RESUMO
The development of a particle-enhanced turbidimetric immunoassay for C-reactive protein is described. The method demonstrates excellent precision, with the calibration curve remaining stable for at least 16 weeks. The method compares well with established techniques and there is no interference from a variety of autoantibodies, endogenous serum constituents or commonly used drugs.
Assuntos
Proteína C-Reativa/análise , Imunoensaio/métodos , Autoanticorpos/imunologia , Soluções Tampão , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Hiperlipidemias/sangue , Icterícia/sangue , Látex , Microesferas , Nefelometria e Turbidimetria , Paraproteínas/imunologia , Polietilenoglicóis , Fator Reumatoide/imunologiaRESUMO
A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum beta 2-microglobulin is described. The method has a working range of 0.2-40 mg/l, with good precision and a correlation coefficient of 0.97 when compared with an established radioimmunoassay method. One of the major advantages of this assay is the stability of the calibration curve (up to at least 20 months). This, and the fact that no pretreatment of serum samples is necessary, makes the assay ideally suited for all types of routine determination.
Assuntos
Imunoensaio/métodos , Nefropatias/sangue , Nefelometria e Turbidimetria/métodos , Microglobulina beta-2/análise , Animais , Soluções Tampão , Calibragem , Bovinos , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Icterícia/sangue , Látex , Lipídeos/sangue , Paraproteínas/metabolismo , Polietilenoglicóis , Reprodutibilidade dos Testes , Fator Reumatoide/sangue , Albumina SéricaRESUMO
The use of chromium dioxide particles as a solid support for very sensitive and rapid immunoassays, is the result of the combination of large surface area (40 m2) and high protein uptake capacity (40 mg/g) allowing rapid capture kinetics and high binding capacity. Magnetic and physical properties of these particles give a rapid separation, a complete resuspension and a rapid high-efficiency washing, highly desirable characteristics for efficient automation of immunoassays. Good precision and accuracy, exemplified by excellent recovery, parallelism and correlation were demonstrated. Test results prove that the technology is highly flexible and applicable to a variety of assay formats.
Assuntos
Compostos de Cromo , Cromo , Imunoensaio/métodosRESUMO
A difference spectrophotometric method for the rapid determination of equilibrium constants for protein--ligand interactions has been developed. The method requires no knowledge of the extinction coefficient of either reactants or products. Furthermore the method allows rapid determination of the temperature dependence of a reaction and thus leads to rapid determination of thermodynamic parameters. The method has been tested by following the interactions of ligands with hemerythrin, the nonheme iron, oxygen storage protein isolated from Phasocolopsis gouldii. The reactions were studied at various temperatures and ionic strengths, and standard thermodynamic parameters were determined. The standard thermodynamic parameters for the conversion of metaquohemerythrin to methydroxyhemerythrin were found to be delta H degrees = 5.8 +/- 1.3 kcal mol-1 and delta S degrees = -11.5 +/- 1.5 cal mol-1 deg-1. For the reaction of metaquohemerythrin with thiocyanate ion to produce metthiocyanatohemerythrin delta H degrees = --13.0 +/- 1.6 kcal mol-1 and delta S degrees - --25.3 +/- 5.5 cal mol-1 deg-1. For the reaction of thiocyanate ion with methydroxy-hemerythrin delta H degrees = --6.6 +/- 0.8 kcal mol-1 and delta S degrees = --38.3 +/- 4.0 cal mol-1 deg-1. Perchlorate ion decreases the affinity of metaquohemerhythrin for thiocyanate ion. This is reflected in both the entropy and enthalpy being more unfavorable for the reaction in the presence of perchlorate ion.
Assuntos
Hemeritrina , Metaloproteínas , Proteínas , Animais , Anelídeos , Ferro , Ligantes , Matemática , Concentração Osmolar , Espectrofotometria , TermodinâmicaRESUMO
We describe a rapid particle-enhanced turbidimetric immunoassay for albumin in urine. Intra- and interassay CVs were less than 5% and less than 10%, respectively, the detection limit is 2 mg/L, and the working range extends to 200 mg/L. Mean analytical recovery of albumin added to centrifuged urines was 100% (SD 10.6%), and, when results were compared with those by the Pharmacia RIA, the correlation coefficient was 0.99. The working reagents are stable for at least six months; thus this assay is suited for both batch and urgent analysis.
Assuntos
Albuminúria/urina , Imunoensaio , Humanos , Imunoensaio/estatística & dados numéricos , Nefelometria e Turbidimetria , Controle de Qualidade , RadioimunoensaioRESUMO
Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].
