Influence of autoantibodies to creatine kinase-BB on assays for MB isoenzyme

We describe the influence of autoantibodies that bind creatine kinase BB (CK-BB) on the methods for MB isoenzyme. If these autoantibodies are present in patients' sera, they cause the formation of macro CK type 1 (immunoglobulin-linked CK-BB). In some of these cases they can bind not only endogenous CK-BB but also CK-MB without significantly affecting enzyme activity. Although these antibodies show distinctly less affinity for CK-MB than for CK-BB, they nevertheless bind CK-MB in these particular sera, because their concentration exceeds that of CK-BB isoenzyme. If a person with such autoantibodies has an acute myocardial infarction, the immunoinhibition method for CK-MB, which does not discriminate between CK-MB and CK-BB, will recognize the increase and peak of CK-MB with time, although persistent macro CK activity will be superimposed on the typical isoenzyme pattern. However, isoenzyme electrophoresis and recently introduced immunoenzymometric assays for CK-MB in these cases may be less sensitive for detecting myocardial infarctions, because the typical increase in CK-MB activity may be identified later in the progression of symptoms, or even be missed.     

Activity concentration and mass concentration (monoclonal antibody immunoenzymometric method) compared for creatine kinase MB isoenzyme in serum

Results of the "Tandem-E CKMB" immunoenzymometric procedure (y) for creatine kinase (CK; EC 2.9.3.2) were compared with electrophoresis (x) for 160 serum samples from patients suspected of having sustained myocardial infarctions. The results correlated well: y, microgram/L (Tandem assay) = 1.3x-6.3 U/L(electrophoresis) (r = 0.95). CK-MB mass measurement was more stable than enzyme activity after storage and appeared to be more sensitive. Sera from 86 other people, which had no detectable CK-MB upon electrophoresis, gave a mean CK-MB value of 1.1 microgram/L (SD 1.3, range 0-8) with the Tandem assay. To determine whether these low values represented actual isoenzyme, we tested for possible interference by heterophile antibodies in the patients' sera by preincubating the samples with mouse serum before the Tandem assay. The mouse serum did not interfere with the assay of sera that had substantial quantities of CK-MB by electrophoresis. However, in five of six samples that were negative by electrophoresis, the CK-MB values were substantially smaller, indicating that the values measured were false-positives caused by the presence of heterophile antibodies directed against mouse proteins, an interference that could be eliminated by pretreatment with mouse serum.     

Evaluation of a new automated system for the determination of ck-mb isoforms

We evaluated a new analyzer (Cardio REP) specifically designed for cardiac CK-MB isoenzyme and isoforms activity, with a performance time of 24 minutes. Ten AMI patients, with times elapsed between the onset of chest pain and admission to hospital ranging from 30 minutes to 4 hours, were monitored every 3–4 hours until the 16th hour of hospitalization. In each serum sample, in addition to total CK-MB and CK-MB isoforms measured by the Cardio REP analyzer, we also assayed total CK activity, CK-MB activity by immunoinhibition method, CK-MB mass concentration, CK-MB isoforms by REP method, troponin T, and myoglobin. The precision study demonstrated acceptable within assay and between assay CVs% for total CK-MB (8.1 and 10.4), MB₁ (9.1 and 14.2), and MB₂ (9.1 and 8.2) isoforms. The method was found to be linear up to 371 U/L for MB₂ isoform fraction and up to 516 U/L for total CK-MB. Results for CK-MB obtained with the Cardio REP correlated well with those for CK-MB activity obtained with the immunoinhibition method (r = 0.869) and those of CK-MB mass concentration (r = 0.923). The sensitivity of the Cardio REP CK isoforms method was found to be greater than that of the REP CK isoforms method. Time to first increased value of MB₂/MB₁ ratio and MB₂ isoform was earlier in comparison to that for CK-MB mass concentrations and similar to that for myoglobin, a marker that, however, lacks specificity. The diagnostic efficiency of CK-MB isoforms and the availability of a real-time, fully automated method for their measurement suggest the utilization of this biochemical marker in emergency for the early diagnosis of AMI.     

