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An immunochemical procedure for determination of creatine kinase 31 (serum-specific) isoform in human serum evaluated
Clin Biochem, Vol. 23, pp. 225-228, 1990 Printed in Canada. All right8 reserved.
0009-9120/90 $3.00 + .00 Copyright v 1990 The Canadian Society of Clinical Chemists.
An Immunochemical Procedure for Determination of Creatine Kinase 31 (Serum-Specific) Isoform in Human Serum Evaluated MAURO PANTEGHINI, ROBERTO BONORA, FRANCA PAGANI, and OLGA ALEBARDI 1st Laboratory of Clinical Pathology, Spedali Civili, Brescia, Italy Changes in the proportions of individual isoforms of creatine kinase (CK) in serum promptly reflect both myocardial infarction and coronary reperfusion. A new commercial kit has been introduced for measuring CK-31 isoform in serum (ISOFOR-MM, International Immunoassay Labs.). This is an immunochemical assay containing CK-31 specific monoclonal antibody, bound to magnetizable particles, used to immunoextract this isoform. The CK activity of the sample is measured before and after immunoextraction and the difference in the two values gives the measure of CK-31. Extraction of CK-31 was complete at <-1200 U/L. Analysis of between-day imprecision gave CV between 2.9-7.9%. The method was not susceptible to interference by CK-32 and OK-3 3 isoforms, CK-2 isoenzyme, or mitochondrial CK. Reference interval for CK-3~ (expressed as percent of total CK-3) was 42-69%. Correlation between percent CK-31 by isoform electrophoresis (x) and evaluated procedure (y) was y = 0.83x + 7.6, with r = 0.957 (n = 40). The ISOFORMM performed well enough in this evaluation to replace electrophoresis or isoelectric focusing for measurement of CK-31 isoform.
KEY WORDS: creatine kinase isoenzyme; isoforms of isoenzyme; immunochemical assay; "kit" method. Introduction
igh resolution separation techniques have shown multiple forms of creatine kinase (CK; ATP: H creatine N-phosphotransferase, EC 2.7.3.2) isoenzymes in human serum (1). Specifically, five isoforms (subforms derived from the same isoenzyme that exhibit the same specific catalytic activity and slightly different isoelectric points), three of CK-3 (CK-31, CK-32, CK-33), and two of CK-2 (CK-21 and CK-22), can be observed in human sera after electrophoresis on agarose (2), polyacrylamide gel (3), cellulose acetate membrane (4,5), isoelectric focusing (3,6), anion exchange liquid chromatography (7), and chromatofocusing (8). Only CK-33 and CK-22 (the geneencoded forms) are present in extracts of muscle
Correspondence: Dr. Mauro Panteghini, I Laboratorio Analisi Chimico-Cliniche, Spedali Civili, 25123 Brescia, Italy. Manuscript received October 30, 1989; revised January 21, 1990; accepted January 26, 1990. 1Presented in part at the 8th International Congress of Clinical Enzymology, Toronto, Canada, May 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, J U N E 1990
tissue (9), whereas, CK-32, CK-31, and CK-21 represent the additional forms produced post-translationally by the hydrolysis of the carboxyterminal lysine residue on the native M subunit by plasma carboxypeptidase N (10). The clinical utility of CK isoform determination has been actively investigated (11). In particular, separating the isoforms in the serum of patients with acute myocardial infarction permits one to time the onset of myocardial necrosis (4), to have an earlier detection of acute enzyme release from necrotic myocardium (12), and to distinguish between reperfused and nonreperfused patients after thrombolytic therapy (13). However, all methods developed thus far are too slow or cumbersome for routine use in making clinical decisions (14). Therefore, a method for determining CK isoforms, suitable for routine analysis, is desirable. Significant improvement would be expected with immunoassay methods. Preparation of monoclonal antibody, specific for inhibiting CK-33 (the pure gene product), has been described (15). In addition, a new commercial diagnostic reagent, using CK-31 isoform specific monoclonal antibody to immunoextract this serum isoform, has been recently introduced (16). The CK activity is measured before and after extraction; the difference in the two values gives the measure of CK-31 (the pure postsynthetic form). In this report, we have evaluated the analytical performance of this newly commercialized test kit and compared results with those obtained by an electrophoretic technique. Materials a n d m e t h o d s ASSAY OF TOTAL CK ACTIVITY
CK activity was measured at 37 °C by the method recommended by the Scandinavian Committee on Enzymes (17), with reagents from Boehringer, Mannheim, F.R.G., and a Cobas Bio analyzer (F. HoffmanLa Roche and Co., Ltd., Basel, Switzerland). Results were expressed as U (~LmO1• min-1) per liter. Upper reference limits were 160 U/L for women and 190 U/L for men. Day-to-day CV, calculated for monthly control periods, never exceeded 2.1% (mean 171 U/L). 225
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Figure 1--Effectiveness of immunochemical assay with specimens containing high CK-3 activities. A strongly positive CK-3 serum (CK-31 at isoform electrophoresis, 88%), with undetectable CK-2, was serially diluted with the zero diluent supplied with the kit. ASSAY OF CK ISOFORMS
Electrophoresis Serum CK isoforms were separated by the technique described previously (4,13). The isoform bands were quantified by fluorometric scanning of the tracings with a Helena "Cliniscan" densitometer (Helena Labs., Beaumont, TX 77704). Specimens with total CK activity exceeding 500 U/L were diluted before analysis. The interassay CV of CK isoform activities was <12% in lower, and <5% in higher ranges of activity. Isoform nomenclature followed accepted practice (1).
