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At least 433 records · Page 24Linked to original sources

PubMed1980

Detection and quantitation of hepatitis-B surface antigen immune complexes (HBsAg-ICs) by an antigen-specific method. I. Detection and quantitation of in vitro prepared HBsAg-ICs.

An antigen-specific method has been developed for direct detection and quantitation of HBsAg-ICs. The method involves (1) precipitation of HBsAg-ICs with 3.5% PEG; (2) dissociation of the PEG precipitated ICs by treatment with NaSCN, NaI, KBr, low or high pH buffers, trypsin or papain; and (3) detection and titration of HBsAg and/or anti-HBs liberated from ICs. Treatment with 2 M and 3 M NaSCN, papain or trypsin liberates HBsAg, while following treatment with 3 M and NaI free anti-HBs is detectable. Trypsin digestion (2 mg/ml, 30 min at 37 degrees C) proved to be most effective for disrupting HBsAg-ICs formed at equivalence as well as in excess of antigen or antibody. After trypsin digestion of the sample RPHA, RIA and ELISA may be used as a third step. Th

Antibodies↗

PubMed1986

Detection of spatial beats: non-linearity or contrast increment detection?

We measured sensitivity of human observers to the 1 c/deg beat between sinusoidal gratings of 9 and 10 c/deg, at different contrasts of the 2 components. Raising the contrast of one component increases the contrast required in the other component to detect the beat. This is consistent with the hypothesis that the beat is detected because of the local increments in contrast which it produces, but not with the hypothesis that it is detected when a difference-frequency distortion product generated by non-linear transduction of luminance reaches threshold.

Discrimination, Psychological↗

PubMed1981

Immunochemical detection of ds-RNA in healthy and virus-infected plants and specific detection of viral ds-RNA by hybridization to labelled complementary DNA.

Double-stranded (ds) RNA was detected in nucleic acid preparations from virus-free leaf tissues of several plant species by radial double-diffusion tests with antiserum raised against polyinosinic : polycytidylic acid [poly(I) : poly(C)]. No significant differences in the concentrations of ds-RNA were detected by such tests in virus-free French bean leaves and those infected with tobacco mosaic virus or tobacco ringspot virus. However, virus-specific ds-RNAs as well as genomic RNAs of both viruses were readily detected and estimated quantitatively by hybridization to 3H-labelled complementary DNA probes. It is concluded that it is not feasible to screen plant material infected with single-stranded RNA viruses by immunochemical tests for virus-associated ds-RNAs.

Fabaceae↗

PubMed1996

Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system.

The development of a nested polymerase chain reaction (PCR) assay to detect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers located in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genital HPVs (types-6, -11, -18, -31 and 33). This assay was carried out in 96-well plates utilising internal primers labelled with dinitrophenol (DNP) and biotin so that amplicons can be captured onto streptavidincoated plates and detected using an alkaline phosphatase-labelled monoclonal antibody to DNP. The assay was effective for detecting HPV-16 DNA in plasmids, cell-lines and, both freshly collected or archival (formalin-fixed/paraffin embedded) clinical specimens. This system is therefore suitable for epidemiological studies to identify individuals infected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.

DNA Primers↗

PubMed1994

Enhanced separation and detection of serum bilirubin species by capillary electrophoresis using a mixed anionic surfactant-protein buffer system with laser-induced fluorescence detection.

The four major bilirubin species in serum are separated by capillary electrophoresis and detected using laser-induced fluorescence detection. The optimum buffer system consists of 40 mM sodium dodecyl sulfate (SDS)-0.012 mM bovine serum albumin (BSA). The use of the SDS-BSA mixture in the mobile phase allows for the separation of four major bilirubin species at physiological pH with untreated capillaries. The results show that the use of BSA as a run buffer modifier in SDS solution improves separation efficiency and increases sample solubility via pH changes of the run buffer. The limits of detection for the bilirubin species using laser-induced fluorescence are between 30 and 150 nM, depending on the bilirubin species; not only is this approximately two orders of magnitude lower than with visible-light absorption methods, it allows the bilirubin species in normal sera to be quantitatively measured without sample pretreatment.

Adult↗

PubMed1987

HPLC detection of choline and acetylcholine in serum and urine by an immobilized enzyme reactor followed by chemiluminescence detection.