Analytical and clinical evaluation of creatine kinase MB mass assay by IMx: comparison with MB isoenzyme activity and serum myoglobin for early diagnosis of myocardial infarction.

We analytically and clinically evaluated Abbott's IMx assay for creatine kinase (CK) isoenzyme MB (CK-MB) in serum. Over a 1-year period, the method was more specific but less precise than catalytic isoenzyme measurements by electrophoresis or immunoinhibition. Sera from different individuals without electrophoretic evidence of CK-MB but containing macro CK type 1 (n = 20), mitochondrial CK (n = 5), or CK-BB (n = 5) were scored as CK-MB negative by the IMx. Likewise, CK-MB-negative by the sera remained so after addition of purified human CK-MM (< or = 7600 U/L) or CK-BB (< or = 8100 U/L). For 39 patients admitted for suspicion of uncomplicated acute myocardial infarction (precordial pain for < or = 4 h), the diagnostic performance of the IMx CK-MB assay on admission and 4 h later was superior to that of total CK activity and compared well with that of CK-MB activity measured by electrophoresis or immunoinhibition. An admission, myoglobin showed a higher diagnostic sensitivity, specificity, and predictive value than did CK-MB and was the most informative test. Diagnostic performance on admission and 4 h later was further improved by considering positivity for myoglobin and for CK-MB by IMx and for the change in each over the first 4 h of hospitalization as criteria. Twelve hours after admission, diagnostic performance was further improved for all CK and CK-MB methods but began to decline for myoglobin.     

Immunoenzymometric assay of creatine kinase with monoclonal antibodies to the MB isoenzyme compared with electrophoresis

We compare a "second-generation" immunoenzymometric assay (Tandem-E CKMB II) for creatine kinase (EC 2.7.3.2) MB with its electrophoretic (Beckman Paragon system) determination. In the former, two monoclonal antibodies are directed against the B and M subunits. We evaluated 502 samples from 253 patients. Precision, linearity, and analytical recovery for both assays were excellent. The two methods correlated well (r = 0.936). The reference interval for individuals with no suspected cardiac disorder was 0-6.0 micrograms/L; that for non-infarct patients was 0-18.0 micrograms/L. Peak CK-MB values determined by the two assays agreed for 95% of the patients, in terms of exceeding the normal reference interval or not. Diagnostic efficiencies were 86% (Tandem) and 88% (electrophoresis). The immunoenzymometric assay showed no cross reaction with other CK isoenzymes. Both assay methods performed well in detecting CK-MB, although there were some false positives by both methods, as judged from electrocardiographic results. When total CK for the Tandem assay exceeds 2000 U/L, we recommend calculation of a ratio (CK-MB, micrograms/L:total CK, U/L).     

Mass concentration of creatine kinase MB isoenzyme and lactate dehydrogenase isoenzyme 1 in diagnosis of perioperative myocardial infarction after coronary bypass surgery

Recent advances in methodology allow the mass concentration of creatine kinase MB isoenzyme (CK-MB), and of lactate dehydrogenase isoenzyme 1 (LD1) to be determined quickly and easily as routine, emergency tests. We evaluated these tests as diagnostic criteria of perioperative myocardial infarction (PMI) after coronary bypass surgery. These tests were compared with the usual measurements of CK-MB activity by immunoinhibition and LD1 by electrophoresis and with other biological markers of myocardial infarction such as total CK, total LD, and aspartate aminotransferase. Sixty-one patients who underwent coronary bypass grafting were followed pre- and postoperatively by enzyme determinations and electrocardiography; a subgroup was monitored by myocardial scintigraphy. CK-MB mass appeared to be the best marker of PMI during the first 48 h, although LD1 was the marker of choice from days 2 to 4.     

