Highly sensitive enzyme immunoassay for human creatine kinase BB isozyme

A sensitive sandwich-type enzyme immunoassay method for measurement of brain-type isozyme of human creatine kinase (CK-BB) was developed using purified antibodies specific to the B subunit. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and 1 pg of CK-BB was measurable. The assay was specific to the B subunit of creatine kinase (CK-B), and it cross-reacted about 25% with CK-MB, the heart-type isozyme. However, the assay showed no cross-reactivity with CK-MM, the muscle type-isozyme or with neuron-specific gamma gamma enolase. Coefficients of variation in within-run and between-run precision studies for serum CK-B were less than 8%. Serum CK-B levels in healthy adults of various ages (16-59 yr old) ranged from 0.25-1.44 ng/ml, whereas the CK-B concentrations in children (less than 10 yr old) were relatively high, ranging from 1.3-7.4 ng/ml. The CK-B levels in the cerebrospinal fluids (CSF) could be determined by the present method, and they ranged from 0.10-0.76 ng/ml in the samples from patients with non-neuronal disorders. Determination of immunoreactive CK-B in the extracts of various human tissues confirmed previous reports that CK-B was distributed at high concentrations in the central nervous tissue, prostate, uterus, bladder, gastrointestinal tract and heart muscle.     

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.     

Distribution of immunoreactive carbonic anhydrase III in various human tissues determined by a sensitive enzyme immunoassay method

A sensitive sandwich enzyme immunoassay method for measurement of carbonic anhydrase III (CA-III) was established by use of purified antibodies to CA-III. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from E. coli. The assay was highly sensitive and pg levels of CA-III were measureable. Coefficients of variation in within-run and between-run precision studies for serum CA-III were less than 10%. Serum CA-III levels in healthy subjects of various ages ranged from 0.8 to 24 ng/ml. Concentrations of immunoreactive CA-III in the extracts of various human tissues were also determined. Tissues composed of striated muscle contained more than 10 micrograms/mg protein of CA-III, whereas other tissues, including heart muscle, contained less than 0.5 microgram/mg protein. These results were consistent with other data showing that serum CA-III levels were raised in patients with progressive muscular dystrophy but not in those with acute myocardial infarction.     

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.     

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.     

Creatine kinase isoenzymes in the diagnosis of acute cranio-cerebral trauma

The cerebrospinal fluid (CSF) creatine kinase (CK) activity and isozymic spectrum (EC 2.7.3.2) have been examined in patients with craniocerebral injuries of varying severity. The CK activity has been elevated in all the patients. Three isoforms have been detected: CK-BB, CK-MB, and CK-MM. CK-BB has been detected in all the patients in the presence of the total CK activity; this is explained by the isozyme release from the brain tissue during the injury and as a result of functional and structural impairment of the cellular membranes in intensification of lipid peroxidation. The CK-MM activity is due to blood admixture in the CSF and to impaired hematoencephalic barrier during the injury. The presence of CK-MB in the CSF of patients without cardiac symptoms probably results from a recombination of CK-BB and CK-MM isoforms and is of no diagnostic significance. Measurements of the total and isozymic CK activity in the CSF of patients with craniocerebral injuries may become a test for the laboratory diagnosis of the trauma severity and course.     

Macro creatine kinase type 1 with electrophoretic mobility identical to that of the MB isoenzyme

We describe the case of an elderly woman whose symptoms and electrocardiographic pattern initially suggested acute myocardial infarction. The value for total serum creatine kinase (EC 2.7.3.2; CK) was 737 U/L (reference interval: 22-269 U/L), and electrophoresis for CK isoenzymes demonstrated two bands, the more anodal migrating to the CK-MB region and the second migrating between the CK-MB and CK-MM regions. The above-normal total CK and electrophoretic pattern persisted during her 11-day hospital course. The QuiCK-MB (International Immunoassay Labs.) and Tandem-E CK-MB (Hybritech) immunoassays, however, showed CK-MB mass measurements within the normal range. In further investigation with a mixture of patient's serum and human-serum-based control containing all CK isoenzymes, the electrophoretic mobility of only CK-BB was altered, proving that the patient had antibody to the B unit of CK in her serum. Immunofixation revealed the more anodal band to be a CK-IgA lambda complex, and the more cathodal band, a CK-IgG kappa complex. Mixing the patient's serum with polyclonal antibody specific for CK-B slowed the electrophoretic mobility of only the more anodal band. Polyclonal antibody specific for CK-M had no effect on either band. Evidently, this patient had two different types of macro CK type 1, both containing CK-BB. We conclude that macro CK type 1 can mimic CK-MB and be a source of confusion.     

