RÉSUMÉ
We describe the certification of a mass concentration value in the already prepared creatine kinase-2 reference material (BCR 608). Creatine kinase-2 was purified from human heart. The purified enzyme was diluted in order to measure its protein concentration by the Doetsch method. A protein concentration value of 124.30+/-13.17 mg/l was assigned to the stock solution of purified creatine kinase-2. This stock solution was diluted in 25 mmol/l piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) pH 7.2, containing 2 mmol/l ADP, 5 mmol/l 2-mercaptoethanol, 154 mmol/l sodium chloride and 50 g/l human albumin to obtain a stable liquid standard of known creatine kinase-2 mass concentration (80.36 microg/l). This standard was then used to recalculate the creatine kinase-2 mass concentration measured in the BCR 608 material by immunoassay. The mass concentration of creatine kinase-2 in samples of reconstituted BCR 608 was certified to be 93.30+/-9.65 microg/l.
Sujet(s)
Creatine kinase/analyse , Isoenzymes/analyse , Calibrage , Attestation , Chromatographie d'échange d'ions , MB Creatine kinase , Électrophorèse sur gel de polyacrylamide , Stabilité enzymatique , Humains , Myocarde/enzymologie , Normes de référence , Valeurs de référenceRÉSUMÉ
The Elecsys Troponin T third generation assay uses recombinant human cardiac troponin T as standard material. The new assay has a linear calibration curve. Thus, linearity problems observed with the second generation assay have been eliminated. The assay has high precision, especially at the low end of measuring range (inter-assay CV < 10% at 0.1 microgram/L). The new standardization does not change the cut-off value of 0.1 microgram/L. The use of recombinant human cardiac troponin T as standard material enables a reproducible and reliable standardization of troponin T assays.
Sujet(s)
Chimie clinique/normes , Maladie coronarienne/diagnostic , Troponine T , Marqueurs biologiques , Calibrage , Maladie coronarienne/sang , Humains , Modèles linéaires , Protéines recombinantes , Valeurs de référence , Reproductibilité des résultats , Troponine T/analyse , Troponine T/sangRÉSUMÉ
Elecsys assays for the cardiac markers Troponin T (cTnT) and CK-MB have been evaluated in an international multicenter study on the random access analyzer Elecsys 2010 to characterize their clinical performance and their comparability with respective established routine methods. In method comparison studies of Elecsys Troponin T (TnT) with Enzymun-Test TnT, good correlations (r > or = 0.95) and a high degree of correspondence (slopes in 4 laboratories between 0.95 and 1.05) were found. The method comparison studies of Elecsys CK-MB with various CK-MB routine methods lead to good correlations but some systematic deviation in the slopes due to varying standardization. In a reference population of 350 persons upper reference limits (97.5th percentile) of 0.03 milligrams/l for Elecsys TnT and 3.1 milligrams/l for Elecsys CK-MB were found. In cardiosensitivity studies the equivalent diagnostic information of the new Elecsys assays to routine methods was confirmed in the early diagnosis of acute myocardial infarction (AMI), the detection of minor myocardial damages in patients with unstable angina pectoris (UAP) and in time course data monitoring of AMI and bypass surgery patients. The superior sensitivity of cTnT versus CK-MB has been established in a screening situation where in 29 patients with cardiac diseases only cTnT, but not CK-MB, was found pathologically increased; this was due either to the larger diagnostic window of cTnT in AMI or to the more sensitive recognition of minor myocardial damage. In the same study, the cardiospecificity of Elecsys TnT was found to be at least 99.5%. This has also been demonstrated in an earlier study for Enzymun-Test TnT. Further cardiospecificity testing, e.g. in renal failure patients, showed results equivalent to those of Enzymun-Test TnT. An extended clinical study involving 294 patients with chest pain, of whom 58 had a final diagnosis of AMI, revealed highly comparable sensitivity and specificity for the Elecsys assays and routine methods. Thus, the already recommended clinical cut-off values of 0.1 milligrams/l for cTnT and 5 milligrams/l for CK-MB are also valid for the Elecsys assays. The slightly improved sensitivity of Elecsys TnT in the lower range even allows the recognition of pathological increase at cTnT concentrations below 0.1 milligrams/l in special situations with sufficient additional clinical information. Summarizing, provide the two cardiac markers on the Elecsys 2010 at least equivalent or even superior diagnostic information in various clinical situations of cardiac disease compared with routine methods. The short turn-around time and reliable performance qualify the Elecsys assays as new methods of choice for routine and emergency use.
