ABSTRACT
BACKGROUND: Circulating osteocalcin is a well-known marker for bone formation, but none of the commercial kits currently available can be used in automated systems. Here we present the first semiautomated assay for human serum osteocalcin. METHODS: Polystyrene beads were coated with antibodies against the COOH terminus of osteocalcin and used in the COBAS((R)) EIA System. Osteocalcin was detected with peroxidase-conjugated antibodies against the osteocalcin NH(2) terminus. RESULTS: The time required to analyze an unknown sample was 60 min, with a lower detection limit of 4.5 microg/L and a linear dose-response curve between 4.5 and 100 microg/L. The intraassay imprecision (CV) was 5-8% (n = 21); the interassay variation was 6-9% (n = 14). In samples from human volunteers and patients, data generated with the newly developed assay were comparable to those obtained with standard microtiter plate-based assays. CONCLUSIONS: The coated beads assay may be implemented on fully automated analyzers, which not only may further reduce imprecision but may also substantially increase the applicability of osteocalcin as a marker for bone metabolism in the routine clinical setting.
Subject(s)
Osteocalcin/blood , Adult , Age Factors , Aged , Aged, 80 and over , Biomarkers/blood , Bone and Bones/metabolism , Female , Humans , Immunoenzyme Techniques/methods , Male , Microspheres , Middle Aged , Osteocalcin/immunology , Osteoporosis, Postmenopausal/blood , Osteoporosis, Postmenopausal/drug therapy , Polystyrenes , Reagent Kits, Diagnostic , Sex FactorsSubject(s)
Carrier Proteins/blood , Fatty Acids/blood , Myelin P2 Protein/blood , Myocardial Infarction/diagnosis , Neoplasm Proteins , Tumor Suppressor Proteins , Adult , Biomarkers/blood , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Humans , Immunoassay , Microspheres , Middle Aged , Nephelometry and Turbidimetry , Reference Values , Reproducibility of ResultsABSTRACT
Human serum osteocalcin is a well known bone formation marker. On the basis of their different affinities for hydroxyapatite, the total immunoreactive osteocalcin may be separated into two fractions. Six commercial test kits for osteocalcin were compared. All kits reacted with both osteocalcin fractions but the absolute amounts found in the same serum samples differed widely. During serum storage at room temperature, there was no significant loss of osteocalcin during the first 6 h. After longer storage periods, the recorded decrease of osteocalcin depended on the system used: with two kits, over 80% of the original immunoreactive antigen was left after 9 days. It is considered that the different osteocalcin fractions may become useful as markers for different metabolic bone processes. A more precise definition of the various circulating osteocalcin fractions, and the development of separate tests for each fraction, are requirements for the optimal use of osteocalcin as a diagnostic tool for metabolic bone disorders.
Subject(s)
Osteocalcin/blood , Osteocalcin/chemistry , Adult , Blood Preservation , Drug Stability , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoradiometric Assay , Male , Osteocalcin/metabolism , Radioimmunoassay , Reagent Kits, Diagnostic , Temperature , Time FactorsABSTRACT
A cost effective method for emergency and single assays is presented. By this method, a serial (consecutive) determination of reagent blank, sample, standard, and control serum is performed in one single cuvette and with only one pipetting of reagent. Compared to a sequential determination of sample, standard, and control serum, this is specially economical with regard to reagents and cuvettes. A particular application is seen in the field of prefilled cuvette tests, where the consecutive method allows analysis in the sample and in the control serum, and determination of the required standard absorption in a single test. The results found in the determination of glucose and of aspartate aminotransferase show that the consecutive measuring principle is feasible and even results in a more expressive control of accuracy. If the value found in the assay exceeds the measuring range due to too high a substance concentration or activity, or for photometry reasons, it is immediately seen from the incorrect value for the control serum. The same is not possible in the conventional technique, as it uses two separate tests for the sample and for the control serum.
Subject(s)
Aspartate Aminotransferases/blood , Blood Glucose/analysis , Emergencies , Hexokinase , Humans , Reference Values , Spectrophotometry/methodsABSTRACT
The direct molybdate method for the determination of inorganic phosphorus was adapted to the (Cobas) Bio centrifugal analyzer. We evaluated test characteristics such as reaction time, linearity range, accuracy and precision within series and from day to day. Accuracy was checked using control sera, three different types of recovery experiments, and by experiments running in parallel. Because the results of the determination of inorganic phosphorus depend greatly on the method and the procedure used (with/without deproteinization), we checked the accuracy not only by experiments running in parallel, but by comparison with the following methods: the malachite green method without and with two different techniques for deproteinization, the molybdate/p-methylaminophenol sulfate method with and without deproteinization, and the molybdate/vanadate method with deproteinization. Using patients' sera as well as control sera, it was shown that the preferred molybdate method gave, within the normal range of errors, the same results as the malachite green method without deproteinization.
Subject(s)
Phosphorus/blood , Centrifugation/instrumentation , Chemical Phenomena , Chemistry , Coloring Agents , Humans , Methods , Molybdenum , Rosaniline Dyes , Vanadates , VanadiumABSTRACT
Measurement in cuvettes with their longitudinal dimension parallel to the light beam, while under the influence of a centrifugal force, has many advantages that are not immediately apparent. This survey summarizes the advantages in comparison to cuvettes standing with their long axis perpendicular to the light beam: in the latter the pathlength is fixed by the geometry, but if the cuvette is lying longitudinal to the light beam the pathlength is given by the filling volume. Because of the dependence of the pathlength on the filling (reaction) volume when cuvettes are lying longitudinal to the light beam, the calculation formulas show that the calculation factors, both for enzyme and substrate determination, become independent of the reagent volume form. This is demonstrated by simple equations and confirmed in practice by use of the (Cobas) Bio centrifugal analyzer in assays for glucose and aspartate aminotransferase.
Subject(s)
Spectrophotometry/methods , Aspartate Aminotransferases/analysis , Glucose/analysis , Light , MathematicsABSTRACT
The kinetic determination of urea based on the urea/glutamate dehydrogenase method was adapted for the LKB Reaction Rate Analyzer System 8600. A ratio of sample to reagent volume of 1:50 ensures linearity up to 33.3 mmol/l with a day to day precision of 5%. Parallel studies with the urease/glutamate dehydrogenase method were performed with the CentrifiChem System and with the manual Berthelot/Salicylate method.
Subject(s)
Urea/analysis , Autoanalysis/methods , Glutamate Dehydrogenase , Indicators and Reagents , UreaseABSTRACT
In the method described, by modification of the measurement according to changing reagent blanks (7 g albumin/100 ml + reagent for serum, and water + reagent for urine), excellent agreement with the results obtained by the Frankonit method was found. 262 parallel determinations with serum and 103 with urine were carried out. With a correlation of 0.99, the regression line y = x was almost maintained.
Subject(s)
Creatinine/analysis , Creatinine/blood , Creatinine/urine , Evaluation Studies as Topic , Humans , Kinetics , Methods , Regression AnalysisABSTRACT
The determination of iron was adapted to the CentrifiChem System. The iron bound to transferrin is freed with a detergent, reduced to Fe++ with sodium dithionite, and determined with bathophenanthroline disulphonate. The operation consists of one run for the blank value and one analytical run. Although the actual reaction time is extremely short, a reaction time of 8--10 min is recommended for both the blank value and the analysis. This ensures adequate clearing during centrifugation under the influence of Teepol. 2 times 100 mul serum are required, and 80 determinations per hour are possible.
