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At least 253 records · Page 14Linked to original sources
PubMed1997

A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces.

The determination of total protein is often a key step for the quantitative analysis of various parameters in tissue and general biochemical research. The classical protocols are restricted to a few compatible buffers, and protocols for the determination of protein in solutions containing protein agglomerates or of protein immobilized on solid surfaces are not available. In such cases, quantification may be complicated. Here, we describe a simple sensitive method for protein quantification circumventing all these restrictions. Proteins in solution or suspension in any buffer are spotted onto cellulose acetate, dried, and stained with Amido Black. After washing off the excess dye, bound Amido Black is solubilized in an acidic solution and determined photometrically. Tissue slices (fixed or native), adherent cell cultures, or Western blots can also be stained and their protein content determined irrespective of the supporting material. A micro-version of the protocol for proteins in solution allows large numbers of samples to be evaluated at a time in microtitration plates and requires only 1-2 microl per sample. A linear concentration dependency (r2=0.950-0.999) was obtained for all samples in all cases investigated. The method presented here permits the exact determination of soluble protein in a large variety of buffers, of insoluble or immobilized protein present on a wide variety of supports, and even of whole cells or tissue slices.

Amido Black↗
PubMed1997

Quantification and genotyping of serum HCV-RNA in patients with chronic hepatitis C undergoing interferon treatment.

Quantification of serum HCV-RNA and HCV genotyping was studied in 27 patients with chronic hepatitis C undergoing interferon treatment. Pretreatment serum HCV-RNA levels were quantified using competitive RT-PCR and compared to a quantitative RT-PCR assay based on co-amplification of HCV-RNA with a synthetic RNA standard. HCV genotyping was performed using a line probe reversed hybridisation assay or direct solid-phase sequencing. This study shows the feasibility of performing HCV-RNA quantification. RT-PCR based on co-amplification HCV-RNA titer less than 6 x 10(4) genome equivalents/ml serum did correlate with a complete sustained response to alpha interferon in chronic hepatitis C. HCV genotype 1b was alpha predominantly associated with a high non-responder rate. Future prospective trials will be required to evaluate quantitative HCV-RNA levels and HCV genotyping as response predicting parameters for interferon-treatment.

Adult↗
PubMed1991

Comparison of thallium-201 SPECT and planar imaging methods for quantification of experimental myocardial infarct size.

To compare single photon emission computed tomography (SPECT) and planar thallium-201 (TI-201) myocardial perfusion imaging methods for quantification of left ventricular infarct size, 12 dogs with 6 to 8 hours of closed-chest coronary occlusion and 5 normal dogs were studied. After intravenous administration of TI-201, SPECT and three-view planar images were obtained. After the animals were put to death, hearts were sliced and stained with triphenyltetrazolium chloride (TTC) for planimetric determination of left ventricular infarct size. Infarct size on each SPECT slice and planar image was defined as the percentage of circumferential count profiles falling below the limits derived from normal dogs. Infarct size as a percentage of left ventricular mass was determined from SPECT and planar images before and after correcting for differences in myocardial slice mass from apex to base. The correlation coefficients, the concordance correlation coefficients (reflecting closeness to the line of identity), and the mean absolute deviations of the four methods versus TTC staining were 0.83, 0.77, and 5.1% (SPECT, no correction); 0.85, 0.84, and 3.7% (SPECT with correction); 0.81, 0.42, and 12.9% (planar, no correction); and 0.75, 0.49, and 10.4% (planar with correction). The regression lines did not differ from the line of identity for SPECT, whereas they differed significantly for planar imaging. Thus both SPECT and planar imaging are well suited for quantification of left ventricular infarct size. SPECT, however, appears to be superior to planar imaging, since its regression line more closely approximates the line of identity.

Animals↗
PubMed1975

Diagnosis and quantification of arrhythmias in ambulatory patients using an improved R-R interval plotting system.

An improved technique for identification, diagnosis and quantification of arrhythmias during rest or ambulatory electrocardiographic recording is described. With simultaneous plotting of the R-R interval and the QRS duration and QRS vector measurement of each beat versus time, all periods of arrhythmias or abnormal complexes can be identified and characterized. Analog electrocardiographic samplings are used to confirm the diagnosis of the arrhythmia and to exclude artifact. The availability of a permanent record for the characterization of each QRS complex enables the physician to check the technician's analysis of the recording and to relate all events to the patient's heart rate and clinical symptoms. This technique also provides data for quantification of ventricular arrhythmias.

Ambulatory Care↗
PubMed1981

Quantification of left to right cardiac shunts by multiple deconvolution analysis.