Comparison of enzymatic characteristics of creatine kinase BB from human healthy and tumor stomach tissues

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2, CK) BB isoenzyme from stomach tumor tissue was partially purified and its characteristics were compared with those from healthy tissue. Molecular mass of tumor CK-BB was estimated to be 82 000 by polyacrylamide gel electrophoresis. Tumor CK-BB was separated into 2 main subbands around pH 4.5 and 11, minor subbands around pH 5-7.5 by agarose isoelectric focusing. The isoenzyme reacted with anti-human brain CK-BB antibodies and formed a hybrid, CK-MB, with CK-MM prepared from healthy human skeletal muscle. The above physicochemical and immunological characteristics of tumor CK-BB were the same as those of normal CK-BB from normal stomach tissue. Optimum pH of tumor CK-BB was more acidic than that of normal CK-BB. Affinity for creatine phosphate and heat sensitivity of tumor CK-BB were slightly lower than those of normal CK-BB. Tumor CK-BB was more stable after iodoacetamide and urea treatments.     

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.     

Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme MM

Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CM-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 microliter of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3--17% with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 microgram/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms.     

Determination of creatine kinase isozymes in sera and tissues of patients with various lung carcinomas

Three creatine kinase isozymes (CK-BB, CK-MB and CK-MM) were estimated by immunoassay in tumor tissues and in sera of patients with various lung carcinomas. CK-BB was increased in small cell carcinoma, but not in other lung carcinomas. CK-MM and CK-MB were not increased in any types of carcinoma. Serum CK-BB was increased in all types of lung carcinoma examined, while serum CK-MM and CK-MB were within normal limits in all patients. Serum CK-BB of healthy adults was estimated as 0.32 +/- 0.14 (mean +/- SD) ng/ml, ranging from 0.11-0.68 ng/ml. If CK-BB values above 1.0 ng/ml were considered abnormal, elevation occurred in 28/40 (70%) of patients with small cell carcinoma, 25/67 (37%) with adenocarcinoma, 21/51 (41%) with squamous cell carcinoma, 4/11 (36%) with other carcinoma of the lung and 10/42 (24%) with lung tuberculosis. Since serum CK-BB with lung cancer changed in parallel with the clinical course, this isozyme may be a marker for monitoring the clinical course, especially in small cell carcinoma of the lung.     

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.     

Sensitive enzyme immunoassay for human Cu/Zn superoxide dismutase

A highly sensitive enzyme immunoassay method for measurement of human Cu/Zn superoxide dismutase (SOD) was established. Antisera were raised in rabbits by injecting SOD purified from human erythrocytes, and antibodies to SOD were purified by the use of Cu/Zn SOD-coupled Sepharose 4B. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive, and the minimum detection limit was 3 pg or 0.1 fmol/assay tube. Serum Cu/Zn SOD levels of normal healthy subjects (36.3 +/- 15.6, n = 120, 16-64 years old) were not related to age and sex. It was confirmed that Cu/Zn SOD levels in erythrocytes and blood plasma were significantly enhanced in patients with Down syndrome. Immunoreactive Cu/Zn SOD was detectable in all the tissues examined and was present at high concentrations in brain, adrenal gland, liver, heart and kidney.     

