Clinical assessment of specific enzyme immunoassay for the human cardiac myosin light chain II (MLC II) with use of monoclonal antibodies

A highly specific enzyme-linked "sandwich" immunoassay was developed for determining cardiac myosin light chain II (MLC II) in serum by using an anticardiac MLC II monoclonal antibody and a solid phase consisting of glass rods coated with another monoclonal antibody. We can detect as little as 0.2 ng of cardiac MLC II per assay. The measurable range of cardiac MLC II concentration in serum is 1 to 30 micrograms/L. The assay demonstrated no cross-reactivity with a skeletal muscle MLC within the measurable range. The mean coefficients of variation were 6.1% within assay and 5.1% between assay. The concentration of cardiac MLC II in sera from healthy subjects ranged from 0 to 4.0 micrograms/L (mean 0.75 micrograms/L and median 0 micrograms/L). The concentrations of cardiac MLC II in serum of patients with skeletal muscle disease due to various causes (n = 15) and patients with effort angina (n = 25), in general, were not significantly elevated above normal. In all patients with myocardial infarction, the concentrations of cardiac MLC II were over 4.0 micrograms/L at 12 h after onset. The mean (+/- 1 SD) peak concentration of cardiac MLC II was 16.2 (+/- 4.4) micrograms/L at 90 h (mean) after onset. On the 5th day, the cardiac MLC II concentrations in all patients with myocardial infarction were significantly elevated above normal; none showed abnormal MB-creatine kinase (CK-MB) activity at this time. Thus, the measurement of cardiac MLC II concentration in serum may be useful to provide a specific and sensitive diagnosis of myocardial necrosis at any time period following myocardial infarction.     

Concentration of Mn-superoxide dismutase in serum in acute myocardial infarction

Serum concentrations of manganese-containing superoxide dismutase (Mn-SOD; EC 1.15.1.1), a mitochondrial enzyme present in high concentrations in the heart, were measured successively by enzyme immunoassay in 18 patients with acute myocardial infarction (AMI) and in eight with angina pectoris. Results were compared with creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) concentrations in the same specimens. The mean (and SD) serum Mn-SOD concentration in 120 healthy adults was 77.5 (18) micrograms/L. Mn-SOD concentrations exceeding 150 micrograms/L (greater than mean + 4 SD) were considered above-normal. In patients with AMI, the Mn-SOD concentration was 80 (16.8) micrograms/L on admission and increased gradually after the first hospital day. The peak, 260 (109) microgram/L, occurred on the fourth hospital day, at which time the Mn-SOD concentrations in all AMI patients were above normal. Thereafter the Mn-SOD concentration decreased slowly, but still was above normal in 14 of 18 patients on the seventh hospital day. On the other hand, in patients with angina pectoris, the Mn-SOD concentration was 78 (11) micrograms/L on admission and did not increase significantly [peak value 97.5 (42) micrograms/L on the fourth hospital day]. The serum concentration of Mn-SOD is a potentially useful marker for estimating cardiac mitochondrial damage and for diagnosing AMI, especially in the late phase.     

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)     

S-100ao protein in serum during acute myocardial infarction

Concentrations in serum of S100ao protein (alpha alpha form of S-100 protein, which is present at high concentrations in heart muscle) were successively measured by enzyme immunoassay in 21 patients with acute myocardial infarction (AMI) and six with angina pectoris (ANP). Results were compared with measurements of creatine kinase isoenzyme MB (CK-MB) concentrations in the same specimens. Mean S100ao concentrations in sera from 100 healthy adults were 0.12 (SD 0.08) microgram/L. In patients with AMI, S100ao concentrations were 4.74 +/- 5.27 micrograms/L at admission, peaked 8 h after admission (23.5 +/- 27.7 micrograms/L), then decreased gradually. Among nine AMI patients who were admitted within an hour after their attack, eight showed abnormally high concentrations of S100ao in serum (greater than 0.5 microgram/L), whereas only four showed abnormally high CK-MB concentrations (greater than 5 micrograms/L) in sera at the time of admission. Serum S100ao concentrations remained within the normal range in all six patients with ANP; however, serum CK-MB concentrations were increased in two of them. Therefore, serum S100ao is useful not only for detection of AMI but also for differentiating AMI from ANP.     

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.     