A new method for quantification of left to right cardiac shunts was studied in 17 patients scheduled for cardiac catheterization who had also undergone radionuclide angiocardiography. The observed pulmonary transit curve was deconvoluted in two different ways: (1) by the superior vena caval ("bolus") time-activity curve, to yield the deconvoluted pulmonary transit curve, which represented the theoretical pulmonary transit curve with a perfect bolus injection, and (2) by the right ventricular time-activity curve, to yield the pulmonary transfer function, which represented the theoretical pulmonary transit curve with a perfect bolus injection an with no intracardiac shunts. The pulmonary transfer function was superimposed on the deconvoluted pulmonary transit curve, and the area A under it obtained. The pulmonary transfer function was then subtracted from the deconvoluted pulmonary transit curve. The pulmonary transfer function was scaled to fit the resulting shunt recirculation peak in the difference curve, and the area B under this scaled pulmonary transfer function obtained. Shunt size was quantified as the pulmonary (QP) to systemic (QS) flow ration QP/QS = A/(A-B). The method correlated closely with oximetry (r = 0.93). Use of this multiple deconvolution analysis technique provides accurate shunt quantification and reduces subjective operator decisions.

Adolescent↗
PubMed1985

Digital quantification eliminates intraobserver and interobserver variability in the evaluation of coronary artery stenosis.

A leading problem with subjective interpretation of coronary angiography is high intraobserver and interobserver variability. Four experienced angiographers independently determined percent diameter narrowing of 36 stenoses using 3 methods: by subjective analysis of single-frame cine film images (film), by subjective analysis of digitized nonenhanced single-frame images (digital), and by using a semiautomated digital caliper quantification system (Corona). The reproducibility of interpretations was assessed by comparison of estimated intraclass correlation coefficients. Digital and Corona readings correlated well with subjective interpretation of film (r greater than 0.85 for both). In contrast to Corona, the angiographers systematically overestimated the magnitude of stenoses in the intermediate (50 to 75%) range. Corona markedly improved intraobserver (p less than 0.005) and interobserver (p less than 0.001) reproducibility. Corona less frequently misclassified individual observations than did film when categories of less than 50%, 50 to 75% and more than 75% diameter stenosis were used (3.7% vs 31.5%, p less than 0.001). Our results suggest that digitization of a coronary angiogram in a 512 X 512 matrix has no significant adverse effects on the perception and quantification of stenosis by angiographers. Additionally, automatic measurement of coronary stenosis has 2 major advantages: It is accurate compared with a group of experienced angiographers and for the practical purpose of clinical decision-making, it eliminates intraobserver and interobserver variability.

Angiography↗
PubMed1993

Comparison of magnetic resonance imaging studies with enzymatic indexes of myocardial necrosis for quantification of myocardial infarct size.

To evaluate the potential of gadolinium-diethylene triamine pentaacetic acid (DTPA)-enhanced magnetic resonance imaging (MRI) in the quantification of infarct size in patients with a first acute myocardial infarction, 24 patients with a first acute myocardial infarction were studied by electrocardiographic gated MRI at a mean of 4.3 days after the acute event. Multislice, single-phase, T1-weighted, spin-echo MRI in the true short-axis plane was performed 20 minutes after intravenous injection of gadolinium-DTPA (0.15 mmol/kg of body weight). Circumscript myocardial regions of increased signal intensity on gadolinium-DTPA-enhanced images were considered to be infarcted. Infarct size (in g) was determined using Simpson's rule, and was compared with that based on cumulative release of alpha-hydroxybutyrate dehydrogenase activity in plasma and with peak creatine kinase-MB level in plasma. Infarct size quantified with MRI correlated well with "enzymatic" infarct size (in g equivalents) (y = 0.99 x + 0.71; r = 0.93; p = 0.0001) and peak creatine kinase-MB levels (r = 0.72; p = 0.002). It is concluded that gadolinium-DTPA-enhanced MRI enables accurate quantification of infarct size in patients with a first acute myocardial infarction.

Adult↗
PubMed1983

A rapid and specific enzymatic method for the quantification of phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol in amniotic fluid.

