CLIN.CHEM.3214,657-663 (1986)
DirectMeasurementof CreatineKinase-MBActivityin Serum after Extraction
witha MonoclonalAntibodySpecificto the MB Isoenzyme
Hemant C. Valdya, Yvonne Maynard, David N. Dletzler, and Jack H. Ladenson1
Fusionof splenocytesfrom NJ mice immunizedby creatine
MaterIals and Methods
kinase (EC 2.7.3.2)-MB isoenzyme (CK-MB) with SP2/OPreparation of Antigen
Agi 4 myeloma cell line generated hybridomas producing
monoclonal antibodies specific to CK-MB. One of these
CK-MM and CK-MB were purified from human skeletal
monoclonalantibodies(“Conan-MB”)was used to developa
and heart muscle as described previously (10,11). CK-BB,
purified from human brain, was obtained from Behring
direct assay for CK-MB activity. In the assay, serum is
Diagnostics,
La Jolla, CA 92037. Purified human heart
incubatedfor 30 mm at room temperaturewith “Conan-MB”
mitochondrial
CK was supplied by Dr. Fred Apple. Hybrid
immobilizedon latex beads. The beads are then washed,
CK-MB was prepared
as described by Jacobus (12), as
and CK-MB activityboundto the antibodyis measuredafter
follows.
Subunits
of
OK-MM
and CK-BB were dissociated by
incubationwith CK enzyme. reagent for 30 mm at 37#{176}C.
incubation
for
30
mm
in
a
6.5
mol/L solution of guanidine
Resultswith the assay correlatedwell (r = 0.997) with those
hydrochloride. The M and B subunits were then allowed to
forCK-MB concentrationas measuredby a two-siteimmunoassociate to form hybrid CK-MB, CK-MM, and CK-BB after
assay. Neither CK-MM, CK-BB, mitochondrialCK, nor a
dialysis
overnight against Tris buffer (10 mmol/L, pH 8.0)
hemolysate of erythrocytes interfered. Use of this unique
containing 100 mmol of 2-mercaptoethanol
per liter. The
monoclonalantibodyallows rapid, precise, and directdeterabove solution was further dialyzed overnight against the
minationof CK-MB activity.
same buffer containing 10 minol of 2-mercaptoethanol
per
Measurement
of creatine kinase-MB (CK-MB, CK2; variant of ATP:creatine
N-phosphotransferase,
EC 2.7.3.2) is
well established
as a diagnostic aid in identification
and
determination
of the extent of myocardial infarction.2 The
properties, clinical use, and methods for analysis of OK-MB
have been extensively reviewed
by various authors (1-9).
The analytical
procedures
utilized are qualitative,
semiquantitative,
or quantitative,
and have various degrees of
specificity. The most common procedures consist of measuring the enzymatic
activity of CK-MB after it has been
separated from the other CK isoenzymes on the basis of
differences in charge by electrophoresis or ion-exchange, or
by inimunoinhibition,
or by a combination of immunoinhibition and iinmunoprecipitation.
Alternatively,
the mass of
CK-MB has been measured by immunoassay,
either with
use of one antibody for the assay of B subunit (CK-MB plus
CK-BB) or by two-site antibody techniques. In the latter,
one antibody, usually to the B subunit, is attached to a solid
phase to extract the isoenzymes containing that subunit;
after the solid phase is washed, a labeled (with enzyme or
ss1) antibody to the other subunit is added.
We describe here the use of a unique monoclonal antibody
(“Conan-MB”)
that reacts with CK-MB but not CK-BB or
CK-MM. We use the antibody
to extract
CK-MB from
serum, thus allowing direct measurement
of OK-MB catalytic activity.
Division of Laboratory Medicine, Departments of Pathology and
Medicine, Box 8118, Washington University School of Medicine,
660 S. Euclid Ave., St. Leuis, MO 63110.
‘Author to whom conespondence should be addressed.
2NoJ
abbreviations: CK-MB, CK-MM, CK-BB, isoenzymea of creatine kinase; PBS, phosphate-buffered saline; TBS,
Tris-buffered saline.