Sujet(s)
Creatine kinase/sang , Dosage immunologique/instrumentation , Mesures de luminescence , Infarctus du myocarde/diagnostic , Traitement du signal assisté par ordinateur/instrumentation , Troponine/sang , Adulte , Sujet âgé , Pontage aortocoronarien , Femelle , Humains , Isoenzymes , Mâle , Adulte d'âge moyen , Infarctus du myocarde/enzymologie , Complications postopératoires/diagnostic , Complications postopératoires/enzymologie , Valeur prédictive des tests , Valeurs de référence , Troponine TRÉSUMÉ
We report on the evaluation of the second-generation assay for cardiac troponin T (cTnT) on the Enzymun system. This new assay is completely specific for the cardiac isoform of TnT, utilizing two cardiospecific monoclonal antibodies. The assay time is reduced to 45 min. The interassay precision shows a median CV of 5.5%; 20% interassay CV was found between 0.05 and 0.1 microg/L. The cardiosensitivity of the second-generation cTnT assay in patients with ischemic myocardial injury appears equivalent when compared with the first-generation assay. We found no falsely positive results in patients with skeletal muscle damage including multitraumas, surgery patients, and marathon runners who showed highly increased values with the unspecific first-generation assay. In Duchenne disease cTnT was still increased, but to a much lower extent. cTnT remains increased in renal failure, but to a lesser degree than with the first-generation assay. The cause of this increase remains unclear. Although a cross-reactivity of skeletal muscle TnT in the second-generation assay could be excluded by our findings, minor myocardial damage or expression of the cardiac isoform of TnT in regenerating muscles cannot be ruled out in those cases with apparently falsely increased cTnT values. The second-generation cTnT assay is a step forward in the combination of cardiosensitivity and cardiospecificity in biochemical markers for diagnosis of heart disease.
Sujet(s)
Ischémie myocardique/diagnostic , Myocarde/composition chimique , Troponine C/analyse , Troponine/analyse , Artéfacts , Marqueurs biologiques/analyse , Humains , Muscles squelettiques/composition chimique , Reproductibilité des résultats , Sensibilité et spécificité , Troponine TRÉSUMÉ
The first generation of troponin T ELISA (TnT 1) can yield false-positive results in patients with severe skeletal muscle injury. Therefore, a cardiac-specific second-generation troponin T ELISA (TnT 2) was developed, in which the cross-reactive antibody 1B10 has been replaced by a high-affinity cardiac-specific antibody M11.7. No cross-reactivity of TnT 2 was observed with purified skeletal muscle troponin T (1000 micrograms/L) or in test samples from 43 marathon runners and 24 patients with rhabdomyolysis and highly increased creatine kinase. TnT 2 was increased > 0.2 microgram/L in 5 of 40 patients with renal failure and in 4 of 20 muscular dystrophy patients. The detection limit is 0.012 microgram/L. Day-to-day imprecision (CV) within the range 0.19-14.89 micrograms/L was < 5.8%. In 4955 patients without myocardial damage, 99.6% had TnT < 0.10 microgram/L. Assay comparison (TnT 1 vs TnT 2) over the whole concentration range (i.e., in 323 samples from AMI-suspected patients) showed a slope, intercept, and standard error of estimate (Sey) of 1.18, 0.01 micrograms/L, and 0.81 microgram/L, respectively.
Sujet(s)
Anticorps monoclonaux , Test ELISA/méthodes , Troponine/immunologie , Animaux , Anticorps monoclonaux/immunologie , Spécificité des anticorps/immunologie , Marqueurs biologiques/analyse , Creatine kinase/analyse , Réactions croisées/immunologie , Défaillance rénale chronique/diagnostic , Souris , Souris de lignée BALB C , Muscles squelettiques/composition chimique , Muscles squelettiques/immunologie , Dystrophies musculaires/diagnostic , Infarctus du myocarde/diagnostic , Myocarde/composition chimique , Myocarde/immunologie , Reproductibilité des résultats , Rhabdomyolyse/diagnostic , Sensibilité et spécificité , Troponine/isolement et purification , Troponine TRÉSUMÉ
We describe a new one-step enzyme immunoassay of troponin T that uses two specific monoclonal antibodies and streptavidin-coated tubes as the solid phase. The monoclonal antibodies were obtained by conventional hybridoma technology, with human troponin T as antigen. The identity of the cardiac troponin T antigen was confirmed by analysis of the amino acid composition and by partial sequence analysis. The specificity of the monoclonal antibodies for cardiac troponin T was proved by immunoblot analysis and displacement curves. The capture antibody is labeled with biotin. The second antibody is conjugated to horseradish peroxidase (EC 1.11.1.7). The assay [Enzymun-TestR system (Boehringer Mannheim GmbH)] is performed in only 90 min at room temperature; the measuring range for troponin T is 0.1 to 15 micrograms/L. The assay shows excellent between-run precision (CV = 3.3-4.9%).
Sujet(s)
Techniques immunoenzymatiques , Muscles/composition chimique , Myocarde/composition chimique , Troponine/analyse , Séquence d'acides aminés , Animaux , Anticorps monoclonaux/immunologie , Spécificité des anticorps , Protéines bactériennes , Bovins , Humains , Techniques immunoenzymatiques/normes , Techniques immunoenzymatiques/statistiques et données numériques , Données de séquences moléculaires , Streptavidine , Troponine/sang , Troponine/composition chimique , Troponine TRÉSUMÉ
For clinical studies with erythropoietin (EPO), enzyme-linked immunosorbent assays for the determination of EPO and EPO antibodies were developed. Using polyclonal and monoclonal EPO antibodies in a sandwich technique, serum EPO levels greater than 10 pg/ml (corresponding to 1 mU/ml, calibrated with the 2nd WHO IRP EPO) can be determined. In 103 healthy blood donors, a mean (+/- SD) value of 36 +/- 19 pg EPO/ml was found. Very high EPO concentrations were found in patients suffering from myelodysplastic syndrome and aplastic anemia; elevated levels were associated with rheumatoid arthritis and myelomatosis. No EPO antibodies were detectable in EPO-treated patients.