obtained from Calbiochem Corp., La Jolla, CA 92037 (CK-33 isoform); from Aalto, Escondido, CA 92025 (CK-32 and CK-31 isoforms); and from Scripps Labs., San Diego, CA 92131 (CK-2 isoenzyme). The composition of these commercially available materials was assayed by isoform electrophoresis (see above) and the homogeneity of each preparation was confirmed. Mitochondrial CK was prepared according to Wevers et al. (18). Results and discussion LINEARITY AND DETECTION LIMIT
CK-31 immunochemical assay The kit assay (ISOFOR-MM, International Immunoassay Labs., Inc., Santa Clara, CA 95054; product no. 204-1) was performed according to the manufacturer's direction. This is an immunochemical assay containing CK-3~ specific monoclonal antibody, bound to magnetizable particles used to immunoextract this isoform from serum. This antibody reagent (100 ~tL) is added to serum sample (100 jxL), to bind CK-31 during a 15 min incubation, on a horizontal rotator set, at room temperature. The magnetizable particles are then separated from the liquid phase on a magnetic rack and the supernatant is assayed for CK activity (see above). This final result, when subtracted from total CK, is used to calculate CK-3~ activity and percentage. CHARACTERIZATION OF HUMAN C K ISOFORMS A N D ISOENZYMES
The purified C K isoenzymes and isoforms, employed for specificity and recovery studies, were
226
The ability of the procedure to completely remove various amounts of CK-31 from serum is shown in Figure 1. As can be seen, extraction of CK-31 was complete at -<1200 U/L. We assessed analytical sensitivity by 10 replicate measurements of the zero diluent (human serum containing no detectable CK activity) supplied with the kit. The minimal detectable activity of CK-31, defined as mean zero signal + 1.42 SD (19), was estimated to be 0.6 U/L. IMPRECISION
Imprecision studies are shown in Table 1. Withinrun imprecision was determined by 20 replicate extractions of CK-31 isoform from three different serum samples in one assay. Between-day imprecision was determined by 10 measurements of the same serum samples; between each experiment, the sera were stored at - 2 0 °C. The CV for intra- and
CLINICAL BIOCHEMISTRY, VOLUME 23, J U N E 1990
IMMUNOCHEMICAL ASSAY FOR CREATINE KINASE 31 DETERMINATION TABLE 1
100 90
y: 0.83X + 7.6 r : O. 957 Syx : 5.4
80
n:
Within-run and Between-day Imprecision Total CK, U/L 655 336 320
CK-31 Isoform, U/L 122 (18.7) a 123 (36.6) 214 (66.9)
CV~ % Within-run Between-day (n = 20) (n = 10) 2.4 1.7 0.9
7.9 4.1 2.9
aNumbers in parentheses refer to CK-31 percentage. interassay imprecision were between 0.9-7.9% for the samples tested. RECOVERY
Analytical recovery studies were performed by adding various quantities of purified h u m a n CK-31 to h u m a n serum which contained a CK-31 activity of 153 U/L. The CK-31 activity was then measured with the immunochemical assay. Analytical recovery was calculated by the following formula: ([amount found - control amount]/amount added) x 100. Recovery with added amounts of 125,250, 500, and 750 U/L averaged 104.9, 99.2, 98.0, and 91.1%, respectively. Average percentage recovery was 98.3% (CV, 5.8%). INTERFERENCE STUDIES