Received November 18, 1985; accepted January 13, 1986.
liter. The hybrid CK-MB was purified by chromatography
on a column of diethylarnmnoethyl-Sepharose
(11). Its purity
was confirmed by electrophoresis on agarose gel (10). Protein concentration was estimated by the procedure of Lowry
et al. (13) with bovine serum albumin as a standard. We
labeled OK-MB with im1, using the Bolton-Hunter
reagent
[succimmidyl 3-(p-hydroxy[’I}iodophenyl)propionate;
New
England Nuclear, Boston, MA 02118] as described previously (14).
Production and Characterization of Monoclonal
Antibodies
Immunization
protocol. We injected eight-week-old female
AJJ mice, H2H haplotype (Jackson Laboratories,
Bar Harbor, ME 04609), intraperitoneally
with 25 zg of human OK
MB emulsified in an equal volume of complete Freund’s
adjuvant (Sigma Chemical Co., St. Louis, MO 63178). Four
weeks and eight weeks later, we similarly administered the
same amount of antigen in incomplete Freund’s adjuvant
and phosphate-buffered
saline (PBS; 50 mmol of sodium
phosphate and 150 minol NaC1 per liter, pH 7.2). A final
booster intraperitoneal
injection of 25 g of antigen in PBS
was given four days before fusion, at least three weeks after
the third injection.
Fusion technique. Spleens were removed aseptically
from
the immunized mice. Splenocytes (108) were fused with
SP2/0-Ag14 cells (10), a Balb/c myeloma cell line, in the
presence
of polyethylene glycol (Mr 1500), essentially
as
described
by K#{246}hler
and Milstein (15).
Screening
procedure.
Using
solid-phase
radioimmunoas8ay,
we screened for hybridomas
that produced
antibodies against OK-MB. Goat anti-mouse IgG antibodies
(Pel-Freez Biologicals, Rogers, AR 72756), were coated onto
96-well round-bottom microtiter plates (Dynatech, Alexandria, VA 22314) by incubating 100 1zL of a 2 mg/L solution of
antibody in sodium borate buffer (100 mmolJL, containing
150 mmol NaOl per liter, pH 8.5) overnight at 4#{176}C
or for 2 h
at 37#{176}C.
The plates were washed with Tween-saline
[0.5
CLINICALCHEMISTRY,Vol. 32, No.4, 1986 657
mL of polyoxyethylene
(20) sorbitan monolaurate CFween20), 8.77 g of NaCl, and 0.02 g of NaN3 per liter]. We then
added 100 1zL of hybridoma supernate, incubated the mixture, and washed as above.
We detected the antibodies to human CK-MB in the
superuates as follows. We added 100 000 counts/mm of
radiolabeled
antigen per well and incubated overnight at
4#{176}C.
The radiolabeled OK-MB was diluted in PBS containing 10 g of bovine serum albumin and 1 mmol of 2mercaptoethanol
per liter. The plates were washed and
dried, and the radioactivity
of the bound radiolabeled CKMB was counted in a Packard gamma-counter.
The results
were considered positive if the counts bound to the well
exceeded by at least twofold those for the negative control
wells containing unrelated hybridoma supernates. The positive control wells contained appropriately
diluted subunitspecific monoclonal antibodies to human CK-M and CK-B
(Hybritech, San Diego, CA 92121). Hybridomas producing
antibodies to CK-MB were cloned in soft agar and stored
under liquid nitrogen in Dulbecco’s modified Eagle’s medium (K.C. Biological Inc., Lenexa, KS 66215) containing 100
roL of dimethyl sulfoxide and 300 mL of horse serum per
liter.
Purification of monoclonal antibody from ascites. CAF1/J
mice (Jackson
Laboratories)
primed with pristane
(2,6,10,14-tetramethyl-pentadecane,
Sigma) were injected
intraperitoneally
with i06 hybrid cells. We collected ascites
fluid one to two weeks later. Ascites fluids produced by each
cell line were pooled and stored at -20#{176}C
after the cell
debris was removed by centrifugation. We used a Protein A
affinity column system (&rs’;
Bio-Rad, Richmond, CA
94804) to purify monoclonal antibody from the ascites fluid.
Fractions obtained from the column containing the antibody
were concentrated
with an ultrafiltration
cell (Amicon
Corp., Lexington, MA 02173). The purity of the monoclonal
antibodies was checked by electrophoresis on agarose gel.
Agarose gel electrophoresis.
