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At least 901 records · Page 50Linked to original sources
PubMed1995

Quantification and mapping of antigenic determinants of serum amyloid A (SAA) protein utilizing sequence-specific immunoglobulins and Eu3+ as a specific probe for time-resolved fluorometric immunoassay.

Serum amyloid A (SAA) protein, the most prominent amongst acute-phase proteins, is the specific precursor protein of secondary reactive amyloidosis. The fact that SAA once released into the circulation as a 'free' protein rapidly associates with lipoproteins of the high-density range indicates a specific role in lipoprotein metabolism. In this study a new sensitive assay for quantification of human SAA protein in biological specimens using affinity-purified polyclonal antibodies and Eu3+ as a specific probe for time-resolved fluorometric immunoassay is presented. Both purified SAA and SAA-rich high-density lipoprotein particles served as reliable standards in the indirect and the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). The detection limit of the DELFIA technique presented was 4-10 ng after sample dilution of 1/2500. The intra-assay coefficient of variation averaged 4.3% whereas the inter-assay coefficient of variation averaged 6.2%. Comparison with the nephelometric assay, a widely and commonly used assay for SAA quantification in plasma, revealed correlation coefficients of 0.9428. In addition to polyclonal anti-human SAA antibodies sequence-specific antibodies raised against synthetic peptides corresponding to region; 1-17, 14-30, 27-44, 40-63, 59-72, 68-84, 79-94, and 89-104 of the human SAA amino acid sequence were studied. Sequence-specific antibodies raised against epitopes 27-44, 59-72, 68-84, and 89-104 recognize human SAA protein in the DELFIA assay whereas antibodies raised against epitopes 1-17, 14-30, 40-63 and 79-94 failed to recognize the corresponding epitopes. Results obtained from these studies indicate that the N-terminal domain (1-30) as well as epitopes 40-63 and 79-94 of human SAA are apparently masked by the environment of the lipoprotein particle. From our studies it is proposed that the epitopes 31-39, 64-78, and 95-104 may be responsible for the interaction of SAA-rich high density lipoprotein particles with peripheral cells.

Antibodies, Monoclonal↗
PubMed1987

Methods for the metabolic quantification of regional myocardial ischemia.

An adequate balance between oxygen supply and demand is a basic requirement for normal cardiac function. When oxygen supply does not meet the demand, progressive cellular damage occurs leading to cardiac dysfunction and, ultimately, tissue death. While traditionally "ischemia" has been defined as decreased oxygen supply secondary to a decrease in blood flow, and "hypoxia" as decreased oxygen supply secondary to a decrease in oxygen tension, this review defines ischemia in its broader sense, namely as a pathophysiologic state in which there is a lack of oxygen relative to the demand for it. In a large number of experimental studies involving the heart, there is need to promptly recognize the ischemic state, to monitor its course in vivo, and to quantify it. Because of cardiac autoregulatory mechanisms, research methods which attempt to quantify supply (e.g., measurement of myocardial blood flow) and/or demand (e.g., measurement of myocardial oxygen consumption) do not necessarily reflect the status of the balance between supply and demand. An imbalance between myocardial supply and demand is more likely to be reflected by metabolic fluxes and by the accumulation of products specific to the ischemic state. Thus, the purpose of this review is to summarize the various methods available to the cardiac surgical investigator today for the metabolic quantification of myocardial ischemia. Due to the complexity of the heart and its inherent regional differences, myocardial ischemic changes are frequently regional in nature. Thus, this review will address metabolic methods for the regional quantification of myocardial ischemia.

Animals↗
PubMed1985

Doppler echocardiographic diagnosis and quantification of valvular heart disease.

Doppler echocardiography is a logical companion to ultrasound imaging in the diagnosis and quantification of valvular heart disease. Accurate noninvasive identification and quantification of valvular heart disease is now possible if both techniques are utilized together. The continued application of our knowledge of hemodynamics should extend our current uses of Doppler echocardiography to allow more intelligent management of patients with known or suspected valvular heart disease.

Blood Flow Velocity↗
PubMed1995

A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules.

A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, CD4, CD8, HPRT and beta-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding beta-actin, IL-10, IFN-gamma and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.

Animals↗
PubMed1992

Immunochemical quantification of procion red HE-3B used as ligand in affinity chromatography.

The quantification of Procion Red HE-3B used as a ligand in affinity chromatography for proteins is reported. It's based on an enzyme-linked immunosorbent assay using antibodies against the dye. Polyclonal antibodies were classically prepared after conjugation of the dye on KLH and injection into rabbits. The development of the assay was based on the competitive inhibition between hemoglobin-dye complex and free dye. The sensitivity of this method was about 1000-times higher than a classical spectrophotometric assay, and was modulated by some chemical substituents attached on the native dye. It was demonstrated that the assay was applicable to the determination of dye traces that may be released from dye affinity sorbents. Moreover, the quantification of the dye was successfully applied to proteins that are being purified from a dye affinity column.

