CLIN.CHEM. 36/3,550-553 (1990)
A Two-Tube Immunochemical Method for Determination of CK-MB Isoenzyme in Serum
Evaluated
Mauro Panteghlnl, Roberto Bonora, and Franca Paganl
A new commercial kit (lmpres-MB; International Immunoassay Labs.) recently was introduced for measuring the MB
isoenzyme of creatine kinase (CK-MB) based on the use of
monoclonal antibodies. After antibodies to CK-MM isoenzyme are added to precipitate the CK-MM, antibodies to
CK-M monomer are added to precipitate the M-subunit
isoenzymes of CK. Subtracting the enzymatic activity of the
second supernate from the residual activity in the first yields
the activity of CK-MB. Results are not affected by CK-BB,
mitochondnal CK, or adenylate kinase. However, the antiCK-MM antibodies precipitated only about 98% of serum
CK-MM and may have partly precipitated CK-MB isoenzyme
(average analytical recovery of CK-MB, 86.6%). Comparison
between lmpres-MB ( and electrophoresis (x) yielded the
following linear-regression equation: y = 0.79x + 3 (r =
0.982, n = 97). Data for CK-MB temporal kinetics, obtained
from patients with myocardial infarction, correlated significantly in both methods; however, peak activity values of
CK-MB were significantly different, confirming that the difference between the new method and the electrophoretic
method averages 20%.
AdditIonal Keyphrases: “kit” methods
temporal changes in
myocardial infarction . monoclonal antibodies
reference interval
Creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) is
widely measured in serum as a diagnostic indicator of acute
myocardial
infarction
(AMI)
(1).’ Accordingly,
different
commercial methods for quantifying
this isoenzyme have
been developed, which vary in specificity, sensitivity, and
precision (2). The procedures most recently described for
quantifying CK-MB in sera are immunologically based (3).
The widely used immunoinhibition method for CK-M subunit, with subsequent quantification of the still-active B
subunits, is in many instances diagnostic of AMI (4), but
quantification of CK-MB is impaired by the presence in
serum of macro CK, mitochondrial CK, or CK-BB isoenzyme (5, 6). The immunoinhibition-immunoprecipitation
method effectively eliminates interference from CK variants as well as problems associated with increases
in
CK-BB (7) but is less efficient (it is difficult to automate)
(4). Some recently introduced immunometric procedures for
specific detection of CK-MB mass concentration instead of
its catalytic
activity
are not suitable for use in a “stat”
(emergency)
laboratory
because of long turnaround time
(8-11). Therefore, a simple, fast CK-MB-specific
assay is
still clearly needed (12).
10Laboratorio Analisi Chimico-Cliniche, Spedali Civili, 25123
Brescia, Italy.
Nonstandard abbreviations: CK, creatine
kinase (ATP:creatme N-phosphotransferase;
EC 2.7.3.2); AM!, acute myocardial
infarction; K,,, maximum
rate of enzymatic activity increase; and
Kd, rate of fractional disappearance of enzyme.
Received July 7, 1989;accepted November 20, 1989.
550 CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990
Here we describe the analytical
performance of a new
based
on the use of monoclonal antibodies and recently available
from International
Immunoassay Labs. We compare resultswith those by an electrophoretic procedure, routinely
used in our laboratory,
for samples from AM! patients.
commercial
assay (Impres-MB)
immunoprecipitation
Materials and Methods
Blood Samples
We studied 21 patients, admitted because of AM! to the
Department of Anaesthesia and Reanimation of the Civil
Hospital of Brescia. Assessment of myocardial necrosis was
based on the diagnostic criteria of the World Health Organization (13): clinical symptoms, electrocardiographic
findings, and typical increase and decrease in enzyme and
isoenzyme activities in serum. Peripheral venous blood
samples were obtained immediately after admission to the
hospital, every 4 h for the first 24 h, and every 8 h for the
following 48 h.
In addition,140 apparentlyhealthypeople, ages 20 to 70
years, with normal resultsfor serum biochemical and
hematological testsand withoutclinical
evidenceofcardiac
and muscle diseases, were studiedto establishthe reference interval for CK-MB isoenzyme activity in serum.
All serum samples were assayed immediately for total CK
activity; electrophoresis
and immunochemical
assay were
performed within 12 h after collection with interim refrigerated storagein hermeticallysealedplastic containers.
Assay Methods
Measurement of total CK. CK activity was measured by
the method recommended by the Scandinavian Committee
on Enzymes (14), with reagents from Boehringer, Mannheim, F.R.G., and a Cobas Bio analyzer (F. Hoffmann-La
Roche and Co., Ltd., Basle, Switzerland).Results were
expressed as U (mol mm 1) Upper referencelimitsfor
non-AM! were 160 UIL forwomen, 190 U/L formen.
