Immunological Assay of CKMB

TEST
Immunological Assay of CKMB
THEORETICAL CONSIDERATIONS
TEST EVALUATION
Sara Bodner, M.D.
A combination of creatine kinase (CK) and lactate dehydrogenase isoenzymes
provide the best laboratory indication of myocardial infarction, and the classic
method of determining these isoenzymes is electrophoresis (1). Recently developed
immunologic assays for these isoenzymes are quicker and less expensive than
electrophoresis (2). One such assay is an immunochemical method for CKMB
determinations combining an immunoinhibition step with a immunoprecipitation step
(3).
Creatine kinase (CK), found in high concentrations in muscle, catalyzes the
phosphorylation of ADP from phosphocreatine, creating ATP and creatine (Fig. 1).
Phosphocreatine is an energy storage form in muscle and creatine kinase (CK)
catalyzes the rapid replenishment of ATP from phosphocreatine for use in muscle
contraction. In serum, CK activity consists of at least 4 different isoenzymes, as
well as a cross reactive enzyme, adenylate kinase, and other miscellaneous CK
activities (Fig. 2). Structurally, CK is a dimer and 3 forms of the enzyme consist of
3 combinations of a "M" and a "B" monomer, CKMM, CKMB, and CKBB. Other CK
activities include a mitochondrial CK, and a complex containing CKBB and
immunoglobin. Creatine kinase MM is the predominent form in heart and skeletal
muscle, although CKMB is found in substantial quantities in cardiac muscle.
Creatine kinase BB is found in the brain, bladder, GI tract, and neonatal serum (4).
The distribution of isoenzymes in tissues is shown (Table 1). Creatine kinase MB
isoenzyme appears in serum 4 to 6 hours after an infarction, reaches a maximum at
18 to 24 hours, and gradually declines to baseline levels at 48 to 72 hours post
infarction (4). Total CK activity may be used in the estimation of the size of an
infarction.
Total CK activity in serum may be measured using NADH disappearance (340 nm) or
the liberation of phosphate at a quantifiable endpoint. If, however, certain of the
isoenzymes are removed from serum the remaining CK activity reflects only those
isoenzymes which have not been inactivated, removed, or destroyed. Specific CK
isoenzyme activity may be destroyed in 2 ways, immunoinhibition, or
immunoprecipitation. In immunoinhibition, antibody directed against the CKM
monomer inhibits all CKMM activity, one half of the CKMB activity, and none of
the CKBB, mitochondrial CK, adenylate kinase, or other CK activity (Fig. 3,
Step II). In immunoprecipitation anti-CK-M-monomer-antibody, followed by anti-
Fc-segment-antibody cause CKMM and CKMB to precipitate out of solution (Fig. 3,
Step HI). The basis of the "immunochemical" method of CKMB determination is the
subtraction of activity remaining after immunoprecipitation from the activity
remaining after immunoinhibition (Fig. 3, Step IV). This assay is a substantial
improvement over previous assays which employed only an immunoinhibition step
(5-7,10), thus erroneously including CKBB and adenylate kinase activity in the
measurement of CKMB.

