles, termed particle-enhanced turbidimetric immunoassay
(PETIA) (7)(8)(9)(10). These techniques do not require specific instruments, merely a general
automated analyzer, and have the advantages of decreased operating time
and reduced costs together with an overall integration with other
clinical chemistry analyses. Here we describe a PETIA for the
measurement of IgE adapted for use in an ILab®
automatic analyzer, and compare it with a well-established MEIA.
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Materials and Methods
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apparatus
IgE measurements were performed in an ILab 900, an
automated, random-access, discrete clinical chemistry analyzer
(Instrumentation Laboratories). This instrument facilitates the
performance of spectrophotometric and turbidimetric reactions at
37 °C, using one or two reagents, in bar-code primary tubes. Up to
50 different techniques can be processed simultaneously on-line in the
same sample. For comparison studies, we used the MEIA technique from
Abbott Diagnostics in an IMx® immunoassay
automatic analyzer (Abbott). The analyzer is a batch immunoassay system
capable of producing 40 results per hour following an initial delay of
30–50 min. The MEIA technique is a type of enzyme immunoassay in which
the sample reacts with antibody-coated latex microparticles; after
incubation at 34 °C, the bound and unbound components are separated
using a glass-fiber matrix (11).
reagents and procedures
The IgE PETIA was performed with commercial reagents obtained from
Biokit (Quantex IgE kit; Biokit S.A.). The assay required two reagents.
In the first step, 5 µL of serum was diluted with 200 µL of 0.05
mol/L glycine buffer (pH 8.3) and incubated for 3.4 min. In the second
step, the diluted sample was reacted with a suspension of 100 µL of
polystyrene latex particles of uniform size coated with anti-human IgE
monoclonal antibody. The resulting agglutination was read at 570 nm
twice, at 36 s and at 5.4 min after the addition of the latex
reagent. Both reagents are liquid and do not require any preparation
before the analysis.
MEIA technique was performed according to the manufacturer's
instructions.
calibrators and control materials
The five human liquid calibrators (Quantex IgE Standard
multipoint; Biokit) were between 50 and 1000 kIU/L and were traceable
to the WHO 2nd International Reference Preparation of Human Serum IgE
(75/502).
Control levels 1 (IgE, 55 ± 7 kIU/L) and 3 (IgE, 430 ± 43
kIU/L) were from Biokit. Control 2 was the Immunoassay Control, level 2
(228 ± 23 kIU/L) from Dade®. Three pools
of sera were also used for performance studies and were designated as
"low" (19 ± 1 kIU/L), "medium" (289 ± 3 kIU/L), and
"high" (866 ± 7 kIU/L). The sera were obtained from routine
clinical samples, mixed, and frozen. When required they were thawed,
mixed gently for 20 min on a Coulter mixer (Coulter Electronics), and
assayed 20 times by the MEIA technique.
samples
For comparison purposes, blood samples were obtained from 117
patients referred to the Clinical Laboratory of Hospital Universitari
de Sant Joan de Reus. We chose samples from patients with a range of
clinical conditions to include low, normal, and high concentrations of
serum IgE. Blood was allowed to clot at 37 °C, and serum was
obtained by centrifugation at 1000g for 10 min and processed
the same day. All procedures were in accordance with the ethics
standards of our Institution, in which anonymity of data was
guaranteed.
statistical methods
Data are presented as means ± SD. Mean values for IgE by the
two methods were compared by the Student t-test. Statistical
significance was set at P <0.05. The association between
variables was measured by linear regression analysis. The degree of
agreement between both methods of measurement was estimated by the
Bland-Altman graphical procedure (12). Statistical
calculations were performed with the SPSS statistical program
(13).
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Results
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performance evaluation of IgE MEASUREMENT
Calibration curves and kinetics.
Changes in the turbidity of
calibrators are shown in Fig. 1
.
We observed no significant changes in the calibration curves over 10
consecutive calibrations. Kinetic studies for a 1000 kIU/L calibrator,
a high serum sample, and a reagent blank are shown in Fig. 2
. The best-fit model (r 
0.996) for these curves was
quadratic (curves with the general model: y =
b0 + b1x +
b2x2). The upper
limit of the assay was set at 1000 kIU/L. Samples with higher IgE
values were automatically flagged and diluted (1:3) by the analyzer.
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Figure 2. Kinetics of the agglutination reaction.
The increase in absorbance at 3.4 min corresponds to the latex
addition. •, 1000 kIU/L calibrator;  , 2245 kIU/L serum sample;
 , reagent blank.
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Imprecision.
Intraassay imprecision was determined with 20
replicate analyses of the three commercial controls. To assess
interassay imprecision, aliquots of these controls stored at -20 °C
were analyzed over 20 consecutive days. The CV values are shown in
Table 1
.
Total error.
Total error was calculated by adding the
systematic error and the random error as described previously
(14). Results were as follows: 8.5% for control 1, 4.1%
for control 2, and 8.4% for control 3.
Mixing experiment.
When we added 100 µL of the medium
pool to identical volumes of the three commercial controls, the
(measured IgE/theoretical IgE) x 100 for quadruplicate
measurements was 104% ± 3% for control 1, 99% ± 2% for control 2,
and 104% ± 3% for control 3.