We electrophoresed ascites
fluid and purified antibodies on agarose gel (Corning agarose film no. 470100; American
Scientific Products, Palo
Alto, CA 94306) with diethylbarbital buffer (“PHAB”; Corning no. 470180) for 40mm at 90 V. We similarly separated
CK isoenzymes by electrophoresis for 30 miii, using Corning
agarose film (no. 470104) and N-morpholino-2-hydroxypropane sulfonate (“Morso”; Corning no. 470046) buffer. To
stain the proteins, we fixed and stained the gels in a 200-mL
mixture of methanol/acetic
acid/water (40/10/50 by vol)
containing 1.25 g of Coomassie Brilliant Blue per liter. The
background was destained in the same mixture (without the
dye) before air-drying.
Determination of isotype. We determined the isotype of the
monoclonal antibody by the Ouchterlouy double-diffusion
technique, with use of discs and mouse isotype-specific
antisera from Miles Scientific, Naperville, IL 60566.
Determination of specificity. We determined the specificity
of the monoclonal antibodies by competitive RIA. This assay
was similar to the RIA used for screening hybridoma
supernatant
fluid except that the radiolabeled OK-MB was
allowed to bind to the monoclonal antibodies in the presence
of various concentrations
of purified OK isoenzymes. One
hundred microliters of affinity-purified
monoclonal antibody
(2 rng/L in PBS containing 10 g of bovine serum albumin)
was bound to the goat anti-mouse IgG immobilized on the
microtiter plate. We then added 100 iL of a mixture of
labeled CK-MB (100 000 counts/mu) and 0 to 1000 ng of
competing isoenzyme of CK per well and incubated the
658 CLINICAL CHEMISTRY, Vol. 32, No.4, 1986
mixture overnight at 4#{176}C.
After washing and drying the
plates, we measured the radioactivity
of the bound radiolabold CK-MB in a Packard ganims’-counter.
The specificity of the “Conan-MB” antibody was confirmed by allowing radiolabeled “Conan-MB” to bind to OK
isoenzymes separated
on agarose gel by electrophoresis.
After electrophoresis, we fixed the gels in a 200-mL mixture
of isopropanol/acetic
acid/water (25/10/65 by vol) for 30 miii
and then washed twice with 200 mL of de-ionized water for
the same time. We then incubated the gels for 4 h at room
temperature in 50 mL of PBS containing, per liter, 10 g of
bovine serum albumin and 500 000 counts/mu
of ‘Ilabeled “Conan-MB”. Unbound radioactivity was removed
by washing with PBS. We then exposed XAR-5 x-ray films
(Eastman Kodak Co., Rochester, NY 14650) to the dried gels
for 24 h at -70 #{176}C
with an intensifier screen and developed
them in an automatic film processing unit.
Clinical Samples and Standards
Serum samples submitted to the Barnes Hospital Chemistry Laboratory or the Coronary Care Unit Laboratory for
CK-MB analysis were stored at 4#{176}C
after the addition of 2mercaptoethanol (final concentration, 10 mmoJJL) for stabilization, and assayed within five days. Samples were assayed by our direct CK-MB procedure (described below) and
also by a two-site enzyme imniunoassay
(Enzygnost OKMB; Behrung Diagnostics, La Jolla, CA 92037). We prepared
heat-inactivated
pooled serum (56 #{176}C
for 30 mm) from excess
sera from the chemistry laboratory and stored it at -70 #{176}C.
The heat treatment was sufficient to inactivate CK activity
so that we could use the serum pool as a matrix for OK-MB
standards and for dilution of samples with high OK-MB
activity. OK-MB used as standard was purified as previously described (11). We determined the enzyme activity of the
high standard kunetically with a Flexigem
centrifugal
analyzer at 37#{176}C,
utilizing a modified procedure of Rosalki
(16) in which creatine phosphate is used as the substrate
and hexokinase and glucose-6-phosphate
dehydrogenase are
coupling enzymes (Electronucleonics
Inc., Fairfield, NJ
07006). We used calibrator 4 (which is in a bovine serum
albumin matrix) from the Enzygnost OK-MB assay and
serum pools of low and high CK-MB activity as controls. All
other reagents were purchased from Sigma Chemical Co.