Chromatography, Affinity↗
PubMed1996

A zone immunoelectrophoresis assay method for quantification of apolipoprotein D in human cerebrospinal fluid.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in human unconcentrated cerebrospinal fluid (CSF). The apo D concentrations of samples of the serum, plasma and CSF were directly proportional to the migration distances of the corresponding zones of immunoprecipitates developed during electrophoresis in glass capillaries filled with antibody-containing agarose gel. A linear standard curve, between about 1 and 12 mg of apo D/1 was obtained using a commercial serum preparation. Seronorm, as apo D standard. The coefficients of variation of the ZIA were below 8% (n = 5 x 6) and 10% (n = 8) for within-run and between-run reproducibility, respectively. Quantification experiments with disulfide-reducing agent, mixtures of CSF and urine as well as frozen and stored CSF samples indicated parallelism between the precipitate-forming immunologic reactions of apo D in different sample matrices when performed with ZIA. Application of this method to quantify apo D of CSF and plasma samples from 51 normal healthy men aged 16-72 years yielded means +/- SD of 5.3 +/- 1.5 mg/l and 128.4 +/- 22.7 mg/l, respectively. No correlation was found between the CSF and plasma apo D concentrations.

Adolescent↗
PubMed1987

Quantification of synapse turnover in cell culture.

The on and off rates of synaptogenesis can be quantitated using a cell culture system in which embryonic retinal cells form functional synapses with striated muscle cells. Quantification showed that synapse formation and termination can occur simultaneously in culture. Quantification also revealed that some retina-muscle synapses are transient, terminating within 8 h, while other synapses are much more stable. Relatively stable and transient synaptic pairs could be identified prospectively based on the strength of evoked transmission across the retina-muscle synapse.

Action Potentials↗
PubMed1996

Quantification of C-ERB-B2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis.

Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells wih low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as parameters that permit a rapid, objective, and accurate estimation of gene copy number, is presented. This procedure is independent of the signal overlapping and results in a more accurate quantification and characterization of tumor cell heterogeneity.

Breast Neoplasms↗
PubMed1992

Bacterial quantification--a necessary complement for the comprehension of middle ear inflammations.

Quantification of bacteria in various types of middle ear effusion (MEE) obtained during current acute otitis media (AOM), otitis media with effusion (OME) and chronic suppurative otitis media (COM) was performed. The bacteria were stained with acridine orange and their number per ml effusion evaluated under the fluorescence microscope according to a method described in detail elsewhere. During AOM, 53% of the MEE samples were culture-positive and contained 10(6)-10(8) bacteria per ml (median value 10(7) per ml). During OME, serous effusion and 78% of the mucoid effusions contained no bacteria whatsoever, whereas the remaining mucoid effusions contained 10(4)-5 x 10(5) bacteria per ml (median value 10(4) per ml). Mucopurulent effusions contained 6 x 10(5)-10(8) bacteria per ml (median value 5 x 10(6) per ml). During COM, purulent MEE had 6 x 10(6)-10(9) bacteria per ml (median value 10(8) per ml). Quantification of bacteria involved in middle ear diseases provides further information about the etiopathogenesis and appropriate management of various pathological conditions of the middle ear.

Acute Disease↗
PubMed1986

Stable isotope dilution quantification of mutagens in cooked foods by combined liquid chromatography-thermospray mass spectrometry.

A method of general applicability for the detection and quantification of mutagens in cooked foods at the ppb level is presented. A minimal sample prefractionation is employed and [Me-2H3]-labeled analogs of the compounds of interest are added for identification and quantification of mutagens by accurate measurement of chromatographic retention (K') in reverse-phase high-performance liquid chromatography (HPLC), and by measurement of the ratio of response of the protonated molecular ions of analyte and internal standard by directly coupled liquid chromatography-mass spectrometry (LC/MS). Initial application is demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in broiled salmon. Measured levels of IQ and MeIQ in broiled salmon flesh were 0.3-1.8 ppb and 0.6-2.8 ppb, respectively, and for the skin of broiled salmon 1.1-1.7 ppb and 1.5-3.1 ppb, respectively. Results on cooked beef and sardine are also reported.

Animals↗
PubMed1993

Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction.

Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Color image analysis in neuroanatomical research: application to senile plaque subtype quantification in Alzheimer's disease.

Many problems in neuroanatomy and neuropathology require the collection of large data sets and would benefit from a method that allows for rapid quantitative analysis to be carried out on a routine basis. An example is the quantification and subtype classification of the number of senile plaques in post-mortem Alzheimer's disease tissue. A method to reliably automate the analysis of plaques and their underlying subtypes would allow more rigorous and quantitative correlations to be investigated. Computer assisted image analysis of data typically utilizes gray scale images. These methods, however, are only applicable to quantification of objects labeled with a single marker. We sought to extend this type of analysis to double-labeled tissue sections so we could quantify dual labels separately based on their peroxidase color characteristics, analyze the resultant occurrence of overlap between the two labels, and classify senile plaques into discrete subtypes. We present a method for semi-automated color image analysis which allows one to identify separate labels based on histogram mapping of hue, saturation and value as well as apply overlapping feature detection algorithms. The technique is application driven, so that a trained observer can set threshold or object criteria and verify the desired results. These methods were able to yield total "amyloid load" and "dystrophic neurite load" values, generate plaque histograms based on total size, and subtype plaques into diffuse/primitive and neuritic/classical categories. By adjusting feature criteria, we were able to achieve promising agreement (Fisher's R to Z correlation of 0.94) between a human observer and the computer algorithm in the classification of plaque subtypes using three AD cases.