影响肌酸激酶同工酶活性检测结果的因素探讨

目的探讨应用酶免疫抑制法检测血清肌酸激酶（CK）同工酶——CK—MB酶活力单位时,引起临床假阳性的因素,以科学合理地解释每一检测结果及解决方法。方法采用回顾性研究,分析125例非心肌梗死患者,应用酶免疫抑制法检测血清标本中CK、CK—MB酶活力单位,分析可能引起检测值CK—MB酶活力单位假性升高的影响因素。结果86例成人非心肌梗死患者中,总CK活力相对较低,癌症患者、脑梗死患者、变态反应疾病患者CK—MB假阳性比例较大,其中CK—MB占CK总活力大于5％有59例,其中53例在6％～21％,6例大于38％;38例新生儿中CK活性范围为145～1974U／L,其中1例新生儿呼吸窘迫综合征患儿CKMB占CK总活力达90％;另1例溶血标本也引起假阳性。结论应用酶免疫抑制法检测非心肌梗死患者CK—MB酶活力单位假性升高的影响因素,可能是血清中存在巨CK、CK—BB、AK等,影响试剂单克隆抗体与肌酸激酶中的M亚基结合,存在未被抑制的CK—M、M亚基同时与B亚基参加酶促反应,而且因计算时结果乘以2,更加扩大误差,是该方法学缺点造成的错误结果,不能作为诊断依据。     

Concordance of creatine kinase-MB activity and mass

The recent availability of monoclonal antibodies that are highly specific for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme should allow for the development of rapid, sensitive, and specific assays of CK-MB mass and activity. However, the relationship between the mass concentration of CK-MB and its activity in plasma has previously been thought by some to be variable. To determine the extent to which discrepancies of potential clinical significance might arise between measurements of activity and mass in plasma, we compared CK-MB activity and concentration in 1298 samples obtained from 226 patients admitted to the cardiac-care unit. CK-MB activity concentration was determined with an immunoadsorption assay, and mass concentration was measured by an automated "sandwich" assay (Magic Lite; Ciba Corning Diagnostics). Both of these assays are based on specific monoclonal antibodies for CK-MB. Values obtained with these assays correlated well (r = 0.94). Normal and abnormal values with the two assays were concordant in 96% of the samples. In all but three instances, differences occurred late after myocardial infarction and were characterized by minimal increases as determined by one method vs values at the upper limit of normal as determined with the other. Thus, measurements of CK-MB mass and activity concentrations in plasma with assays based on these specific monoclonal antibodies are comparable for the detection or exclusion of acute myocardial infarction.     

A sensitive bioluminescent immunoinhibition test for CK-B subunit activity and a CK-MB specific ELISA compared: correlation with agarose electrophoresis and influence of CK-isoenzyme profile on results

Searching for alternatives to the imprecise spectrophotometric tests for low-concentration creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB), we investigated the analytical performance of two potentially superior approaches--a bioluminescent immunoinhibition assay (I, LKB-Wallac) and an ELISA (enzyme-labeled immunosorbent assay) technique (II, Hybritech)--in comparison with an electrophoretic method (III, Beckman). Only I showed good between-day precision (CV 8.3%) at the upper reference limit, allowing reproducible assay of CK-B subunit activity down to at least 3 U/L. In conditions where CK isoenzyme assays remained unaffected by CK-MM concentrations, test results were proportional to the amount of CK-MB in the sample up to at least 50 U/L for I, 120 micrograms/L for II, and 100 U/L for III (r greater than 0.998 by linear regression analysis). For CK-MB-positive samples, the data by I correlated more closely with values by III (n = 24; r = 0.994) than did results by II (n = 15; r = 0.909), but both methods were equally effective in discriminating between samples with or without electrophoretically supranormal CK-MB activity (93% sensitivity). II was entirely CK-MB specific, whereas CK-B activity by I was consistently (18/18) increased in CK-MB-negative samples containing CK-BB (n = 6; r = 0.996) or macro CK, types 1 or 2 (n = 12; r = 0.930). I is highly sensitive for screening for increased non-MM CK activity, the nature of which should be subsequently clarified by electrophoresis.     