Procedures
Immobilization
of antibody on latex beads. We immobilized monoclonal antibody by passive adsorption onto 0.8gm-diameter latex beads (LB-8; Sigma Chemical Co.), as
follows. We diluted the beads 20-fold to give a 5 g/L
suspension in coating buffer (0.1 molfL sodium phosphate,
pH 6.0) and pelleted them in an Eppendorf Microfuge
(15000 x g, 5 miii), then resuspended them to 5 g/L in
coating buffer containing 0.1 g of monoclonal antibody per
liter and incubated overnight at 4#{176}C
with gentle rotation.
The coated beads were washed twice with Ths-buffered
saline (TBS; 20 mmol of Tris and 150 mmol of NaC1 per
liter, pH 7.2) and resuspended to 5 g/L in TBS. We determined the amount of mouse immunoglobulin
left in the
supernate
by a “sandwich” enzyme immunoassay.
The
amount of monoclonal antibody bound to the latex beads
was determined by subtracting
the concentration of monoclonal antibody left in the supernate from that in the coating
solution. We found that more than 80% of the antibody was
bound to the beads under these conditions so that 10 uL of
coated-bead suspension contained approximately
1.0 pg of
monoclonal antibody.
Direct assay procedure for CK-MB. The direct assay for
OK-MB activity consists of two incubations separated by a
washing step. During the initial extraction or immunoadsorption phase, OK-MB in serum is bound to monoclonal
antibody immobilized on latex beads. The adsorbed enzyme
is then exposed to OK-reagent in the enzymatic activity
phase. The resulting absorbance is measured at 340 nm and
is proportional to enzyme activity. We performed the assay
as follows:
1. Pipet 100 pL of serum sample, standard solution, or
control into 1.5-mL polypropylene Eppendorf Microfuge
tubes. [Prepare standards by diluting purified OK-MB in
Assay Buffer (TBS containing 1 mmol of 2-mercaptoethanol
per liter) to an activity of -125 UIL, then further dilute this
solution with Assay Buffer to concentrations ranging from
-4 to 125 UIL. Measure the enzymatic activity of the high
standard kinetically and calculate the activity of the other
standards by using the appropriate dilution factor.] Add 100
pL of heat-inactivated
serum to the tubes containing standards and nonserum
controls. Adjust the volume of all
samples to 1 mL with Assay Buffer.
2. Add 25 L of monoclonal antibody-coated
latex partides (approximately 2.5 pg of monoclonal antibody), then
incubate for 30 mm at room temperature.
3. Place the samples in a 4#{176}C
water bath and pellet the
latex beads by centrifugation (15 000 x g, 5 miii). Wash the
beads twice with 1 mL of Assay Buffer and resuspend them
in 0.25 mL of Assay Buffer. Add 0.5 mL of OK-reagent and
transfer the samples to a 37 #{176}C
water bath for 30 miii.
4. Recentriftige
the beads, then remove 0.5 mL of the
supernate and measure its absorbance at 340 rim (we used
the Flexigem).
5. Calculate OK-MB activity (UIL) by comparison with
the slope of the least-squares
regression of A
and the
activity
of the standards. Re-assay samples having values
>100 U/L alter diluting them two- and fourfold in heatinactivated serum.
Results
Characterization of the Monoclonal Antibody
Preliminary
experiments
showed that immunized
A/J
mice had much higher antibody titers to OK-MB in their
serum than did the Balb/c mice conventionally used. Fusing
the spleen cells of four immunized A/J mice with Balb/c
myeloma cell line SP21O-Ag14 generated
13 hybridomas
secreting antibodies against human OK-MB. By competitive
RIA, we found that eight of the 13 hybridomas produced
antibodies that reacted with human OK-MB but did not
recognize human OK-MM or CK-BB. In addition, we found
four hybridomas producing antibodies
specific to the B
subunit and one that produced an antibody specific to the M
subunit. We produced ascites fluid by injecting one of the
cloned hybridomas producing antibody specific to OK-MB
(“Conan-MB”).