Electrophoresis.
We electrophoretically
separated CK
isoenzymes on Titan Illcelluloseacetatemembrane in a
“Zip Zone” electrophoresis
chamber (Helena Labs.,Beaumont, TX 77704) with CK reagents from Boehringer. CK
isoenzymes were separated according to the manufacturer’s
instructions. The isoenzyme bands were quantified by fluorimetric scanning of the tracings with a Helena “Cmiscan”densitometer.
The enzymatic activity of the MB band
was calculated by multiplying measured total CK by the
percentage of the fluorescence appearing in the MB region.
By this method, CK-MB is undetectablein serum from
healthypeople.
Immunoenzymometric
assay. For measuring CK-MB
mass concentration,we used the Tandem-E CK-MB II
(HybritechInc.,San Diego,CA 92121)immunoenzymometricassay,performed accordingto the manufacturer’scurrent protocol (11). For absorbance measurements
we used
the Hybritech “Photon” immunoassay spectrophotometer.
All measurements were done in duplicate.
-
Immunochemical
assay. The kit assay (Impres-MB, InImmunoassay Labs.,Santa Clara,CA 95054)
was performed according to the manufacturer’s directions
insert. A two-tube approach isused.To serum in tube 1,
monoclonal antibodiesto CK-MM
isoenzyme (reagentA)
are added to precipitateall of serum CK-MM, and the
activity
remaining isthatfrom CK-MB, CK-BB, mitochondrial CK, and macro CK. To serum in tube 2,monoclonal
antibodiesto CK-M monomer (reagentB) are added to
precipitateM-subunit isoenzymes of CK; the remaining
activity
isthus from CK-BB, mitochondrialCK, and macro
CK. The assay requiresa totalsample volume of 200 L
(100 L foreach tube).Magnetizableparticles
coatedwith
the two different
setsofmonoclonal antibodiesare used to
separatethe supernatesin each tube beforeresidualCK
activity
ismeasured with the reagentsystem describedfor
the measurement of total CK. The differencein the CK
activityof the two tubes (multipliedby 2, the dilution
factor)representsthe serum CK-MB activity.
Characterization
of human CK isoenzymes. The purified
CK isoenzymes used for specificity
and recovery studies
were obtainedfrom Calbiochem Corp.,La Jolla,CA 92037
(MM and BB) and from ScrippsLabs.,San Diego,CA 92131
(MB). CK-MM (cat.no. 238407) was purifiedfrom human
skeletalmuscle,CK-BB (cat. no.238397)was purified
from
human brain,and CK-MB (cat.no. C 1224) was purified
from human heart.The possiblecontamination of each
commerciallyobtainedisoenzyme with anotherwas tested
by electrophoresis
on cellulose
acetatemembrane followed
by stainingfor catalyticactivity.
Each preparationwas
electrophoretically
homogeneous and possesseda specific
activity
ofat least500 kU per gram ofprotein(wetweight).
MitochondrialCK was preparedaccordingtoWevers et al.
(15).
Statistical analysis.
We calculated mean, standard deviation, coefficient of variation,
correlation
coefficient,
and
linear-regression
analysis
by standard methods. Timeactivity curves, constructed for each AM! patient, allowed
us to derive peak activity and time required to reach the
peak value for CK-MB isoenzyme (16). The relationship of
two continuous variables was tested by linear least-squares
regression analysis. In particular,
the rate of increase (K,,)
and the clearance rate (fractional disappearance rate, Kd)
for CK-MB activity were calculated by linear-regression
analysisfrom the linear portions of the ascending and
declining slopes of the time-activitycurves, plotted
semilogarithmically.
At leastthree and four values were
used to determine K,, and Kth respectively.
ternational
Results
Linearity
and detection limit. In the standard assay
procedure, the immunochemical
assay gives results that
vary linearly with concentration of serum CK-MB up to
180 U/L. We serially
diluted pooled CK-MB-rich specimens
with the zero diluent(human serum containingno detectable CK activity)supplied with the kit. Six separate
dilutionswere assayed,and each dilutionwas run four
times,in duplicate.
The standard curve showed no significant curvature when testedforlinearityas suggestedby
Burnett (17) (quadraticregression,
y = -0.123 + 177.48x+
3.3442x2, where the coefficient
of x2 did not differsignificantlyfrom zero),and the response was highlylinear(r =
0.9997).
We assessed analytical sensitivity by 10 replicate measurements of the zero diluent in a single run. The minimum
detectable CK-MB activity, defined according to Rodbard
(18), was estimated to be 1 U/L.