Creatine kinase isoenzymes in human cerebrospinal fluid and brain

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.     

The value of serum CK-MB and myoglobin measurements for assessing perioperative myocardial infarction after cardiac surgery

In 41 patients who underwent coronary bypass surgery, creatine kinase (CK)-MB mass concentration was repeatedly measured in serum during and after the intervention using a new two-site immunoenzymetric assay (IEMA). Serum CK-MB activity was determined with the use of four different techniques: immunoinhibition, immunoinhibition-immunoprecipitation, column chromatography and electrophoresis. Myoglobin (Mb) was also measured in each specimen by radioimmunoassay. In the 33 patients who followed a completely uneventful postoperative course, the cumulated CK-MB release was, on the average, 12.2-fold less than after acute myocardial infarction. The CK-MB peak concentrations using the IEMA were 33 +/- 3 micrograms/l (X +/- SEM) and occurred 6.4 +/- 0.5 h after the intervention was started; CK-MB levels had decreased to 2.9 +/- 0.4 micrograms/l at the end of the first postoperative day. The evolution of the CK-MB concentration was parallel to that of the enzyme activity. The serum Mb maximum concentrations (518 +/- 39 micrograms/l) were reached after 3.3 +/- 0.1 h. The other eight patients developed perioperative myocardial infarction (PMI); in this group, the cumulated CK-MB release was higher, and the serum CK-MB postoperative curves were of three different types. The patients with delayed CK-MB peaks (type I pattern) or sustained elevations (type III) of this isoenzyme also showed increased serum Mb levels at the end of the first postoperative day. The PMI patients with early (10 h) CK-MB elevations (type II) did not demonstrate abnormal serum Mb levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct measurement of creatine kinase-MB activity in serum after extraction with a monoclonal antibody specific to the MB isoenzyme

Fusion of splenocytes from A/J mice immunized by creatine kinase (EC 2.7.3.2)-MB isoenzyme (CK-MB) with SP2/0-Ag14 myeloma cell line generated hybridomas producing monoclonal antibodies specific to CK-MB. One of these monoclonal antibodies ("Conan-MB") was used to develop a direct assay for CK-MB activity. In the assay, serum is incubated for 30 min at room temperature with "Conan-MB" immobilized on latex beads. The beads are then washed, and CK-MB activity bound to the antibody is measured after incubation with CK enzyme reagent for 30 min at 37 degrees C. Results with the assay correlated well (r = 0.997) with those for CK-MB concentration as measured by a two-site immunoassay. Neither CK-MM, CK-BB, mitochondrial CK, nor a hemolysate of erythrocytes interfered. Use of this unique monoclonal antibody allows rapid, precise, and direct determination of CK-MB activity.     

Immunochemiluminometric assay of creatine kinase MB with a monoclonal antibody to the MB isoenzyme

Previous two-site immunometric assays for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme have been based on formation of a "sandwich" complex involving CK-MB and antibodies that recognize the CK-MM and the CK-BB isoenzymes. Single-incubation model assays of CK-MB with these antibodies were susceptible to interferences by CK-MM and CK-BB. We produced two anti-CK-MB monoclonal antibodies and studied their suitability for two-site assays. Both antibodies were compatible with anti-CK-MM and anti-CK-BB, but not with each other. Using anti-CK-MB as the tracer antibody eliminated the interference by both CK-MM and CK-BB. Labeling anti-CK-MB with acridinium ester and immobilizing anti-CK-BB on paramagnetic particles, we developed a rapid and highly sensitive chemiluminescent/magnetic separation CK-MB assay. As little as 1 microgram of CK-MB per liter was detectable after 10- or 30-min incubation at room temperature, and the standard curve was linear up to 400 micrograms/L. Results for serum samples by the new assay correlated well (r = 0.94) with those by Corning electrophoretic and the Hybritech Tandem-E immunoenzymometric CK-MB methods. Sera containing macro CK-1 or high concentrations of CK-MM and CK-BB did not interfere. The combined advantages of a more-specific antibody, paramagnetic solid phase, and chemiluminescent label endow this two-site CK-MB assay with performance characteristics and ease of use superior to those of previous assays.     