This monoclonal antibody, as characterized
by immunediffusion, was of the IgG-2b subclass with kappa light
chains. The demonstration of the specificity of “Conan-MB”
by competitive RIA is shown in Figure 1: “Conan-MB” did
not recognize OK-MM or CK-BB even at concentrations up
to 10 mg/L. We confirmed the specificity of “Conan-MB” by
using hybrid OK-MB, which gave an inhibition pattern
CK-MM
z
z
:3
0
or
__
100
CK-BB
0
0
0
80
60
0
F-
z 40
20
102
5xu2
5#{176}103
10
103
COMPETITOR
ADDED(i/L)
Fig. 1. SpecIficity of “Conan-MB” as determined by competitive RIA
Immobilizedmonoclonalantibody“Conan-MB”was mixedwith various concentrations of CK-MM(x), CK-88 (0), CK-MB(#{149}),
and hybridCK-MB(A) alongwith
100000 counts/mm of Mabe4ed CK-MB (see Materialsand Methods). The
amount(counts)of ‘25Habaled CK-MBboundto “Conan-MB”as a peicentage of
the counts bound with no competitor present are plotted against the log
concentrationof the competitor added
identical to the native CK-MB purified from human heart.
Moreover, radiolabeled
“Conan MB” bound to OK-MB but
not OK-MM or CK-BB separated by agarose gel electrophoresin (Figure 2).
Further characterization
revealed that the binding of
“Conan-MB” to OK-MB did not appear to influence the
enzyme activity. This was also true for CK-MB extracted
from solution by “Conan-MB” immobilized on latex beads.
Because of its specificity and its ability to extract enzymatically active OK-MB, we used “Conan-MB” to develop a
clinical assay for the direct measurement of OK-MB activity’
Assay Development
The assay is performed in two steps, each of which we
have optimized for serum concentration, time, and temperature. We chose 0.8-pm-diameter latex partides for the solid
phase because of their potential for high-density coating and
their ready availabifity. At the concentration of antibody
used for coating, 0.1 g/L, >80% of “Conan-MB” is adsorbed
to a 5 g/L suspension of beads. At lower coating concentrations, virtually all of “Conan-MB” was adsorbed but the
CK-BBClC-M8dC-MM -*
-
4
1;
23
a
Fig. 2. Specific btnding ofradlolabeled
“Conan-MB”to CK-MBseparated by agarose gel electrophoresls: A, CoomassieBrilliant Blue stain of
the agarose gel; B, autoradlograph of the agarose gel Incubated with
radlolabeled “Conan-MB”
We applied3 pg of each purifiedlseenzyme: lane 1, CK-MM;
68; 4, mbture of 3 pg of each CK Isoenzyme
CK-MB; 3, CK-
CLINICALCHEMISTRY, Vol. 32, No. 4, 1986 659
final coated concentration
was less. At higher coating concentrations,
the percentage
of antibody bound decreased,
suggesting that the protein-binding
sites on the beads had
been saturated (Figure 3).
The optimum amount of immobilized “Conan-MB” was
determined by adding increasing amounts of a 5 g/L suspenE
C
sion of coated beads to four different activity concentrations
0
(32-256 U/L) of CK-MB in PBS containing 10 g of bovine
4
serum albumin per liter (Figure 4). After an overnight
incubation
at 4#{176}C,
the “Conan-MB”-coated
beads were
pelleted and washed. The enzyme activity adsorbed on the
beads was measured as A
after 30 mm of incubation with
CK reagent at 37#{176}C;
the enzyme activity left in the supernate was measured by kinetic CK assay, also at 37#{176}C.
The
amount of enzyme activity on the beads increased with
increasing amount of immobilized
“Conan-MB” until a
280
B
plateau was approached at 2 pg of”Conan-MB” (Figure 4A).
Greater than 90% of CK-MB could be bound at each activity
240
of enzyme tested. Rsimining the enzyme activity rems%ining
in the supernate led to similsir condusions (Figure 48). We
200
chose to use 2.5 pg of”Conan-MB” per 25 pL of beads in our
assay.
160
When CK-MB was added to normal serum, a smaller
percentage of enzyme activity was adsorbed by immobilized
120
“Conan-MB” after overnight incubation at 4#{176}C,
even at
high doses of antibody (up to 16 pg), but this effect appeared
to be minimi,ed by diluting the serum. We therefore diluted
100 pL of serum to 1000 pL with Assay Buffer. Standards
and nonserum controls were mixed with 100 pL of heatinactivated
serum and then also diluted to 1000 pL with
Assay Buffer. Under these conditions about 70% of the CKMB in the standards is bound at all concentrations of CKCONAN MB(pg) IMMOBILIZED ON LATEX BEADS
MB tested. To assess the analytical recovery of CK-MB we
added 10.2 or 77.9 U of CK-MB per liter to a serum sample
Fig. 4. Enzymatio activity extracted from four solutions of CK-MBby
different amounts of immobilized “Conan-MB”: A, amounts of CK-MB
initially determined to contain 21.7 UIL; we measured
bound to increasing amounts of latex beads coated with 0.1 g/L
106.9% and 94.1%, respectively, of the added CK-MB.