Precision. Results of precision studies are shown inTable
1. Within-run precisionwas determined by 20 replicate
determinationsof three serum samples in one assay.Between-day precision was determined from data on 10 measurements of three different serum samples; between each
experiment, the sera were stored at -20 #{176}C.
The CVs for
intra- and inter-assay
precisionranged between 2.6% and
10% forthe samples we tested, comparing favorablywith
other versionsof immunochemical
assays for CK-MB determination(2, 8).
Recovery and interference studies. Analytical
recovery
was assessed by adding variousquantitiesof purifiedhuman CK-MB to human serum containing a CK-MB activity
of 19 U/L and assaying. The MB activity was then measured with the immunochemical
assay. Analytical
recovery
was calculated as follows: [(amount found
control
amount)/amount added] x 100. Recovery of added amounts
of 15, 35, 75, and 110 U/L averaged 81.8%, 90.9%, 82.1%,
and 91.7% fortube 1,respectively,
with no residualactivity
in supernate 2. The overallaverage percentagerecovery,
86.6% (CV 6.2%), showed a significant
partial loss of
CK-MB activity in the first analyticalstep.A similar
recovery was obtained by measuring CK-MB mass concentration (average, 84.9%). Thus, the first monoclonal antibody appears to partly precipitate the CK-MB isoenzyme.
CK electrophoresis was performed with a serum sample
(total CK activity, 1100 U/L) containing CK-MM (62.4%),
CK-MB
(21.6%), and CK-BB (16.0%) isoenzymes,before
and after incubation with the antibody reagents of immunochemical assay (Figure 1). As shown, the ability of
Impres-MB to effectively precipitate all of serum CK-MM
isoenzyme in the first step was uncertain. Conversely,
complete precipitation
of MM and MB isoenzymes in the
secondstepofthe assay was assuredby the disappearance
ofthesefractions
after the test serum was incubatedwith
the second reagent.Quantitativetestsconfirmedthat the
anti-CK-MM antibodies(reagentA) couldprecipitate
about
98% oftotalCK-MM (foractivities
s500 UIL),independent
of incubationtime (Table 2).Conversely,in allsamples
tested,therewas no residualCK activityin the supernate
oftube 2.To minimize interference
by CK-MM (reflected
in
unexpectedlyhigh resultsforCK-MB), one should appropriately pre-dilute specimens with CK activity >500 U/L.
Addition of increasing quantities of purified human CKBB (up to 1125 UIL) and mitochondrial CK (up to 325 U/L)
isoenzymes to a serum sample containing a known concentration of CK-MB did not alter the assayed response of
CK-MB from this serum sample. We also found no influence from bilirubin (up to 250 p.molJL), triglycerides (up to
10 mmoIJL), hemoglobin (up to 9 g/L), or adenylatekinase
(ATP:AMP phosphotransferase,
EC 2.7.4.3)
(up to5 mg/L).
Correlation with electrophoresis. Figure 2 summarizes
-
Table 1.ImprecIsionof CK-MB Determination by the
Evaluated Methoda
Within-run (n = 20)
Between-day(n = 10)
Moan,U/L
12
41
73
SD, UIL
CV, %
0.8
1.4
1.9
6.7
3.4
2.6
Mean,U/L
10
22
36
SD, U/L
CV, %
1.0
1.8
2.2
10.0
8.2
6.1
All runs performedwith a single lot number of reagent kits.
CLINICALCHEMISTRY, Vol.36,No.3, 1990 551
AIliA
360
yc 0.81x -1.5
._
320
:0.983
Sv= 15.5
280
n71
/
/
240
CK-MM
200
CK-MB
CK-BB
L)
40
+
Fig. 1. Electrophoretogramof patients samplebefore(A) and after
(I, II) incubation with the antibody reagentsof the immunochemical
assay evaluated
Table 2. Effectiveness of Immunochemical Assay to
Precipitate
Purified CK-MM, UIL
250
CK-MM
isoenzyme
preciphatod,%‘
98.0 ± 17b
CK-MM
160
200
240 280 320
360
4W
440
CK-MB (ectrophoresis) [u/i)
Fig. 2. Correlation of results for CK-MB by electrophoresis (x-axis)
with those by immunochemical assay (y-axis)
Dashed line:the observed regression;continuous line: y = x
lated significantly (Table 3). Peak-value times did not differ
significantly according to methodology, whereas peak activity values of CK-MB
were significantly
different
(Wilcoxon rank-sum test,P <0.01), confirming that the
difference between the new and reference methods averaged 20%.