An enzymatic procedure for the quantification of phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol in amniotic fluid is described. By use of this method, choline and glycerol are released enzymatically from phosphatidylcholine and phosphatidylglycerol, respectively, in reactions catalyzed by phospholipase D. The hydrogen peroxide generated from choline (by the action of choline oxidase) and from glycerol (by the combined action of glycerokinase and glycerol-3-phosphate oxidase) is quantified spectrophotometrically after the addition of horseradish peroxidase, aminoantipyrine, and phenol. The phosphatidylcholine concentration in amniotic fluid was found to be approximately 10 to 30 nmol/ml between the twenty-third and thirty-sixth week of gestation and increased sevenfold to eightfold between the thirty-seventh week and term. The procedure can be modified for the quantification of disaturated phosphatidylcholine. The concentration of phosphatidylglycerol was approximately 2 nmol/ml between the twenty-third and thirty-sixth week and increased to 10 to 20 nmol/ml between the thirty-seventh and forty-first week of pregnancy. Since contamination of amniotic fluid with bile pigments does not interfere with either assay, the phosphatidylcholine and phosphatidylglycerol concentrations in amniotic fluid can be determined in samples that are contaminated with meconium.

Amniotic Fluid↗
PubMed1987

Glutamine and glutamate: automated quantification and isotopic enrichments by gas chromatography/mass spectrometry.

A method is described for simultaneous quantification of glutamine and glutamate plasma levels and isotopic enrichments in these compounds. Glutamine and glutamate are analyzed intact as their tertiary-butyldimethylsilyl derivatives. Deuterated glutamine and glutamate are used as internal standards for quantification by reverse isotope dilution. Preparation of plasma samples is accomplished by adding ammonium formate as an ion-pairing agent followed by extraction of the amino acids into 4.3:1 methanol:water. Negligible amounts of glutamine to glutamate conversion are observed during the sample preparation and GC/MS analysis. Since glutamine is analyzed intact, both single and double [15N]glutamine labels can be quantified. [15N]Glutamine at 0.2 to 11 mol% excess was measured in plasma with an average relative standard error of 3.8%, and [15N]glutamate over a range of 0.4 to 9 mol% excess was measured with a mean relative standard error of 12%. At glutamate levels above 1 mol% excess 15N, the mean relative standard error was 6%. Finally, automated sample injection into the GC/MS and automated data reduction are used for the analysis of samples by GC/MS.

Animals↗
PubMed1988

Enzyme-linked immunosorbent assay (ELISA)-based quantification and identification of in vitro enzyme-catalyzed glycosphingolipid synthesis and degradation products with carbohydrate sequence-specific monoclonal antibodies.

A new method has been developed to monitor glycosyltransferase and glycosylhydrolase activities. Reaction product identification and quantification are accomplished simultaneously with an enzyme-linked immunosorbent assay (ELISA) using carbohydrate sequence-specific monoclonal antibodies. beta-Galactosyltransferase and alpha-galactosidase reactions were used to illustrate the salient features of the method. These include simple product identification and quantification, no detergent requirement, consumption of small amounts of reagents, and no use of radioisotopes. Furthermore, it is possible to measure substrate disappearance or product formation with this method. Enzyme characteristics such as Km, Vmax, divalent cation requirement, and pH optimum were investigated with this new method.

Animals↗
PubMed1989

Enzymatic quantification of sphingosine in the picomole range in cultured cells.

An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-[32P]sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/10(6) cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.

Animals↗
PubMed1989

Quantification of proteins in the subnanogram and nanogram range: comparison of the AuroDye, FerriDye, and India ink staining methods.

The usefulness of three sensitive dyes, AuroDye, FerriDye, and India ink, for the quantification of proteins and peptides bound to nitrocellulose paper has been assessed. In general, the staining intensity varies linearly with the logarithm of protein concentrations. The detection limit of small peptides (Mr less than 5000) is higher than that of large peptides and proteins, but the sensitivity is independent of the molecular weight. Oligopeptides of four or less amino acids either stain with very high detection limits or do not stain at all. The detection limit of proteins stained by AuroDye is approximately 1 ng, and in a number of cases even lower. The useful range for quantification of proteins extends to around 100 ng. The FerriDye and India ink staining methods are less sensitive and can be used to quantify proteins over a wide nanogram range. Among the methods tested, the India ink staining method has the highest protein to protein variation in sensitivity.

Biochemistry↗
PubMed1990

A direct method for quantification of non-transferrin-bound iron.

A direct method for quantification of non-transferrin-bound iron has been developed. This assay relies on the use of a large excess of a low affinity ligand (nitrilotriacetic acid, NTA) which removes and complexes all low molecular weight iron and iron nonspecifically bound to serum proteins. Iron bound to transferrin, ferritin, desferrioxamine, and its metabolites is unaffected. The Fe-NTA complex present in the serum ultrafiltrate is then quantified using an automated HPLC procedure where on-column derivatization with a high affinity iron chelator (3-hydroxy-1-propyl-2-methyl-pyridin-4-one) takes place. The iron complexes of desferrioxamine and its metabolites are unaffected by the above-derivatization procedure. With minor modifications, this method is equally applicable for the quantification of low molecular weight iron in other biological fluids.