To confirm the CK-31 isoform specificity of the ISOFOR-MM procedure, we tested the effect of other h u m a n CK-3 isoforms on the assay. The isoform electrophoretogram, obtained with a patient serum containing all three CK-3 isoforms, before and after incubation with the antibody reagent, showed the selectivity of the monoclonal antibody utilized in the evaluated test. The addition of increasing quantities (up to 1500 U/L) of purified h u m a n CK-33 and CK-32 isoforms to serum sample, containing a known concentration of CK-31 isoform, did not alter the assayed response of CK-31 from this serum sample. We also found no influence from CK-2 (up to 200 U/L), or mitochondrial CK (up to 600 U/L) isoenzymes. CORRELATION WITH ELECTROPHORESIS
The results of the analytical comparison between the immunochemical assay and cellulose acetate electrophoresis for 40 individual random samples submitted to our clinical laboratory are shown in Figure 2. Results obtained with the new immunochemical procedure and isoform electrophoresis correlated closely overall. In particular, differences between the new and reference methods averaged less than 5%. REFERENCE INTERVAL
To establish the reference interval, sera from 100
CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
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Figure 2--Correlation of results for CK-31 by electrophoresis (x-axis) with those by immunochemical assay (y-axis). Total CK activities of sera ranged from 79-1049 U/L. healthy subjects with total CK activity within the reference interval for our laboratory, and undetectable CK-2 isoenzyme, were studied. Expressed as a percentage of total CK-3 isoenzyme activity, CK-31 made up 5 6 - 7.7%, range 42-69%. These data agree well with those obtained with different methods (1).
Conclusion In conclusion, the use of the proposed assay offers a simple and reliable method for quantification of CK-31 isoform in plasma; it is a sensitive diagnostic tool in a routine clinical chemistry laboratory. In particular, the results are obtained within 35 min and can be made available during nights, weekends, and holidays in a stat lab. Laboratories could easily provide clinicians with values for CK-31 isoform in emergency situations for prospective patient management. A clinical study to evaluate the diagnostic sensitivity, specificity, and predictive value of CK31 isoform determined with this method is currently under way at our laboratory.
Acknowledgements The authors are indebted to Dr. V.D. Shah (International Immunoassay Labs.) for the supply of ISOFOR-MM immunoassay kits.
References 1. Panteghini M. Serum isoforms of creatine kinase
227
PANTEGHINI, BONORA, PAGANI, AND ALEBARDI isoenzymes. Clin Biochem 1988; 21: 211-8. 2. Wevers RA, Olthuis HP, van Niel JCC, van Wilgenburg MGM, Soons JBJ. A study on the dimeric structure of creatine kinase (EC 2.7.3.2). Clin Chim Acta 1977; 75: 377-85. 3. Chapelle JP, Heusghem C. Further heterogeneity demonstrated for serum creatine kinase isoenzyme MM. Clin Chem 1980; 26: 457-62. 4. Panteghini M, Cuccia C, Malchiodi A, Calarco M, Pagnoni N. Isoforms of creatine kinase MM and MB in acute myocardial infarction: a clinical evaluation. Clin Chim Acta 1986; 155: 1-10. 5. Wu AHB, Gornet TG, Wu VH, Brockie RE, Nishikawa A. Early diagnosis of acute myocardial infarction by rapid analysis of creatine kinase isoenzyme-3 (CKMM) subtypes. Clin Chem 1987; 33: 358-62. 6. Yasmineh WG, Yamada MK, Cohn JN. Postsynthetic variants ofcreatine kinase MM. J Lab Clin Med 1981; 98: 109-18. 7. Wu AHB, Gornet TG. Measurement ofcreatine kinase MM sub-type by anion-exchange liquid chromatography. Clin Chem 1985; 31: 1841-5. 8. Nohara R, Sobel BE, Jaffe AS, Abendschein DA. Quantitative analysis for isoforms of creatine kinase MM in plasma by chromatofocusing, with on-line monitoring of enzyme activity. Clin Chem 1988; 34: 235-9. 9. Wevers RA, Delsing M, Klein Gebbink JAG, Soons JBJ. Post-synthetic changes in creatine kinase isozymes (EC 2.7.3.2). Clin Chim Acta 1978; 86: 323-7. 10. Perryman MB, Knell JD, Roberts R. Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine: mechanism for in vivo production of multiple forms of