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CLINICAL APPLICATIONS
Page 2
Creatine kinase (CK) isoenzyme determinations have a major impact upon the
diagnosis of myocardial infarction, and several studies have been done to determine
whether immunochemical methods are as sensitive, specific, and efficient as
electrophoresis in making this diagnosis (Table 2) (5-7). The standard against which
these tests were compared were multiparameter laboratory determinations
combined with clinical information, and assessed by a panel of experts. While these
studies were imperfect (e.g., lacking a "gold" standard of diagnosis, and occasionally
using electrophoretic CK and LDH determinations to establish the diagnosis) they
indicate that the newer immunochemical method is as useful clinically in the
diagnosis of myocardial infarction as electrophoresis. One study also demonstrates
a 94.4% agreement between CKMB immunochemical and electrophoretic
determinations. Because the CK immunochemical assay performs as well as the
more expensive, more time-consuming electrophoretic method, it is gaining
increased acceptance among pathologists and clinicians (2,8).
Alternative methods for measuring CK isoenzymes include column chromatography,
and radioimmunoassay. Column chromatography is even more time-consuming than
electrophoresis and does not always adequately resolve CKMM, CKMB, and CKBB.
The radioimmunoassay is very sensitive, but expensive, and exposes personnel to the
hazards of radiation (12).
TECHNICAL CONSIDERATIONS
Most methods presently in use for total CK determinations may be adapted for use
in CKMB immunochemical determinations. Serum pretreated with antibody and
centrifugation as outlined in the immunochemical assay (Fig. 3) may be run in the
same way as a total CK, and with a simple arithmetic computation the CKMB result
can be calculated (R CKMB =2X (tube 2 - tube 1) (Fig. 3). Since 2 measured CK
activities are being subtracted, a sensitive spectrophotometer must be used for
h!fh.est accuraey- The small quantities resulting after subtraction may also create
difficulties in precision and accuracy in the lower range of normal. Finally, some
reagents used in total CK determinations are not compatible with substrates and
reagents presently being used in the immunochemical assay. For example: the only
immunochemical CKMB assay now on the market is produced by Roche. It is
compatible with their (Roche) reagents for total CK determinations. Beckman's
assay for total CK using the enzyme module on the Astra is also compatible with the
(Roche) immunochemical assay for CKMB. On the other hand, KDA's reagents for
total CK determinations result in imprecise results when combined with the (Roche)
immunochemical assay for CKMB (13). To ensure highest precision and accuracy,
the appropriate methods for CK determinations and sensitive spectrophotometers
must be used.
CONCLUSION
For many years the gold standard in the diagnosis of acute myocardial infarction has
been the combination of CK and LDH isoenzyme electrophoresis. Recently
f developed immunochemical methods have demonstrated sensitivity, specificity,

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Page 3
efficiency, and predictive values comparable to those of electrophoresis in clinical
trials. These immunochemical methods are also less time-consuming and less
expensive than electrophoretic methods. Because of the economic pressures on
medical care it appears likely that immunochemical methods will at least undergo
further scrutiny, and possibly in part supplant electrophoretic methods in the
diagnosis of acute myocardial infarction.
REFERENCES
1. Galen RS, Ruffel JA, Gambino R. Diagnosis of acute myocardial infarction
relative efficiency of serum enzyme and isoenzyme measurements. JAMA
232(2), 1975.
2. Wu AHB, Bowers GN. Evaluation and comparison of immunoinhibition and
immunoprecipitation methods for differentiating MB from BB and macro forms
of creatine kinase CK isoenzymes in patients and healthy individuals. Clin
Chem 28:2017-2021, 1982.
3. Wicks R, Usatequi-Gomez M, Miller M, Warshaw M. Immunochemical
determination of CK-MB isoenzyme in serum. II. An enzymatic approach.
Clin Chem 28(l):54-58, 1982.
4. Pesa MA. The CK isoenzymes: findings and meanings. Laboratory
Management, October, 1982.
5. Ali M, Laraia S, Angeli R, Fayemi O, Braun EV, Davis E, Palladino PH.
Immunochemical CK-MB assay for myocardial infarction. Am J Clin Pathol
77(5):573-579, 1982.
6. Seckinger DL, Vezquez A, Rosenthal PK, Mendizabal RC. Cardio isoenzyme
methodology and the diagnosis of acute myocardial infarction. Am J Clin
Pathol 80(2):164-169, 1983.
7. Bruno DL, Chitwood J, Koller K, Hill KE, Mostrom J, Savoy J. Creatine
kinase-MB activity: Clinical and laboratory studies of immunochemical
technique with optimized enzymatic assay. Ann Clin Lab Sci 13(l):59-65.
1983.
8. Statland BE. CK isoenzymes. MLO, June 1982, pp 14-15.
9. Galen RS. Tips on technology. MLO, September 1981, pp 13-14.
10. Obzansky D, Lott JA. Clinical evaluation of an immunoinhibitor procedure for
creatine kinase-MB. Clin Chem 26(1):150-152, 1980.
11. Sobel BE, Brenahan GF, Shell WI, et al. Estimation of infarct size in man and
its relation to prognosis. Circulation 46:640-648, 1972.
12. Lott JA, Stung JM. Clin Chem 26:124-150, 1980.
13. Personal communication. Technical representative. Hoffman Roche, Inc.
Nutley, New Jersey.