Sujet(s)
Anticorps/analyse , Érythropoïétine/sang , Hémopathies/sang , Anticorps monoclonaux , Test ELISA/méthodes , Érythropoïétine/immunologie , Hémopathies/immunologie , Humains , Dosage radioimmunologiqueRÉSUMÉ
The co-localization of glutamine synthetase, glial fibrillary acidic protein, galactocerebroside and fibronectin was investigated by immunofluorescence double staining in primary cultures of dissociated brain cells from newborn mice. In cultures, grown in serum-free medium, containing dibutyryl-cAMP, glutamine synthetase was found in about half of the glial fibrillary acidic protein containing astroblasts. After addition of dexamethasone to the cultures, glutamine synthetase appeared also in another cell type, in which no glial fibrillary acidic protein, but fibronectin was detectable. This demonstrates that these cells, which are present in substantial amounts, are not of glial nature. In cultures treated with dibutyryl-cAMP no other cell type was found positive for fibronectin. For cultures grown in serum-containing medium lacking dibutyryl-cAMP, evidence was obtained that glutamine synthetase was induced by dexamethasone only in part of all cells staining for fibronectin. This suggests the presence of two different populations of fibronectin-positive cells. Oligodendrocytes, revealed by staining for galactocerebroside, never contained detectable amounts of glutamine synthetase, irrespective of the presence of dexamethasone. This also holds for cultures grown in serum-free medium containing dibutyryl-cAMP. However, under these conditions, oligodendroblasts are seen only very rarely. Our results demonstrate an unexpected heterogeneity in the cellular composition of primary cultures from newborn mouse brain.
Sujet(s)
Encéphale/physiologie , Glutamate-ammonia ligase/métabolisme , Animaux , Animaux nouveau-nés , Encéphale/enzymologie , Cellules cultivées , Fibronectines/métabolisme , Technique d'immunofluorescence , Galactosylcéramides/métabolisme , Protéine gliofibrillaire acide , Histocytochimie , Protéines de filaments intermédiaires/métabolisme , Souris , Lignées consanguines de sourisRÉSUMÉ
The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.
Sujet(s)
Benzomorphanes/pharmacologie , Cellules hybrides/métabolisme , Morphinanes/pharmacologie , Récepteurs aux opioïdes/métabolisme , Marqueurs d'affinité , Alprostadil , Animaux , Benzomorphanes/analogues et dérivés , Cellules cultivées , AMP cyclique/métabolisme , Relation dose-effet des médicaments , Méthionine-enképhaline/analogues et dérivés , Méthionine-enképhaline/métabolisme , Gliome , Neuroblastome , Prostaglandines E/pharmacologieRÉSUMÉ
Synthesis of carnosine (beta-alanylhistidine) and related peptides by glial cells in primary culture could be demonstrated. After incubation with [3H]beta-alanine, the radiolabeled dipeptides could be isolated from the cell extract and the culture medium. With gamma-amino[3H]butyric acid, however, rapid degradation of the tracer without significant synthesis of homocarnosine (gamma-aminobutyrylhistidine) was observed. Aminooxyacetic acid, a known inhibitor of gamma-aminobutyryl:alpha-ketoglutarate aminotransferase, inhibits the degradation of gamma-amino[3H]butyric acid very strongly and of [3H]beta-alanine partially. After preincubation of the cells with this inhibitor, incorporation of gamma-amino[3H]butyric acid into homocarnosine and related peptides could be demonstrated.
Sujet(s)
Encéphale/métabolisme , Carnosine/biosynthèse , Dipeptides/biosynthèse , Névroglie/métabolisme , Alanine/métabolisme , Animaux , Animaux nouveau-nés , Carnosine/analogues et dérivés , Cellules cultivées , Cinétique , Souris , Acide gamma-amino-butyrique/métabolismeRÉSUMÉ
The cellular distribution of glutamine synthetase was determined by indirect immunofluorescence in cultures of dissociated brain cells from newborn mice. The enzyme could be detected in about 40% of all cells, among which cells with astrocytic morphology were clearly identified. Treatment with the glucocorticoid dexamethasone led to a strong increase in the number of positivity stained cells. Enzyme induction by dexamethasone was maximal after 36 h and at a concentration of 0.1 micrometer. Under these conditions glutamine synthetase specific activity was elevated about six fold. Steroid hormones other than corticosteroids had no effects. The basal activity in these cultures was near that found in brains of newborn mice, but far below the activity in adult brains, showing that in culture the normal development of these cells is disturbed. A comparison of glial and neuronal cell lines showed that glutamine synthetase is present in both types of cell lines at a very low specific activity. Inducibility of this enzyme by dexamethasone was found in glial but not in neuronal cell lines.