228
creatine kinase in plasma. Clin Chem 1984; 30: 662-4. 11. Roberts R. Reperfusion and the plasma isoforms of creatine kinase isoenzymes: a clinical perspective. J A m Coll Cardiol 1987; 9: 464-6. 12. Panteghini M, Calarco M. Serum isoforms of creatine kinase MM and MB in myocardial infarction. An appraisal of quantitative, clinical and pathophysiological information. Scand J Clin Lab Invest 1987; 47: 325-9. 13. Panteghini M, Pagani F. Serum isoforms of creatine kinase isoenzymes in acute myocardial infarction after intracoronary thrombolysis. Clin Chem 1987; 33: 2039-42. 14. Wu AHB. Creatine kinase isoforms in ischemic heart disease. Clin Chem 1989; 35: 7-13. 15. Suzuki T, Tomita K, Totani M, Murachi T. A monoclonal antibody inhibiting creatine kinase-MM3, but not MM 1 sub-type. Clin Chem 1988; 34: 1279. 16. Shah VD, Yen SE, Diorio AF, H a m m e r PA. Two commercial test kits for CK-MM isoforms evaluated for early recognition of acute myocardial infarction. Clin Chem 1989; 35: 493-4. 17. The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recommended method for the determination of creatine kinase in blood modified by the inclusion of EDTA. Scand J Clin Lab Invest 1979; 39: 1-5. 18. Wevers RA, Mul-Steinbusch MWFJ, Soons JBJ. Mitochondrial CK (EC 2.7.3.2) in the human heart. Clin Chim Acta 1980; 101: 103-11. 19. Rodbard D. Statistical estimation of the minimal detectable concentration ("sensitivity") for radioligand assays. Anal Biochem 1978; 90: 1-12.
CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
0009-9120/90 $3.00 + .00 Copyright v 1990 The Canadian Society of Clinical Chemists.
An Immunochemical Procedure for Determination of Creatine Kinase 31 (Serum-Specific) Isoform in Human Serum Evaluated MAURO PANTEGHINI, ROBERTO BONORA, FRANCA PAGANI, and OLGA ALEBARDI 1st Laboratory of Clinical Pathology, Spedali Civili, Brescia, Italy Changes in the proportions of individual isoforms of creatine kinase (CK) in serum promptly reflect both myocardial infarction and coronary reperfusion. A new commercial kit has been introduced for measuring CK-31 isoform in serum (ISOFOR-MM, International Immunoassay Labs.). This is an immunochemical assay containing CK-31 specific monoclonal antibody, bound to magnetizable particles, used to immunoextract this isoform. The CK activity of the sample is measured before and after immunoextraction and the difference in the two values gives the measure of CK-31. Extraction of CK-31 was complete at <-1200 U/L. Analysis of between-day imprecision gave CV between 2.9-7.9%. The method was not susceptible to interference by CK-32 and OK-3 3 isoforms, CK-2 isoenzyme, or mitochondrial CK. Reference interval for CK-3~ (expressed as percent of total CK-3) was 42-69%. Correlation between percent CK-31 by isoform electrophoresis (x) and evaluated procedure (y) was y = 0.83x + 7.6, with r = 0.957 (n = 40). The ISOFORMM performed well enough in this evaluation to replace electrophoresis or isoelectric focusing for measurement of CK-31 isoform.