Table I. Distribution of CK and CK isoenzymes in Various Tissues
Tissue Range of
Total CK
(U/gm tissue)
Range of CK Isoenzymes
M M M B B B
(%)
Skeletal muscle 1080-3050 96-100 0-4 0
Heart muscle 190-692 58-36 15-42 . 0-1
Brain 73-200 0 0 100
Bladder 162 2 6 92
Placenta 250 19 1 80
Gastrointestinal
tract
145 3-4 0-2 96
Thyroid 32-34 4-79 0-6 15-96
Uterus 9-38 2-20 2-20 60-96
Kidney 10-18 10 0 90
Lung 11-13 26-60 0-1 2-14
Prostate 8-9 4-40 3-4 56-93
Spleen 2-7 0-74 0 26-100
Liver 3-4 0-90 0-6 4-100
Pancreas 3 14 1 85

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Table 2. Studies Comparing Electrophoretic and Immunochemical Methods
in the Diagnosis of MI
Ref/Method #Pts 3 Pts w/ MI Sensitivity Specificity PV(+) PV(-) Efficiency Precision
(5)Ali 215 45
Immunochemical Method 95.5 95.3 95.3
Electrophoretic Method 93.3 94.7 94.4
(6) Sukmger
Immunochemical Method
Electrophoretic Method
(7) Bruns
Immunochemical Method
180 36
99 27
100
88
93 78
79
100 97 94 5 to 12%
100
94
95
89
(u = 39)

1 "1 1
Figure I.
Creatinec,^
P h o s p h o c r e a t i n e - | - A n p ^ ^ A T P + C r e a t i n e
Kinase
Figure II.
CK^, • CKtilo , CK
CK Total = olH/IM + UIVMB + olBB + CK(mitochondrial) + Adenylate Kinase
+ O t h e r s

F Ijre III.
ISOMUNE — CK PROCEDURE
I. Add 200 |iI of patient's serum to each of two test tubes
m
qO AK
OQ
Tubel
iQQl
»O0
Tubel
AK
qQ
AK
OB
Tube 2 (Blank tube)
Add 200 ul of solid phase
anti-goat IgG, mix and
incubate 5 min. at ambient
temperature
Tube 2 (Blank tube)
Solid phase
anti-goat IgG
Anti-MM
serum
1.
Add 250 ul of anti-MM serum,
JL mix and incubate for 20 min.
at ambient temperature
/
A
anti-MM
w.
|Q0]
Tubel
AK
1001 AK
■ O g -
QQ
Tubel
Add 50 ul of anti-MM serum,
mix and incubate for 5 min.
at ambient temperature
AK
Tube 2 (Blank tube)
Centrifuge for 5 min. at 1000 x g
Tube 2 (Blank tube)
Subtract the enzyme activity in the supernate of the blank
tube from that of tube 1

Figure II. Fibrinogen Degradation (22)
Fibrinogen
Plasmm
Fibrinogen
Degradation
Products
Thrombin
Fibrin monomer
and macromolecular complexes
non-crosslinked Fibrin
Monomer
Plasmm
non-crosslinked
Fibrin Degradation
Products
Modified from Colman and Hirsch (page 155)
xnia
crosslmked Fibrin
Plasmm
crosslmked
Fibrin Degradation
Products