Reassessment of creatine kinase BB as a marker for cancer of the prostate, breast, and lung

We used an enzyme-linked immunoabsorbent assay to measure creatine kinase (CK; EC 2.7.3.2) BB in the sera of 58 cancer patients. A pre-incubation step with an anti-CK-M antibody-coated bead removed M chain components, and CK-BB was quantified with use of an anti-CK-B antibody-coated tube. No cross reactivity was observed with mitochondrial CK or CK-MM; CK-MB cross reacted slightly (1.6%). Macro CK type 1 was measured as CK-BB. Average analytical recovery of purified CK-BB added to serum was 97.7%. Although the enzyme activity of CK-BB is labile, our studies show that this protein is antigenically stable for 12 months when stored frozen. The upper limit of normal for CK-BB concentration was 0.3 micrograms/L (95th percentile, n = 25). Of the 20 cases of breast cancer of various stages, none showed any increases of serum CK-BB. Only two of 18 patients with prostatic carcinoma (stage D), and two of 10 patients with oat-cell carcinoma of the lung had increased concentrations of CK-BB in the serum. Ten patients with squamous cell cancer of the lung had normal concentration of the enzyme. Thus the CK-BB isoenzyme is not frequently increased in cancers of the prostate, lung, and breast, and it has little apparent value as a tumor marker for these diseases.     

Creatine kinase activity and isoenzyme pattern in various normal tissues and neoplasms

I measured total creatine kinase (CK; EC 2.7.3.2) activity and isoenzyme pattern in normal and neoplastic tissues. CK activity was detected in all of them examined. In various tumors it was greater than, less than, or the same as that in normal tissue, no clear correlation being seen between total activity and growth rate or degree of differentiation. In several cases, there was a greater proportion of the CK-MM isoenzyme, and 15 of 53 cases showed an atypical CK-MM band. The atypical CK-MM band, also reported by others, might be an insensitive and nonspecific tumor marker. The CK-BB isoenzyme, ubiquitous in neoplastic tissues, might accordingly be a nonspecific marker. Total CK activity was very low in most tumor tissues. Presumably a bulky tumor or an advanced stage of malignancy is a requisite to release of routinely detectable CK-BB into the circulation.     

Highly sensitive enzyme immunoassay for human brain aldolase C

A sensitive sandwich-type enzyme immunoassay for brain-type isozyme of human aldolase C4 was developed using purified antibodies specific to the C subunit. The antibodies were raised in rabbits by injecting the purified aldolase C4, and purified by means of immunoaffinity chromatography on a column of aldolase C4-coupled Sepharose. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit of aldolase C4 was 3 pg/tube. The assay was specific to the C subunit of aldolase (aldolase C). It cross-reacted about 60% with aldolase AC3, 30% with aldolase A2C2, and 4% with aldolase A3C, but showed no cross-reactivity with aldolase A4, the muscle-type isozyme. Coefficients of variation in within-run and between-run precision studies for serum aldolase C were less than or equal to 11%. Serum aldolase C levels in healthy adults of various ages (16-59 yr old) and both sexes ranged from 8.74-18.9 ng/ml. Immunoreactive aldolase C in the extracts of various human tissues was determined. It was distributed at high concentrations in the central nervous tissue and heart and at significant levels in liver, adrenal glands and testis. The assay of aldolase C in cerebrospinal fluid or serum by employing this sensitive immunoassay might be useful in the diagnosis of neurological disorders or acute myocardial damage.     

Creatine kinase-inhibiting monoclonal antibodies: preparation and characterization of porcine MM isoenzyme-specific antibodies

Five monoclonal antibodies (CKM-B07, F12, D08, H09 and G01) against porcine creatine kinase (CK; EC 2.7.3.2) MM isoenzyme, which inhibit the enzymatic activity, were prepared. The hybridomas which produced monoclonal antibodies were screened by direct measurement of the inhibitory activity of their culture supernatant. Only two of them, however, were found to be measurable by an enzyme-linked immunosorbent assay with porcine CK-MM as an antigen. CKM-G01 inhibited 100% porcine CK-MM activity, while the others, 73-87%. On the other hand, only CKM-H09 inhibited porcine CK-BB activity (15%). CKM-F12 and D08 inhibited more than 50% CK-MB activity, whereas they did not inhibit CK-BB activity. The monoclonal antibodies were also tested for bovine, rabbit and human CK-MM. All the antibodies inhibited bovine and human CK-MM activity as well. In particular, CKM-G01 was found to exhibit more than 98% inhibition of all CK-MM activity tested, indicating that a common or very similar epitope which affects the activity is present on these enzymes. Admixing of CKM-B07 with other antibodies effected synergisms in inhibition, not only to porcine CK-MM activity but also to human CK-MM activity. A mixture of CK-B07 and G01 inhibited 100% human CK-MM activity, suggesting applicability of these monoclonal antibodies to clinical laboratory diagnosis.     