KEY WORDS: creatine kinase isoenzyme; isoforms of isoenzyme; immunochemical assay; "kit" method. Introduction
igh resolution separation techniques have shown multiple forms of creatine kinase (CK; ATP: H creatine N-phosphotransferase, EC 2.7.3.2) isoenzymes in human serum (1). Specifically, five isoforms (subforms derived from the same isoenzyme that exhibit the same specific catalytic activity and slightly different isoelectric points), three of CK-3 (CK-31, CK-32, CK-33), and two of CK-2 (CK-21 and CK-22), can be observed in human sera after electrophoresis on agarose (2), polyacrylamide gel (3), cellulose acetate membrane (4,5), isoelectric focusing (3,6), anion exchange liquid chromatography (7), and chromatofocusing (8). Only CK-33 and CK-22 (the geneencoded forms) are present in extracts of muscle
Correspondence: Dr. Mauro Panteghini, I Laboratorio Analisi Chimico-Cliniche, Spedali Civili, 25123 Brescia, Italy. Manuscript received October 30, 1989; revised January 21, 1990; accepted January 26, 1990. 1Presented in part at the 8th International Congress of Clinical Enzymology, Toronto, Canada, May 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, J U N E 1990
tissue (9), whereas, CK-32, CK-31, and CK-21 represent the additional forms produced post-translationally by the hydrolysis of the carboxyterminal lysine residue on the native M subunit by plasma carboxypeptidase N (10). The clinical utility of CK isoform determination has been actively investigated (11). In particular, separating the isoforms in the serum of patients with acute myocardial infarction permits one to time the onset of myocardial necrosis (4), to have an earlier detection of acute enzyme release from necrotic myocardium (12), and to distinguish between reperfused and nonreperfused patients after thrombolytic therapy (13). However, all methods developed thus far are too slow or cumbersome for routine use in making clinical decisions (14). Therefore, a method for determining CK isoforms, suitable for routine analysis, is desirable. Significant improvement would be expected with immunoassay methods. Preparation of monoclonal antibody, specific for inhibiting CK-33 (the pure gene product), has been described (15). In addition, a new commercial diagnostic reagent, using CK-31 isoform specific monoclonal antibody to immunoextract this serum isoform, has been recently introduced (16). The CK activity is measured before and after extraction; the difference in the two values gives the measure of CK-31 (the pure postsynthetic form). In this report, we have evaluated the analytical performance of this newly commercialized test kit and compared results with those obtained by an electrophoretic technique. Materials a n d m e t h o d s ASSAY OF TOTAL CK ACTIVITY
CK activity was measured at 37 °C by the method recommended by the Scandinavian Committee on Enzymes (17), with reagents from Boehringer, Mannheim, F.R.G., and a Cobas Bio analyzer (F. HoffmanLa Roche and Co., Ltd., Basel, Switzerland). Results were expressed as U (~LmO1• min-1) per liter. Upper reference limits were 160 U/L for women and 190 U/L for men. Day-to-day CV, calculated for monthly control periods, never exceeded 2.1% (mean 171 U/L). 225
PANTEGHINI, BONORA, PAGANI, AND ALEBARDI
100
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~
E e
60
~
40
¢'O "¢'-'
~" ~
20 I
400
I
800
I
I
I
I
I
1200 1600 2000 2400 2800 Total CK-3 activity (U/L)
Figure 1--Effectiveness of immunochemical assay with specimens containing high CK-3 activities. A strongly positive CK-3 serum (CK-31 at isoform electrophoresis, 88%), with undetectable CK-2, was serially diluted with the zero diluent supplied with the kit. ASSAY OF CK ISOFORMS
Electrophoresis Serum CK isoforms were separated by the technique described previously (4,13). The isoform bands were quantified by fluorometric scanning of the tracings with a Helena "Cliniscan" densitometer (Helena Labs., Beaumont, TX 77704). Specimens with total CK activity exceeding 500 U/L were diluted before analysis. The interassay CV of CK isoform activities was <12% in lower, and <5% in higher ranges of activity. Isoform nomenclature followed accepted practice (1).