97.9
±
750
97.6
± 12b
1000
97.0
±
1.2k’
Discussion
1250
96.9
96.4
953
±
1.0’
±
1 5b
The principle of the method used for Impres-MB is based
on the modification of methodology originally reported by
Wicks et al. (7). The Roche assay measures CK activity
before and after a second antibody is added to precipitate
anti-CK-M along with bound enzyme. The difference between the measured activity in the presence of anti-M
monomer and the residual activity after precipitating antiM is proportional to the CK-MB activity. The Impres-MB
makes use of two different sets of monoclonal
antibodies,
such that the supernate in tube 2 contains only the interfering activities mentioned above. Therefore, subtraction of
the activity in tube 2 from those in tube 1 should provide a
measurement of CK-MB alone. The use of monoclonal
antibodies would presumably have less lot-to-lot variation
than methods involving polyclonal antibodies (19). However, the first set of antibodies was not completely specific
for MM isoenzyme, showing a significant partial precipitation of CK-MB isoenzyme. Furthermore, the same reagent
precipitated only 98% of the total CK-MM in serum, which
1750
± 0.4k
91.9 ± 0.1
2000
a Each point (mean ± SD) was run eight times.b.c Significantly different
(bp
120
500
1500
1.2k’
80
<0.05, P <0.01) from 100%.
the results of the analytical comparison between the immunochemical assay and electrophoresis
for 97 unselected
individual
samples submitted
to our clinical laboratory. In
both methods, samples with total CK activity
>500 U/L
were diluted before analysis to minimize CK-MM interference. Correlation between Impres-MB activity and electrophoresis-measured
activity was good, but CK-MB activity
appeared to be underestimated by the immunoprecipitation test.
Reference interval. We used the immunochemical assay
to measure the activity of MB isoenzyme in sera from 140
nonhospitalized,
apparently
healthy subjects and used the
data for these subjects to calculate the reference interval,
using non-parametric
determination
of percentiles. The
median value was 3.5 U/L and the upper reference limit,
defined as the 95th percentile, was 6 U/L. Given that the
cross-reactivity of CK-MM in the assay was 2% and the
total CK averaged 87 U/L, we estimate the CK-MB activity
in serum of normal adults to be about 1-2 U/L.
Comparison
studies with AM! patients.
To compare
changes in CK-MB activity with time by both the immunochemical and the electrophoretic methods, we analyzed
252 serial serum samples from 21 separate patients clinically diagnosed
as having AMI. The data for CK-MB
temporal kinetics obtained with the two methods corre552 CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990
Tabie 3. Data for CK-MB isoenzyme
Patients
Ka, h1
Peak time,
h
Peak value,
U/L
K
h1
Impres-Ma
0.089a
(0.060/0.242)
20.0
(13.0/26.4)
133
(39/545)
-0.025
(-0.013/-0.032)
Medianvalue (and range).
Kinetics
Etoctrophoresls
0.116
(0.050/0.211)
19.3
(13.0/26.6)
165
(50/592)
-0.031
(-0.016/-0.037)
in 21 AMI
CorrelatIon
r = 0.945
P <0.01
r = 0.976
P<0.001
r=0.980
P <0.001
r = 0.827
P <0.01
is a problem because it makes interpretation
of test results
that are near the decision level very uncertain. Nevertheless, we feel that routine use of this method has several
advantages: direct measurement of enzymatic activity and
comparison of CK-MB to total CK values; satisfactory
precision near the upper reference limit, although the
results are based on a small difference between two low
numbers; good linearity within the dynamic range of the
assay; and adequate sensitivity for detecting the MB concentrations encountered in normal sera. Furthermore, the
results are available within 35 min and during nights,
weekends, and holidays in a stat laboratory,
so that laboratories can conveniently provide clinicians with values for
both total CK and CK-MB in emergency situations.
Our clinical evaluation was planned to emphasize the
agreement and disagreement in results of the two MB
assays (immunoprecipitation
and electrophoretic) rather
than their specificity and sensitivity for the diagnosis of
AMI. The clinical findings demonstrate that the temporal
sequence of CK-MB after AMI was the same by both
methods and that, as with other methods, measuring isoenzyme concentrations at the proper time in the post-AMI
period is absolutely essential (8). On the other hand,
although correlations of patients’ results were excellent,
the significant difference between the peak activity values
obtained with the evaluated immunoassay and the electrophoretic method confirmed the need to use method-specific
reference intervals. Given the possibility
of a defective
recovery of serum CK-MB by the new method, exclusive of
differences in the reaction conditions of the two systems,
one should not try to interpret the CK percentage obtained
for samples determined by Impres-MB in terms of that
obtained by electrophoresis.
The skillful technical assistance of Mrs. Olga Alebardi is gratefully acknowledged.
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CLINICALCHEMISTRY, Vol.36,No.3,1990 553