“Conan-MB” solution and ( the concentrations (U/L) left in the
In assessing the effects of temperature
and time on the
supemates
immunoadsorption
step, we found that the activity of CKThe enzyme activity bound to the beads was assessed by measuring the A
MB was constant for 24 h at either 22#{176}C
or 4#{176}C
in the
alter incubating the washed beads withCKreagent for30 mm at 37 “C. The A
for the solutions containing the two higher concentrations of CK-MB were
supernates obtained after incubation with control beads
measured after four- or twofolddilution.The enzyme activityIn the supemate was
(coated with mouse IgG) (Figure SB); at 37#{176}C,
the enzyme
assessed by kinetic CK assay, also at 37 “C
activity decreased progressively after 30 mm of incubation,
CK-MB activity was mRYimnhly bound to the “Conan-MB”coated beads after 30 miii at room temperature
or 2 hat 4#{176}C temperature. These decreases in bound CK-MB were associated with proportional
increases in CK-MB in the super(Figure 5Aj; after these times, there was a small decrease in
nates, which suggests that the CK-MB dissociated from the
bound CK-MB at 4#{176}C
and a greater decrease at room
monodonal antibody or the CK-MB-antibody
complex dissociated from the latex beads. We chose to incubate samples
with “Conan-MB”-coated
beads for 30 miii at room tempera-n
ture, then to chill them without delay in an ice-water bath
to minimise the release of CK-MB during subsequent washE4
ing steps.
.
After washing and resuspending
the beads at 4#{176}C,
we
1W
initiated
the
second
step
of
the
assay by adding CK-reagent
ZIand transferring
the samples to a 37#{176}C
water bath. The
volume and time of incubation were chosen to produce an
A
of 1.3 for the high-concentration
standard (-125 U/L)
and an A
of 0.16 for a standard containing
CK-MB of 15
U/L, the expected upper reference limit for normal subjects.
The increase
in A
was linear up to at least 128 U/L
100
200
300
400
(Figure 6). The conditions of the second step could be altered
COATING CONCENTRATION (mg/I)
to increase or decrease the sensitivity of the assay.
FIg. 3. Coating of latex beads with monoclonal antibody ‘Conan-MB”
We added 2-mercaptoethanol
to our assay mixture at 1
“Ccnan.M8” Immoblized on 0.8-pm-dIameter latex beads after overnight incubammol/L to enhance enzymatic activity and maintain stabiltion of the beads (5 g/L suspension) at 4 “C with various concentrations of the
ity. Adding it at 10 mmol/L also enhanced the activity but
C.,
83
660
CUNICAL CHEMISTRY, Vol. 32, No.4, 1986
A
4C
a
C
C.)
appeared to increase the dissociation of captured enzyme
from immobilized “Conan-MB.”
In assessing the effect of potentially interfering
substances under our final assay conditions, we found that
serum did not influence the results, even when present at up
to 80% of the initial assay volume. These results, performed
with a 30-mu incubation at room temperature,
were at odds
with those for an overnight incubation at 4#{176}C
(see above).
We continued to use heat-inactivated serum as a matrix for
dilutions and standards because the apparent dissociation of
CK-MB from the latex beads at 22#{176}C
was greater when
serum was present than when only buffer was present (data
not shown). The addition of lysed erythrocytes
as a source of
adenylate kinase (EC 2.7.4.3) did not influence the results
even when the concentration
of hemoglobin (as an assessment of hemolysis) was as much as 246 g/L (Figure 7). The
addition of up to 200 kU of CK-MM or 250 kU of CK-BB per
liter caused only a nonspecific effect at such high amounts
of added enzyme some residual enzymatic activity was not
removed
by two Washes of the latex beads. When we
performed the assay with control beads and deducted the
0.2
small residual enzyme activity (e.g., 36 U/L for the added
200 kU/L CK-MM solution), we found no effect from CK-BB
or CK-MM (Figure 7). We confirmed these results by
repeating the interference studies with four washes of the
latex beads. We also found no influence from mitochondrial
CK.