Non-M CK--a practical measure of creatine kinase isoenzymes in cancer patients

The individual creatinine kinase (CK) isoenzymes CK-BB and CK-Macro II have previously been investigated as potential tumour markers. We believe there is a need for a system to measure those CK forms not usually present in serum. We have studied a CK-MB immunoinhibition kit which measures all residual CK activity following inactivation of M-subunit activity. In 162 patients with cancer we found no difference in grading (+ or -) between detailed isoenzyme studies and the simple non-M assay. In 33 samples with elevated non-M CK, detailed analysis showed BB alone (45%), Macro II alone (9%), or both (36%). Raised activities were mainly found in patients with small cell lung cancer (SCLC) (17/40; 43%) and GI Tumours (6/11; 55%). In patients with SCLC, elevated activities were associated with disseminated disease. Preliminary evidence indicates that Non-M CK may also be a simple means of monitoring initial treatment response.     

Monoclonal antibody inhibiting creatine kinase MM3 but not isoform MM1

Monoclonal antibody CKM-G01 inhibited greater than 99% of the activity of porcine and human creatine kinase(CK)-MM isoenzyme purified from muscle. However, it inhibited only 54% of CK-MM in human serum. Chromatofocusing of serum CK-MM showed that CKM-G01 inhibited 100% of MM3 but not isoform MM1. CKM-G01 inhibited CK-MM2 by 57%. CKM-G01 specifically inhibited only the original CK-M subunit and not the subunit modified by removal of C-terminal lysine by carboxypeptidase N. CKM-G01 can be used for assay of CK isoforms. We devised a new diagnostic reagent involving it, which requires no analytical separation of isoforms, based on the immunoinhibition method, and applied it to early diagnosis of acute myocardial infarction. The "inhibition index," (inhibited CK activity/total CK activity) x 100, increased more rapidly than did total CK and CK-MB. Evidently this diagnostic reagent can be used for easy, early diagnosis of acute myocardial infarction.     

Immunoenzymetric assay for creatine kinase MB with subunit-specific monoclonal antibodies compared with an immunochemical method and electrophoresis

Results of an immunoenzymetric assay (TANDEM-E CKMB) for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme, in which subunit-specific monoclonal antibodies are used, were compared with those by an immunochemical method (Isomune-CK) and electrophoresis (Corning agarose gel). The study involved 200 patients; greater than 500 samples were analyzed by all three methods. The analytical performances were acceptable. Between-method correlation coefficients ranged from 0.881 to 0.975. Two reference intervals were established for the immunoassays: 0-4 micrograms/L (TANDEM) and 0-4 U/L (Isomune) for "normal" patients; 0-9 micrograms/L (TANDEM) and 0-14 U/L (Isomune) for noninfarct patients. Agreement with respect to increased CK-MB as defined by the reference intervals for the noninfarct patient was 96% between TANDEM and electrophoresis, 90% between Isomune and electrophoresis. All three methods are acceptable for use in determining CK-MB, but the overall diagnostic efficiencies for the mass or activity concentration of the isoenzyme and for its proportion of total CK activity, based on the predictive value model, are 92% (electrophoresis, 0-7 U/L), 90% (electrophoresis, 0-4%), 92% (TANDEM, 0-9 micrograms/L), 88% (TANDEM, 0-3% index), 88% (Isomune, 0-14 U/L), and 83% (Isomune, 0-4%). All three methods can detect CK-MB in serum, but its presence is not necessarily diagnostic of acute infarct. We recommend using the actual concentration of CK-MB to evaluate patients with suspected acute myocardial infarct, and the percentage of CK-MB when total CK is very high.     