Adenylate kinase mimics creatine kinase-MM isoenzyme in a CK isoenzyme electrophoresis assay

Adenylate kinase activity (AK) originating from erythrocytes, present in hemolyzed serum behaves like creatine kinase MM isoenzyme (CK-MM) in some CK electrophoresis assays that employ, in their visualization reagent kits, adenosine monophosphate (AMP) as the sole inhibitor of AK, rather than a combination of AMP and a more potent inhibitor of erythrocyte AK, diadenosine pentaphosphate (Ap5A), to inhibit all contaminating-AK activities in serum and quantify only the CK isoenzyme activities in serum following electrophoretic fractionation on agarose gel. This can spuriously overestimate the CK-MM fraction and thereby result in underestimation of CK-MM or CK-BB isoenzymes if present. A hemolyzed serum sample obtained from an elderly patient was erroneously reported as containing low CK-MB due to such overestimation of CK-MM fraction in the sample. Supplementing the AMP already present in the visualization reagent formulation, used to estimate CK isoenzyme concentration in serum, with Ap5A can eliminate or effectively minimize AK interference, especially that caused by hemolysis, and thereby prevent reporting false-negative CK-MB result obtained with CK isoenzyme electrophoresis assays. © 1994 Wiley-Liss, Inc.     

Sensitive enzyme immunoassay for human Mn superoxide dismutase

A sensitive sandwich-type enzyme immunoassay for measurement of human Mn superoxide dismutase (Mn SOD) was developed using purified antibodies specific to Mn SOD. The antisera were raised in rabbits by injecting Mn SOD purified from human liver. The antibody IgG, purified by the use of Mn SOD-coupled Sepharose, showed a single band on the immunoblotting test with a crude liver extract. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg human Mn SOD/assay tube. Serum Mn SOD concentrations of healthy adults (77.5 +/- 18.0 ng/ml (1 SD), n = 120, 16-64 yr old) were not related to age or sex. Immunoreactive Mn SOD was detectable in most tissues examined except for erythrocytes. The concentrations of immunoreactive Mn SOD and Cu/Zn SOD in the cerebral cortex were not different among the patients with Alzheimer's disease, and the age matched and young patients without neurological disorders.     

Macro creatine kinase BB isoenzyme in serum: most likely an antigen-autoantibody complex

Nine sera containing an abnormal creatine kinase BB isoenzyme, "macro CK-BB", were examined. Immunoglobulin precipitation after addition of radiolabelled CK-BB suggested that in eight of the sera the enzyme was linked to an immunoglobulin G. Results obtained with papain-digested and with pepsin-digested IgG suggested that the binding of CK-BB occurred in the antigen-binding region (the "Fab-region") of IgG. Each of the two antigen-binding fragments of IgG, obtained by papain-digestion, were CK-BB specific, since they complexed this isoenzyme equally well when excess CK-MM and CK-MB was added. From Scatchard plots the affinity constant for binding of CK-BB to IgG and the BB-binding capacity of four of the sera was calculated. The affinity constant was high and differed little between the sera (range 0.7 x 10(11)-1.6 x 10(11)1/mol). The BB-binding capacity differed widely (range 21-900 microgram of CK-BB per litre of serum), but in each serum it roughly paralleled the activity of the macro CK-BB complex. The results suggest that in eight of the nine sera examined the BB-binding IgG is an antibody with activity directed towards CK-BB.