Acetates↗
PubMed1990

Quantification of monoclonal antibodies in complex mixtures by protein G high-performance liquid affinity chromatography.

High-performance liquid affinity chromatography (HPLAC) utilizing Protein G as a ligand has been evaluated for rapid quantification of monoclonal antibodies (MAbs) in various solutions. The results obtained by HPLAC agreed to within 10% of a standard enzyme-linked immunospecific assay (ELISA). A standard curve was prepared by injection of known amounts of a purified murine IgG1 with the elution peak area analyzed by computer integration software. Accuracy of quantification was independent of the injection volume, solution compositions, or mouse IgG subclass. A method is described for using Protein G HPLAC to determine murine IgG levels in various complex mixtures within 15 min, compared to the ELISA which required 5 h.

Animals↗
PubMed1992

Quantification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and simultaneous ultraviolet-visible/radioactive detection.

An HPLC-based method for quantification of desferrioxamine (DFO) and its iron chelating metabolites in plasma has been developed. This assay overcomes stability problems associated with DFO by the addition of radioactive iron to convert unbound drug and metabolites to radio-iron-bound species. A dual detection system utilizing uv-vis absorption and radioactive (beta-particle) detector was used to quantify total and radio-iron-bound species. The use of octadecyl silanol solid phase extraction cartridges permits concentration of samples and allows accurate quantification of drug and metabolites down to 0.1 nmol/ml.

Chromatography, High Pressure Liquid↗
PubMed1990

Quantification of biomineralization: an in-vitro tissue culture system and microanalysis of calcium, phosphorus and trace elements by total-reflection X-ray fluorescence.

An in-vitro tissue culture system with folded periostea of 17-day-old fetal chick calvaria was combined with analytical methods to achieve quantification of biomineralization. A scanning electron microscope with an energy-dispersive X-ray detector was applied to show the distribution of calcium, phosphorus and trace elements. Calcium and phosphorus were concentrated in the zone of the mineralized matrix. Strontium was distributed similar to calcium. Zinc was distributed equally in the soft tissue and the mineralized matrix. Total-reflection X-ray fluorescence was used for quantification. Thyroxine in high concentration reduces the calcium content of the samples. One week after incubation magnesiumchloride (1.8 mM) or zincchloride (.1 mM) were found to reduce the calcium content by 38% or 82%, respectively.

Animals↗
PubMed1982

The direct quantification in whole serum of HDL subfractions.

A method is described for the direct electrophoretic separation in human serum of three HDL subfractions and the quantification of the cholesterol content of these by means of gas-liquid chromatography. Used in conjunction with established procedures for the separation of total HDL, VLDL and LDL on agarose gels (with gas-liquid chromatography quantification of cholesterol in each band) the procedure affords comprehensive information on lipoprotein metabolism in patients. Two technicians can process 10 samples per day, making the method suitable for semi-large scale screening of patients such as may occur in clinical trials or to evaluate in greater detail lipoprotein patterns of patients suspected of having lipoprotein abnormalities on the basis of simpler screening methods. Results obtained by the method correlate well with those obtained by means of ultracentrifugation.

Centrifugation, Density Gradient↗
PubMed1983

Quantification of human serum lipoprotein Lp(a): zone immunoelectrophoresis assay, a new sensitive method as compared to electroimmuno assay.

This study aimed at improving the immunological procedures for the quantification of human serum lipoprotein Lp(a). Lipoprotein Lp(a) was estimated by two immunoelectrophoretic methods: electroimmunoassay and zone immunoelectrophoresis assay. Whereas the electroimmunoassay gave curvilinear calibration lines (in peak height versus concentration plots) the zone immunoelectrophoresis assay revealed linearity in the same concentration range. The results obtained are in good agreement with each other (r = 0.975). The inter-assay coefficient of variation for the zone immunoelectrophoresis assay was 12% as determined by the use of a lyophilised reference serum. Zone immunoelectrophoresis assay is recommended for the routine quantification of lipoprotein Lp(a). Although the levels for total cholesterol (p less than 0.0025), HDL-cholesterol (p less than 0.001) and triglycerides (p less than 0.025) differed significantly between male (n = 24) and female (n = 22) individuals, no difference could be detected comparing the frequency distributions of Lp(a)-levels found for the male and the female group.

Adolescent↗