Liquid chromatography and fluorescence polarization immunoassay methods compared for measuring flecainide acetate in serum

We analyzed 99 patients' serum samples for concentrations of a new antiarrhythmic agent, flecainide acetate, by fluorescence polarization immunoassay (FPIA) and "high-performance" liquid chromatography (HPLC). Within-day and between-day coefficients of variation at concentrations in the low and high ends of the therapeutic range were less than 7% for HPLC and less than 9% for FPIA. There was no statistical difference in the mean (+/- SD) concentrations of the clinical serum samples measured by the two methods (607 +/- 334 micrograms/L by HPLC, 602 +/- 344 micrograms/L by FPIA), but results by each differed by a mean of 0.13%. FPIA and HPLC measurements correlated significantly (r = 0.98, P less than 0.05), and were linearly related (slope = 0.970, intercept = 13 micrograms/L) as assessed by orthogonal regression. Both assay methods produced similar concentration measurements and were sufficiently accurate and precise to be used in therapeutic drug monitoring.     

Stratus automated creatine kinase-MB assay evaluated: identification and elimination of falsely increased results associated with a high-molecular-mass form of alkaline phosphatase

We compared the performance of an automated assay of creatine kinase MB isoenzyme (CK-MB) mass (Stratus) with that of a CK-MB enzymatic assay routinely used at our institutions. Both of these assays use the same CK-MB-specific monoclonal antibody to immunocapture CK-MB, thus providing a direct means of comparing a mass assay with an activity assay. Routine CK-MB measurements for 206 samples within the analytical range of both assays revealed the following relationship: Stratus (micrograms/L) = 0.67 (activity U/L) + 0.18 (r = 0.95, Sx.y = 4.45). The linearity, sensitivity, and precision of the Stratus assay were acceptable for routine clinical use. Icteric, lipemic, and hemolyzed samples do not interfere with the assay. During our evaluation we identified a single, clinically significant false-positive sample. Because this patient had alkaline phosphatase values greater than 1100 U/L, we investigated additional samples with increased activities of alkaline phosphatase and found that samples from 12 of 23 patients selected for alkaline phosphatase values greater than 460 U/L produced falsely increased CK-MB values. We determined that a membrane-associated, high-molecular-mass form of alkaline phosphatase was a cause of these falsely increased values and instituted an approach to identify falsely increased Stratus CK-MB values. Samples from 23 of 1933 patients were falsely increased, the increase being clinically significant in samples from 14 of these patients. Consultation with the manufacturer resulted in the successful reformulation of the substrate/wash solution to minimize interferences from high-molecular-mass forms of alkaline phosphatase.     

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.     

Enzyme-linked immunosorbent assay of retinol-binding protein in serum and urine

This highly sensitive method for determining retinol-binding protein in human serum and urine is based on a double-antibody "sandwich"-type enzyme-linked immunosorbent assay. The assayable concentration range is 0.8-48 micrograms/L, the detection limit 0.2 micrograms/L. Within-assay coefficients of variation for 10 determinations at two different concentrations were 5.5 and 5.8%. The corresponding between-assay CVs were 7.9 and 9.2%. We saw no interference from any components of urine or serum. The mean urinary excretion by 30 healthy subjects, as determined by this method, was 101 micrograms/g of creatinine (SD 38.8). The concentration in serum averaged 43 mg/L (SD 12.1).     

S-100ao protein in blood and urine during open-heart surgery

Concentrations of S100a0 protein and CK-MB were measured by enzyme immunoassay in serial samples of arterial and coronary-sinus blood and urine taken from 26 patients who were undergoing mitral valve surgery. The mean concentration of arterial S100a0 in plasma was 0.32 (SD 0.28) ng/mL at the beginning of anesthesia, increased sharply after reperfusion, peaking [14.4 (SD 6.63) ng/mL] after 45 min of reperfusion, then decreased rapidly. The concentration of creatine kinase (CK) isoenzyme MB in arterial blood plasma was greatest 3 h after reperfusion [107 (SD 54.5) ng/mL]. S100a0 concentrations in urine increased dramatically after reperfusion [16,300 (SD 12,000) ng/h vs 44 (SD 32) ng/h], while CK-MB increased slightly [135 (SD 75) ng/h vs 19 (SD 12) ng/h]. These results suggest that S100a0 in cardiac muscle is released into the bloodstream during open-heart surgery and is discharged into the urine more rapidly than is CK-MB. Determination of S100a0 in plasma or urine thus may be useful for estimating damage to heart muscle during open-heart surgery.     

Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody

This semi-automated colorimetric assay for the MB isoenzyme of creatine kinase (EC 2.7.3.2) is based on a monoclonal antibody ("Conan-MB") specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986;32:657-63). A 0.64-cm bead coated with 2 to 3 micrograms of antibody is incubated with 100 microL of serum and 10 microL of 0.2 mol/L beta-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37 degrees C. A solution containing trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB greater than or equal to 12 U/L (n = 347) were verified by electrophoresis on agarose.     

急性心肌梗死患者血浆N-末端脑钠肽前体水平检测及其临床意义

目的探讨急性心肌梗死(AMI)患者住院早期血浆N-末端脑钠肽前体(NT-proBNP)水平与梗死面积、心功能及住院期心脏事件的关系。方法采用ELISA法测定41例AMI患者住院后48小时内血浆NT-proBNP水平,并与患者肌酸激酶(CK)和肌酸激酶同功酶(CK-MB)峰值浓度、左室射血分数(LVEF)、住院期主要不良心脏事件(MACE)对比分析。结果AMI患者血浆NT-proBNP水平明显高于正常对照组(699.44±386.28pg/ml vs 41.75±24.26pg/ml,P<0.001)。血浆NT-proBNP水平与CK、CK-MB峰值浓度呈正相关(r=0.817,P=0.001;r=0.772,P=0.001),与LVEF呈负相关(r=-0.661,P<0.01)。住院期发生MACE患者的血浆NT-proBNP水平明显高于未发生MACE者(971.50±367.01pg/ml vs 393.60±261.16pg/ml,P<0.001)。结论AMI患者血浆NT-proBNP水平与CK、CK-MB峰值呈正相关,与LVEF呈负相关。检测血浆NT-proBNP水平可预测AMI患者梗塞面积、心功能及住院期心血管事件。     

Avidin-biotin enzyme immunoassay of osteocalcin in serum or plasma.

We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).     

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.     

Differentiating muscle damage from myocardial injury by means of the serum creatine kinase (CK) isoenzyme MB mass measurement/total CK activity ratio

We immunoenzymometrically measured creatine kinase (CK) isoenzyme MB in extracts of myocardium and in homogenates of five different skeletal muscles. CK-MB concentrations in the former averaged 80.9 micrograms/g wet tissue; in the skeletal muscles it varied widely, being (e.g.) 25-fold greater in diaphragm than in psoas. CK-MB in skeletal muscles ranged from 0.9 to 44 ng/U of total CK; the mean for myocardium was 202 ng/U. In sera from 10 trauma and 36 burn patients without myocardial involvement, maximum ratios for CK-MB mass/total CK activity averaged 7 (SEM 1) ng/U and 18 (SEM 6) ng/U, respectively. Except for an infant (220 ng/U), the highest ratio we found for serum after muscular damage was 38 ng/U. In contrast, the mean maximum ratio determined in 23 cases of acute myocardial infarction exceeded 200 ng/U. Among seven determinations performed 8 to 32 h after onset of symptoms, each infarct patient demonstrated at least one ratio greater than or equal to 110 ng/U. Ratios observed after infarct were unrelated to treatment received during the acute phase. We propose a CK-MB/total CK ratio of 80 ng/U as the cutoff value for differentiating myocardial necrosis from muscular injury.     

The serum selenium concentration of patients with acute myocardial infarction

Serum selenium concentration was determined in 49 patients with acute myocardial infarction within 4 hours after the beginning of the symptoms. The mean serum selenium concentration of the patients was significantly lower than that of healthy controls (55 +/- 15 micrograms/l vs. 78 +/- 11 micrograms/l). Among the 49 patients with acute myocardial infarction 20 (41%) had serum selenium concentration below the 95% percentile of the healthy control group. It is concluded that the low serum selenium concentration was present in these patients before the acute event and was not a consequence of the myocardial infarction. No relationship was found in this study between the serum selenium concentration and the severity of myocardial infarction if the number of coronary vessels occluded is taken as the criterion of severity. Serum selenium concentration was similar in patients with 1 or more coronary vessels occluded. Patients with anterior or posterior myocardial infarction had similar serum selenium concentrations. A positive correlation was observed between serum selenium concentration and total serum creatine kinase (CK) activity and serum myoglobin (MB). The serum selenium concentration correlated negatively with the ratio CK-MB/total CK activity, which can be interpreted as minor injury of mitochondria during infarction in patients with normal serum selenium concentration.     