Algorithms↗
PubMed1984

Quantification by selected ion monitoring of pipecolic acid, proline, gamma-aminobutyric acid and glycine in rat brain.

A procedure for the simultaneous analysis of brain pipecolic acid, proline, gamma-aminobutyric acid and glycine--amino acids with potent inhibitory actions on the central nervous system--was developed. The identification and quantification of the amino acids were performed with a gas chromatographic--mass spectrometric--computer system using deuterium-labelled amino acids as the internal standards. After separation of the amino acids by high-performance liquid chromatography, the methyl ester heptafluorobutyryl derivatives were prepared. The lower limit of quantification for this method is at the picomole level. The usefulness of this chromatographic procedure has been demonstrated by measurement of trace amounts of pipecolic acid in rat brain.

Animals↗
PubMed1987

Characterization and quantification of PAF-acether (platelet-activating factor) as a heptafluorobutyrate derivative of 1-O-alkyl-2-acetyl-sn-glycerol by capillary column gas chromatography with electron-capture detection.

A gas chromatographic method with a glass capillary column and electron-capture detection is proposed for the characterization and quantification of 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF C16) using the corresponding 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-heptafluorobutyrate derivative and, as an internal standard, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine. The reproducibility was approximately 5% and amounts as low as 20 pg could be measured. The method was specific and allowed the quantification of PAF C16 in supernatants from stimulated human polymorphonuclear neutrophils.

Chromatography, Gas↗
PubMed1987

Quantification of riboflavin, riboflavin 5'-phosphate and flavin adenine dinucleotide in plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method with fluorimetric detection for the quantification of riboflavin (RB), riboflavin 5'-phosphate (FMN), and flavin adenine dinucleotide (FAD) in plasma, whole blood, and urine is described. Under isocratic conditions with a reversed-phase column, the compounds are completely resolved and eluted within 9 min. Plasma proteins are precipitated with acetonitrile followed by shaking the aqueous phase with chloroform. Urine samples are diluted and injected directly. The reproducibility of this method for the quantification of RB in plasma has a between-day coefficient of variation of 6%. The application of this method is illustrated by analyzing plasma and urine samples from a human subject who received an intravenous dose of FMN equivalent to 25 mg of RB.

Adult↗
PubMed1987

Sensitive and specific high-performance liquid chromatographic assay for the quantification of sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites in plasma.

A sensitive and specific procedure using high-performance liquid chromatography for the quantification of sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites in plasma was developed. Sulforidazine was first extracted from basified plasma using a mixture of pentane and 2-propanol. Sulforidazine-5-sulfoxide metabolites were then extracted from the same basified plasma using a second solvent mixture consisting of methylene chloride, pentane and 2-propanol. Each organic extract was subsequently back-extracted separately with 0.1 M hydrochloric acid, basified and re-extracted with the original solvent mixtures. In order to avoid interferences due to hydroxylated metabolites co-eluting with sulforidazine-5-sulfoxides in the more polar extract, this extract was derivatized with N-methyl-(tert.-butyldimethylsilyl) trifluoroacetamide. The two extracts were separately chromatographed on a narrow-bore nitrile column using ultraviolet detection. The quantification limits for sulforidazine and two diastereomeric sulforidazine-5-sulfoxide metabolites were 1.0 ng/ml with mean intra-assay coefficients of variation less than 10%. These methods were applied to the analysis of plasma from a dog following the administration of a single oral dose (25 mg of the base) of sulforidazine hydrochloride.

Animals↗
PubMed1992

Identification and quantification of ergotamine in human plasma by gas chromatography-mass spectrometry.

A highly sensitive and simple gas chromatographic-mass spectrometric method is described for the identification and quantification of ergotamine in plasma or serum. Ergotamine is extracted with chloroform from the alkalinized sample and detected by electron ionization mass spectrometry. This analytical method was selected for an intense high-mass ion ideal for the specific quantification. It shows good linearity in the range from 50 pg/ml to 50 ng/ml for ergotamine in plasma. The practicability of this method is demonstrated by determining the plasma concentration of ergotamine in a sample from a patient.

Chloroform↗
PubMed1992

Quantification of erythrocyte S-adenosyl-L-methionine levels and its application in enzyme studies.

A highly selective high-performance liquid chromatographic method for the quantification of human erythrocyte S-adenosyl-L-methionine levels is described. A strong cation-exchange sorbent with propylsulphonic acid functional groups was used to extract S-adenosyl-L-methionine and S-adenosylethionine (internal standard) from erythrocytes. Quantification of erythrocyte S-adenosyl-L-methionine levels was achieved by using reversed-phase high-performance liquid chromatography and ultraviolet detection at 254 nm. This method was adapted to measure methionine-adenosyltransferase activity in erythrocytes, which enables us to study the possible role of altered methylation in different diseases.

Chromatography, High Pressure Liquid↗