obtained from Calbiochem Corp., La Jolla, CA 92037 (CK-33 isoform); from Aalto, Escondido, CA 92025 (CK-32 and CK-31 isoforms); and from Scripps Labs., San Diego, CA 92131 (CK-2 isoenzyme). The composition of these commercially available materials was assayed by isoform electrophoresis (see above) and the homogeneity of each preparation was confirmed. Mitochondrial CK was prepared according to Wevers et al. (18). Results and discussion LINEARITY AND DETECTION LIMIT
CK-31 immunochemical assay The kit assay (ISOFOR-MM, International Immunoassay Labs., Inc., Santa Clara, CA 95054; product no. 204-1) was performed according to the manufacturer's direction. This is an immunochemical assay containing CK-3~ specific monoclonal antibody, bound to magnetizable particles used to immunoextract this isoform from serum. This antibody reagent (100 ~tL) is added to serum sample (100 jxL), to bind CK-31 during a 15 min incubation, on a horizontal rotator set, at room temperature. The magnetizable particles are then separated from the liquid phase on a magnetic rack and the supernatant is assayed for CK activity (see above). This final result, when subtracted from total CK, is used to calculate CK-3~ activity and percentage. CHARACTERIZATION OF HUMAN C K ISOFORMS A N D ISOENZYMES
The purified C K isoenzymes and isoforms, employed for specificity and recovery studies, were
226
The ability of the procedure to completely remove various amounts of CK-31 from serum is shown in Figure 1. As can be seen, extraction of CK-31 was complete at -<1200 U/L. We assessed analytical sensitivity by 10 replicate measurements of the zero diluent (human serum containing no detectable CK activity) supplied with the kit. The minimal detectable activity of CK-31, defined as mean zero signal + 1.42 SD (19), was estimated to be 0.6 U/L. IMPRECISION
Imprecision studies are shown in Table 1. Withinrun imprecision was determined by 20 replicate extractions of CK-31 isoform from three different serum samples in one assay. Between-day imprecision was determined by 10 measurements of the same serum samples; between each experiment, the sera were stored at - 2 0 °C. The CV for intra- and
CLINICAL BIOCHEMISTRY, VOLUME 23, J U N E 1990
IMMUNOCHEMICAL ASSAY FOR CREATINE KINASE 31 DETERMINATION TABLE 1
100 90
y: 0.83X + 7.6 r : O. 957 Syx : 5.4
80
n:
Within-run and Between-day Imprecision Total CK, U/L 655 336 320
CK-31 Isoform, U/L 122 (18.7) a 123 (36.6) 214 (66.9)
CV~ % Within-run Between-day (n = 20) (n = 10) 2.4 1.7 0.9
7.9 4.1 2.9
aNumbers in parentheses refer to CK-31 percentage. interassay imprecision were between 0.9-7.9% for the samples tested. RECOVERY
Analytical recovery studies were performed by adding various quantities of purified h u m a n CK-31 to h u m a n serum which contained a CK-31 activity of 153 U/L. The CK-31 activity was then measured with the immunochemical assay. Analytical recovery was calculated by the following formula: ([amount found - control amount]/amount added) x 100. Recovery with added amounts of 125,250, 500, and 750 U/L averaged 104.9, 99.2, 98.0, and 91.1%, respectively. Average percentage recovery was 98.3% (CV, 5.8%). INTERFERENCE STUDIES
To confirm the CK-31 isoform specificity of the ISOFOR-MM procedure, we tested the effect of other h u m a n CK-3 isoforms on the assay. The isoform electrophoretogram, obtained with a patient serum containing all three CK-3 isoforms, before and after incubation with the antibody reagent, showed the selectivity of the monoclonal antibody utilized in the evaluated test. The addition of increasing quantities (up to 1500 U/L) of purified h u m a n CK-33 and CK-32 isoforms to serum sample, containing a known concentration of CK-31 isoform, did not alter the assayed response of CK-31 from this serum sample. We also found no influence from CK-2 (up to 200 U/L), or mitochondrial CK (up to 600 U/L) isoenzymes. CORRELATION WITH ELECTROPHORESIS
The results of the analytical comparison between the immunochemical assay and cellulose acetate electrophoresis for 40 individual random samples submitted to our clinical laboratory are shown in Figure 2. Results obtained with the new immunochemical procedure and isoform electrophoresis correlated closely overall. In particular, differences between the new and reference methods averaged less than 5%. REFERENCE INTERVAL
To establish the reference interval, sera from 100
CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990
/
40
70
~z
60
~E 50 ,-.T
60 30 20 10 J
10
20
30
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60
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80
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CK-3~ lelectrophoresis )
100
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Figure 2--Correlation of results for CK-31 by electrophoresis (x-axis) with those by immunochemical assay (y-axis). Total CK activities of sera ranged from 79-1049 U/L. healthy subjects with total CK activity within the reference interval for our laboratory, and undetectable CK-2 isoenzyme, were studied. Expressed as a percentage of total CK-3 isoenzyme activity, CK-31 made up 5 6 - 7.7%, range 42-69%. These data agree well with those obtained with different methods (1).