Clinical and analytical evaluation of different methods for measurement of creatine kinase isoenzyme MB

We evaluated the clinical and analytical performance of the new immunochemiluminometric assay (ICMA; Ciba Corning) for measurement of creatine kinase isoenzyme MB (CK-MB), and compared it with three other methods: immunoradiometric assay (IRMA; International Immunoassay Labs); immunoinhibition assay (Seradyn); and an immunoinhibition/column method (Du Pont). Intra-test precision for all kits was good. We evaluated 32 patients' samples by all four methodologies. Only one of the four methods (aca, Du Pont) showed evidence of linearity. Efficiency in the diagnosis of myocardial injury in our study ranged from 53% (Seradyn) to 96% (Du Pont). We evaluated serial specimens from 20 separate patients by the IRMA and the ICMA to determine whether myocardial injury could be diagnosed earlier by the ICMA. In patients with acute myocardial infarction, the ICMA displayed positive values earlier and longer than the IRMA, suggesting that the ICMA is suited for screening for myocardial damage in hospitalized patients.     

Serum creatine kinase isoenzyme MB concentration after endomyocardial biopsy

Serum total creatine kinase (CK), CK-MB and myoglobin (Mb) were serially determined in 17 patients who underwent endomyocardial biopsy. Mean total CK levels increased from 36 +/- 27 U/l 30 min before biopsy to a maximum of 112 +/- 77 U/l 8 h following the procedure (p less than 0.05). Similarly, Mb concentrations rose from 57 +/- 55 micrograms/l to 119 +/- 57 micrograms/l 30 min after biopsy (p less than 0.05). Normalization of total CK and Mb levels occurred within 16 and 8 h, respectively. A new immunoenzymetric assay (IEMA) was used to measure the mass concentration of the CK-MB molecule. The initial CK-MB levels were 0.2 +/- 0.4 microgram/l; a small but significant elevation was recorded as early as 2 h after biopsy (1.6 +/- 1.5 micrograms/l, p less than 0.05). CK-MB returned to initial concentration 16 h after the beginning of the procedure. Comparison with the maximum CK-MB levels recorded in 16 myocardial infarction patients (258 +/- 172 micrograms/l, range 90-680 micrograms/l) indicated that the modest increase of CK-MB level detected after biopsy probably reflects a limited endomyocardium lesion at the sampling site, excluding any significant myocardial damage. Total CK and Mb, which showed more pronounced elevations than CK-MB, are likely to originate from other sources than the myocardium.     

Analytical patterns and biochemical properties of macro creatine kinase type 2

Here we describe our findings for 105 patients' sera containing macro creatine kinase (CK) type 2, as confirmed by exclusion chromatography. Depending on the technique used for determining isoenzyme CKs (electrophoresis, ion-exchange chromatography, immunoinhibition), this variant CK shows characteristic patterns and interferes in CK-MB assays by different mechanisms and to various degrees, thus complicating test interpretation. Macro CK type 2 evidently is not of cytoplasmic origin; rather it is a separate CK activity of human serum, characterized by its heat stability and, especially, by its increased molecular mass and high energy of activation. These latter characteristics have never been associated with the normal-size, dimeric cytoplasmic CK isoenzymes, but are typical for mitochondrial CK isolated from human tissues. We conclude that mitochondrial CK released after severe cell damage usually appears in blood in macromolecular forms (macro CK type 2), not in a dimeric form.     

A biochemical and clinical comparison of two commercially available creatine kinase iso-enzyme MB assay kits suitable for use in the routine medical laboratory.

Two methods for the measurement of plasma creatine kinase MB (CK-MB) activity were compared for analytical performance, cost, practicality, and diagnostic correlation with clinical and electrocardiographic findings in patients admitted to the coronary care unit of a district general hospital. The methods were column chromatography and immunoinhibition. Both methods were found acceptable, and the method to be adopted would depend on the staff arrangements and resources available in the laboratory.