The within-assay (n = 10) CV, assessed twice, was 5.3%
and 9.5% for a low CK-MB serum pool (13.0 UIL) and 3.2
and 1.2% for a high CK-MB serum pool (105.1 UIL). The
a
C
C.)
between-assay
CV was 13.2% for the Enzygnost calibrator 4
(12.8 U/L), 18.7% for the low serum pool, and 4.5% for the
high serum pool (11 different assays each).
The results for 50 samples with our direct assay for CKMB (y) compared well with the two-site immunoassay
used
by the Barnes Hospital clinical laboratories (x). The linear
regression
equation was y = 0.915x + 0.35 (r = 0.997)
(Figure 8).
TIME (h)
Fig. 5. Effect of incubation time and temperature on the measurement
of CK-MBby the direct assay
CK-MBactivitywas measured as A after 30 mm of Incitation withCK reagent
at 37 “C. PanslA shows enzymaticactivityardracted by incitation at 4 “C (S)
“C (I), or 37 “C (A) wIth 2.5 pg of “Conan-MB” immobilizedon latex beads.
Pansl B shows the erytne activity remaining In solution after Incubation with
“Conan-MB-coated beads (sow sjei*da) or with control beads coated with
mouse IgO (ar, s}tnbOIs). Nonspecific binding to mouse gO-coated beads was
<0.05 ‘ unite for all eanles
Discussion
A wide range of analytical procedures are utilized for the
determination
of CK-MB, each with various degrees
of
specificity toward the other two isoenzymea, CK-MM and
CK-BB. In addition, interference by adenylate kinase, macro CK-1 (CK-BB associated
with immunoglobulin),
and
macro CK-2 (mitochondrial
CK) have been encountered.
Each of the various procedures has its advocates, but
HEMOGLOBIN(gIL)
-s
18
15.4
61.5
246
CK-MM
iof__*-
\#{149}#{149}5
50
U
0
‘z
CK-M5
(U/LI
Fig. 6. Standard curve for the direct assay of CK-MB:4
(mean ±
iSO) from the assay of standards on five dIfferentdays, ass function of
the CK-MB activity concentration
o
#{149}..,,,.
4,,,
,,..,
i
n,,
102
10’
10’
105
INTERFERING SUBSTANCE (CKACTIVITY. U/LI
FIg. 7. Effect of CK-MM,CK-BB, mitochondrial CK, and hemolysis on
the direct assay of CK-MB
me controlCK-MBvaluewas 62 U ci purifiedCKper liter.The hemolysatewas
prepared by freezingwashed human erythrocyles(ABC)red ieee than 24 h.
Weused purifiedCK-MM,CK-B8,and mlsschondflalCK Values hi the presence
of the higher concentrationof lsoenzymes are comecled cr a small residual
aciMlypreeent slier twu washes of the latex beads,
with conSul
beads: 35.9 W. forCK-MMof 200 kUt and 25.0 Ut forCK-B8of 250 ktk1
CLINICALCHEMISTRY, Vol. 32, No.4,1986
661
350#{149}
site enzyme immunoassay. Precision was adequate but can
probably be improved by the use of larger particles or
alternative solid-phase supports. Likewise, use of some of
the other monoclonal antibodies we have developed might
increase the extraction efficiency of CK-MB from serum and
allow the use of shorter incubations.
Perhaps a rapid, selfindicating assay with monoclonal antibodies, suitable for
on-site testing, can be developed for CK-MB, as has been
reported for choriogonadotropin
(38).
300
250
4
This work was supported by the Monsanto/Washington University Hybridoma Contract. We thank Dr. Fred Apple for his gift of the
purified human heai,t mitochondrial creatine kinase. The fusion of
eplenocytes with the myeloma cell line was performed by the
Hybridoma Center at the Washington University School of Medisine, St. Louis.
200
-
(50
100
U
References
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50
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I
I
50-
100
I
I
150
I
I
200
I
I
I
250
300
I
I
350
CK-MB (,g/L)
FIg. 8. ComparIson of CK-MB activity (Li/t) determined by direct assay
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Fiftyaanlee were measuredby bothassays. The linearregressioneadon was
y 0.915x + 0.35, wItha correlationcoeliclent of 0.997
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onto 0.8-pm-diameter
latex particles showed no interferences and gave excellent agreement with results of a two662
CLINICAL CHEMISTRY,Vol.32,
No.4, 1986
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