Assuntos
Escherichia coli/enzimologia , Glutamato-Amônia Ligase/metabolismo , Adenilil Imidodifosfato/metabolismo , Sítios de Ligação , Calorimetria , Glutamatos/metabolismo , Ácido Glutâmico , Cinética , Ligantes , Substâncias Macromoleculares , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/metabolismo , Espectrometria de FluorescênciaRESUMO
We describe the development and validation of a fully automated homogeneous immunoassay for serum beta 2-microglobulin on the Dade aca clinical analyzer. The assay employs latex enhanced immunoturbidimetry, with an affinity purified polyclonal antibody covalently coupled to a 40 nm latex particle. The assay working range is < 0.5 to 20 mg/l with no evidence of loss of signal due to antigen excess at concentrations up to 120 mg/l. The assay sensitivity is 0.2 mg/l; within run and between run imprecision showed coefficients of variations of < 7%, across the assay range 1-20 mg/l. A method comparison with an established RIA procedure gave a regression equation of (aca) = 1.14 (RIA)-0.26, r = 0.996, n = 109. Good analytical recovery (98-101%), no evidence of a lack of parallelism and no interference from rheumatoid factor (tested up to 1.2 x 10(6) U/l) were observed. The low method to be considered as an effective means of monitoring seroconversion in HIV infected subjects and treatment of patients with myelomatosis.
Assuntos
Nefelometria e Turbidimetria/instrumentação , Microglobulina beta-2/análise , Artrite Reumatoide/sangue , Soluções Tampão , Infecções por HIV/sangue , Humanos , Técnicas Imunológicas/instrumentação , Mieloma Múltiplo/sangue , Nefelometria e Turbidimetria/métodos , Polietilenoglicóis , Controle de Qualidade , Radioimunoensaio/métodos , Insuficiência Renal/sangue , Sensibilidade e EspecificidadeRESUMO
A fully automated, random access method for the determination of cannabinoids (UTHC) was developed for the Dimension AR and XL clinical chemistry systems. The method utilizes Abuscreen ONLINE reagents and a multianalyte liquid calibrator containing 11-nor-Delta(9)-THC-9-carboxylic acid. Within-run and total reproducibility, determined using NCCLS protocol EP5- T2, was less than 0.6% and 1.6% CV, respectively, at all concentrations. Calibration stability was retained for at least 30 days. An extensive evaluation of non-structurally related drugs and various physiological substances indicated lack of interference in the method. No sample carry-over was observed following a specimen containing 1886 ng/ml 11-nor-Delta(9)-THC-9-carboxylic acid. A 99.1% agreement (N = 445 samples) was found between an EMIT based method on the aca discrete clinical analyser and the Dimension UTHC method.Dimension clinical chemistry system and aca discrete clinical analyser are registered trademarks of Dade International.
RESUMO
The increasing interest in the measurement of serum C-reactive protein in relation to the risk stratification of patients with chest pain has demonstrated the need for more sensitive routine methods of measurement and an accurate definition of the reference range. We report the determination of a reference range in serum samples from 491 blood donors using a particle enhanced turbidimetric immunoassay that has been modified to offer better imprecision within the reference range. The median values were found to be 2.40 and 2.20 mg/l for males and females, respectively with 95th percentile range of 1.20-5.20 and 0.40-5.40 mg/l, respectively.
Assuntos
Doadores de Sangue , Proteína C-Reativa/metabolismo , Espalhamento de Radiação , Adulto , Feminino , Humanos , Luz , Masculino , Pessoa de Meia-Idade , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Available assays for cardiac troponin I (cTnI) yield numerically different results. The aim of this study was to compare patient values obtained from four cTnI immunoassays. METHODS: We studied the Stratus(R) II assay, the Opus(R) II assay, the Access(R) assay, and a research-only cTnI heterogeneous immunoassay that uses the Dade Behring aca(R) plus immunoassay system equipped with two new noncommercial monoclonal antibodies. Because the aca plus cTnI assay is for research only, we first evaluated and analytically validated it for serum and citrated plasma. Initially, each method was calibrated using the method-specific calibrator supplied by each manufacturer; however, the aca plus cTnI assay was calibrated using patient serum pools containing cTnI and selected on the basis of increased creatine kinase MB isoenzyme and with values assigned by use of the Stratus cTnI assay. For method comparisons, individual patient sample cTnI values were determined and compared with the Stratus II assay. RESULTS: Passing and Bablock regression analysis yielded slopes of 1.44 (r = 0.96; n = 72) for the Opus II vs Stratus II assays; 0.07 (r = 0.91; n = 72) for the Access vs Stratus II assays; and 0.90 (r = 0.91, n = 72) for the aca plus vs Stratus II assays. The recalibration of each method with a Stratus II-assigned serum pool improved, but did not entirely eliminate, the slope differences between the different assays (range, 1.00-1.16). The observed scatter in the correlation curves remained. CONCLUSION: There is a need to further explore the specificities of these assays with respect to the different circulating forms of cTnI.