Conclusion In conclusion, the use of the proposed assay offers a simple and reliable method for quantification of CK-31 isoform in plasma; it is a sensitive diagnostic tool in a routine clinical chemistry laboratory. In particular, the results are obtained within 35 min and can be made available during nights, weekends, and holidays in a stat lab. Laboratories could easily provide clinicians with values for CK-31 isoform in emergency situations for prospective patient management. A clinical study to evaluate the diagnostic sensitivity, specificity, and predictive value of CK31 isoform determined with this method is currently under way at our laboratory.
Acknowledgements The authors are indebted to Dr. V.D. Shah (International Immunoassay Labs.) for the supply of ISOFOR-MM immunoassay kits.
References 1. Panteghini M. Serum isoforms of creatine kinase
227
PANTEGHINI, BONORA, PAGANI, AND ALEBARDI isoenzymes. Clin Biochem 1988; 21: 211-8. 2. Wevers RA, Olthuis HP, van Niel JCC, van Wilgenburg MGM, Soons JBJ. A study on the dimeric structure of creatine kinase (EC 2.7.3.2). Clin Chim Acta 1977; 75: 377-85. 3. Chapelle JP, Heusghem C. Further heterogeneity demonstrated for serum creatine kinase isoenzyme MM. Clin Chem 1980; 26: 457-62. 4. Panteghini M, Cuccia C, Malchiodi A, Calarco M, Pagnoni N. Isoforms of creatine kinase MM and MB in acute myocardial infarction: a clinical evaluation. Clin Chim Acta 1986; 155: 1-10. 5. Wu AHB, Gornet TG, Wu VH, Brockie RE, Nishikawa A. Early diagnosis of acute myocardial infarction by rapid analysis of creatine kinase isoenzyme-3 (CKMM) subtypes. Clin Chem 1987; 33: 358-62. 6. Yasmineh WG, Yamada MK, Cohn JN. Postsynthetic variants ofcreatine kinase MM. J Lab Clin Med 1981; 98: 109-18. 7. Wu AHB, Gornet TG. Measurement ofcreatine kinase MM sub-type by anion-exchange liquid chromatography. Clin Chem 1985; 31: 1841-5. 8. Nohara R, Sobel BE, Jaffe AS, Abendschein DA. Quantitative analysis for isoforms of creatine kinase MM in plasma by chromatofocusing, with on-line monitoring of enzyme activity. Clin Chem 1988; 34: 235-9. 9. Wevers RA, Delsing M, Klein Gebbink JAG, Soons JBJ. Post-synthetic changes in creatine kinase isozymes (EC 2.7.3.2). Clin Chim Acta 1978; 86: 323-7. 10. Perryman MB, Knell JD, Roberts R. Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine: mechanism for in vivo production of multiple forms of
228
creatine kinase in plasma. Clin Chem 1984; 30: 662-4. 11. Roberts R. Reperfusion and the plasma isoforms of creatine kinase isoenzymes: a clinical perspective. J A m Coll Cardiol 1987; 9: 464-6. 12. Panteghini M, Calarco M. Serum isoforms of creatine kinase MM and MB in myocardial infarction. An appraisal of quantitative, clinical and pathophysiological information. Scand J Clin Lab Invest 1987; 47: 325-9. 13. Panteghini M, Pagani F. Serum isoforms of creatine kinase isoenzymes in acute myocardial infarction after intracoronary thrombolysis. Clin Chem 1987; 33: 2039-42. 14. Wu AHB. Creatine kinase isoforms in ischemic heart disease. Clin Chem 1989; 35: 7-13. 15. Suzuki T, Tomita K, Totani M, Murachi T. A monoclonal antibody inhibiting creatine kinase-MM3, but not MM 1 sub-type. Clin Chem 1988; 34: 1279. 16. Shah VD, Yen SE, Diorio AF, H a m m e r PA. Two commercial test kits for CK-MM isoforms evaluated for early recognition of acute myocardial infarction. Clin Chem 1989; 35: 493-4. 17. The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recommended method for the determination of creatine kinase in blood modified by the inclusion of EDTA. Scand J Clin Lab Invest 1979; 39: 1-5. 18. Wevers RA, Mul-Steinbusch MWFJ, Soons JBJ. Mitochondrial CK (EC 2.7.3.2) in the human heart. Clin Chim Acta 1980; 101: 103-11. 19. Rodbard D. Statistical estimation of the minimal detectable concentration ("sensitivity") for radioligand assays. Anal Biochem 1978; 90: 1-12.
CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990