Circulating antibodies to mouse monoclonal immunoglobulins in normal subjects--incidence, species specificity, and effects on a two-site assay for creatine kinase-MB isoenzyme

With a two-site CK-MB assay, we screened serum samples from 1008 blood donors for the presence of antibodies to mouse monoclonal immunoglobulin. These antibodies were capable of cross-linking the labeled antibody with the solid-phase antibody in the two-site assay, thus generating a falsely high apparent CK-MB concentration. In 92 (9.12%) of the blood donors tested, apparent CK-MB concentrations of 10-1000 micrograms/L decreased to less than 3 micrograms/L when re-assayed with non-immune mouse serum (10 mL/L) included in the assay reagent. We tested the ability of non-immune sera from other animal species to lower the concentration of apparent CK-MB in 58 of the 92 samples. Bovine and ovine serum were almost as effective as mouse serum; feline, canine, and rabbit serum were less effective. Of the samples tested, 12% (1.1% of the original population screened) showed apparent CK-MB values that either were not depressed by bovine serum or were only partly depressed. We discuss the possible etiology of these antibodies in normal subjects and recommend that all mouse monoclonal two-site assays should contain non-immune mouse serum (or a suitable "irrelevant" mouse monoclonal antibody) to prevent false-positive results.     

Pharmacokinetics of ceftazidime in serum and suction blister fluid during continuous and intermittent infusions in healthy volunteers.

The pharmacokinetics of ceftazidime were investigated during intermittent (II) and continuous (CI) infusion in eight healthy male volunteers in a crossover fashion. The total daily dose was 75 mg/kg of body weight per 24 h in both regimens, given in three doses of 25 mg/kg/8 h (II) or 60 mg/kg/24 h with 15 mg/kg as a loading dose (CI). After the third dose (II) and during CI, serum and blister fluid samples were taken. Seven new blisters were raised for each timed sample by a suction blister technique. Blisters took 90 min to form. Samples were then taken from four blisters (A samples) and 1 h later were taken from the remaining three (B samples). The concentration of ceftazidime was determined using a high-performance liquid chromatography method. After II, the concentrations in serum immediately after infusion (t = 30 min) and 8 h after the start of the infusion were 137.9 (standard deviation [SD], 27.5) and 4.0 (SD, 0.7) micrograms/ml, respectively. The half-life at alpha phase (t1/2 alpha) was 9.6 min (SD, 4.6), t1/2 beta was 94.8 min (SD, 5.4), area under the concentration-time curve (AUC) was 285.4 micrograms.h/ml (SD, 22.7), total body clearance was 0.115 liter/h.kg (SD, 0.022), and volume of distribution at steady state was 0.178 liter/kg (SD, 0.023). The blister fluid (A) samples showed a decline in concentration parallel to that of the concentrations in serum during the elimination phase with a ratio of 1:1. The t1/2 of the A samples was 96.4 min (SD, 3.2). The concentration of ceftazidime in the B blister fluid samples was significantly higher (27%) than in the A samples over time. This shows that blisters may behave as a separate compartment and establishes the need to raise new blisters for each timed sample. The mean AUC/h during continuous infusion was 21.3 micrograms . h/ml (SD, 3.0). The total body clearance was 0.113 liter/h . kg (SD, 0.018), the urinary clearance was 0.105 liter/h . kg (SD, 0.012), and the ceftazidime/creatinine clearance ratio was 0.885. The mean AUC of blister fluid per hour was 84.5% (18.0 micrograms . h/liter; SD, 3.6) compared with that of serum. The A samples did not differ significantly from the B samples. The implications of continuous infusion of beta-lactams for treatment of serious infections are discussed.     

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).     

Quantification of cotinine in plasma and saliva by liquid chromatography

Measurement of cotinine, a nicotine metabolite, has been studied as a method for monitoring smoking behavior and determining smoking status. We describe a specific, sensitive method for quantifying it in plasma and saliva by reversed-phase paired-ion liquid chromatography and detection by absorbance at 257 nm. The cotinine is extracted with methylene chloride, and 2-phenylimidazole is the internal standard. Cotinine peak heights are linearly related to the amount on the column from 0 to 500 ng. The mean (+/- SD) concentration of cotinine in plasma of 31 passively exposed nonsmokers was 2.1 +/- 1.6 micrograms/L (range, 0-7.9 micrograms/L). The regression of saliva cotinine concentration (y) on plasma cotinine concentration (x) at 0, 24, and 48 h in 10 smokers who refrained from smoking for 48 h was y (micrograms/L) = 1.155x (micrograms/L) + 0.245 (r = 0.986). The efficiency of cotinine as a biological marker was determined at 0, 24, and 48 h of smoking abstinence. Within-run CVs were 3.5% (n = 5) and day-to-day CVs 4.4% (n = 6) at 150 micrograms/L.