Linearity and detection limit.
Linearity was assessed by
quadruplicate measurements of serial dilutions of the high pool (from
undiluted up to a dilution of 1:256). The regression line of observed
vs expected values was: y = 0.996x + 2.1
(r = 0.9998). To determine the detection limit, the
absorbance of the reagent blank was measured 20 times, the mean ±
SD was calculated, and the detection limit was defined as the IgE
concentration corresponding to an absorbance equal to the mean of
the reagent blank value + 2 SD. The detection limit thus calculated was
4 kIU/L.
Interference.
The interference from triglycerides, hemoglobin,
and bilirubin was assessed as described previously (15). The
low, medium, and high sera pools were supplemented with chylomicrons,
hemoglobin, or bilirubin at various concentrations. The results are
shown in Fig. 3
. There was no substantial interference from hemoglobin up to 16
g/L or from bilirubin up to 250 µmol/L. Hypertriglyceridemia
(triglycerides 5 mmol/L) increased measured IgE values, particularly
in samples with low IgE concentrations, which indicates that
lipemic samples should be treated in an alternative manner.
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Figure 3. Interference of hemoglobin, bilirubin, and triglycerides
in the IgE assay.
(A), free hemoglobin added up to 65 g/L;
(B), bilirubin added up to 250 µmol/L;
(C), chylomicrons added up to 40 mmol/L.
(C), the three plots are shown separately to show the
higher effect of lipemia on low (•) than on medium () and high
() serum pools.
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Effect of paraproteinemia.
The effect of nonspecific
aggregation and/or cross-reactivity from paraproteinemia on the IgE
determination was assessed as described (15). Serial
dilutions (500 µL) in physiologic saline of sera from patients with
IgG, IgM, and IgA myeloma were added to identical volumes of the high
pool, gently mixed on a Coulter mixer for 20 min, and analyzed, and the
recovery was calculated. The measured IgE concentrations in these
paraproteinemic sera were <10 kIU/L. Recovery results are shown in
Table 2
and indicate that the recovery was not influenced by the
paraprotein concentrations (mean recovery, 103%; range, 94–119%).
comparison of methods
For 117 samples, the mean values for IgE were 249 kIU/L for the
turbidimetric assay and 256 kIU/L for the MEIA assay (r
= 0.998; Sy|x = 26.0). The equation of the
regression line was y = 1.011x -
9.685; the SDs of the slope and the intercept were 0.007 kIU/L and
2.930 kIU/L, respectively (Fig. 4
A). The degree of agreement between methods was assessed using
the Bland-Altman graphical technique (Fig. 4B
). The mean difference was
-6 ± 26 kIU/L. Of the eight samples with values >2 SD from the
mean difference, seven had IgE concentrations >600 kIU/L, indicating a
proportionally low percentage of error.
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Discussion
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The results for all of the performance characteristics of the
PETIA were acceptable when compared with recommended values
(16). The determination of IgE in serum by this assay is an
efficient and convenient alternative to MEIA. As demonstrated, this
assay may be easily adapted to general analyzers, reducing the need for
dedicated instruments and, therefore, helping to reduce laboratory
costs. Apart from the ILab 900, adaptations of this technique have been
described by the manufacturers on the following general analyzers:
Monarch® 2000, ILab 500 and 600 (all from
Instrumentation Laboratories), BM/Hitachi® 704,
717, 917, 747, and 902 (Boehringer Mannheim), Cobas
Mira® (Roche Diagnostics),
Synchron® CX7 (Beckman Instruments), and
Olympus® AU 600 and 800 (Olympus). This
adaptation may allow a much faster laboratory response (9 vs 30 min to
process a single sample) because incubation times are shorter and the
random access to an analyzer preempts the need to wait for a complete
batch of samples to be analyzed before obtaining the results.
Measurements can be performed in bar-code-labeled primary tubes, which
reduces the possibility of mistakes and avoids the need to aliquot or
pipette the samples into special cuvettes. IgE can be measured together
with other immunoglobulins or other general chemistry tests, decreasing
the total volume of blood that must be drawn from a patient, an aspect
that has special relevance in children, who constitute a high
percentage of patients suffering from allergies. Furthermore, in our
laboratory, this method has decreased by 43% the reagent cost of
performing any one, single IgE measurement.
In conclusion, the present study demonstrates that this PETIA is an
effective, reliable, and readily automated method for measuring IgE in
serum, thus contributing substantially to reduced laboratory costs and
efficient sample processing.
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Acknowledgments
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We thank Izasa S.A. and Biokit S.A., Barcelona, Spain, for
gifts of reagents and financial support. Natàlia Ferré was
the recipient of a grant from Fundació Privada Reddis (1998).
Technical assistance was by Peter R. Turner of t-SciMed (Reus, Spain).
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Footnotes
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1 Nonstandard abbreviations: Ig, immunoglobulin; MEIA, microparticle immunoassay; and PETIA, particle-enhanced immunoturbidimetric assay. 
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Abstract
Introduction