A method using HPLC has been developed for the detection of choline (Ch) and acetylcholine (ACh) using an immobilized enzyme reactor which converts Ch and ACh into hydrogen peroxide and betaïne. The formed H(2)O(2) is quantified by means of a solid-state peroxyoxalate chemiluminescence detector based on an immobilized fluorophore and addition of oxalate from a solid bed. The conditions necessary for chemiluminescence detection are obtained by using a make-up flow of acetonitrile after the enzyme reactor. Precipitation problems due to the poor solubility of salts in the final acetonitrile-water mixture are circumvented by adding a crown ether to the make-up flow. The reproducibility of the method was calculated to be 3.4-3.7% RSD. Detection limits are in the sub-picomole range and a linear range of at least three orders of magnitude is found. Measurements in urine and serum reveal no matrix effects.

Journal Article↗

PubMed1990

Liquid chromatographic separation and stereoselective detection of L- and D-amino acids with catalytic reaction detection using immobilized enzymes.

A post-column LC detection system is described for the stereoselective detection of L- and D-amino acids. The effluent of the LC column passes an immobilized enzyme reactor (IMER) containing either L- or D-amino acid oxidase. The hydrogen peroxide formed in this reactor by the oxidation of the amino acids is then transported to a second IMER containing horse-radish peroxidase. With the addition of 4-aminophenazone and dichlorophenosulphonyl chloride to the carrier, the hydrogen peroxide is reacted to form a red-coloured complex which is detected in a flow-through photometric cell at 514 nm. Applications to the analysis of amino acids in bovine and human sera are described.

Amino Acid Oxidoreductases↗

PubMed1994

Detection of 13C labelled compounds by gas chromatography coupled to atomic emission detection--application to caffeine metabolites.

This paper illustrates the use of gas chromatography coupled to atomic emission detection (GC-AED) and of the stable isotope tracer 13C for the determination of drug metabolites. After administration of a parent drug labelled with 13C and extraction of the metabolites from the biological fluids, a 13C chromatographic profile is determined using the specific detection of the 13C atomic emission and subtraction of the 13C natural abundance. Thus, only the compounds which are metabolites with a 13C enrichment over the natural abundance are detected. [1,3,7 trimethyl-13C3]xanthine which is extensively metabolized by the liver is used as an example.

Caffeine↗

PubMed2005

Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection.

Immuno-PCR (IPCR) has been studied to increase the detection sensitivity of current enzyme-linked immuno-sorbent assays (ELISA) as a novel approach for the early detection of Rotavirus infection, a major source for serious diarrhoea for susceptible risk groups. IPCR utilizes specific antibody-DNA conjugates with subsequent amplification of the marker-DNA. An antibody-DNA conjugate specific for Rotavirus antigen VP6 was synthesized and used in combination with a commercially available Rotavirus-ELISA kit. IPCR was carried out using reagents and protocols of the standardized Imperacer system. Real-time PCR monitoring of the marker-DNA amplification was compared to endpoint quantification of amplified haptene-labeled PCR products, using a microtiterplate-based PCR-ELISA. In spiked calibration samples, as few as 100 virus particles/ml could be clearly detected using the IPCR method and either real-time or end-point quantification compared to about 100,000 virus particles/ml in ELISA. Rotavirus positive and negative stool samples were correctly identified by IPCR with a clear separation even of a 10,000-fold dilution of the positive stool samples from the negative control.

Computer Systems↗

PubMed2005

A combination of magnetic permeability detection with nanometer-scaled superparamagnetic tracer and its application for one-step detection of human urinary albumin in undiluted urine.

A rapid (6.5 min) and simple one-step magnetic immunoassay (MIA) has been developed for analysis of human urinary albumin in near patient settings. Polyclonal rabbit anti-human albumin was used as a capture antibody and monoclonal mouse anti-human albumin as a detection antibody in a two-site immunometric assay requiring no additional washing procedures. The polyclonal anti-human albumin was conjugated to silica microparticles (solid phase) and the monoclonal antibody to dextran-coated nanoscaled superparamagnetic particles (tracer). Quantification of human albumin in undiluted urine was performed by adding 2 microL urine to a measuring vial containing solid-phase, superparamagnetic tracer and reaction buffer and then inverting the vial by hand for 20 s. The measuring vial was allowed to stand for 6 min prior to detection, in order for the solid-phase sediment to form at the bottom of the vial. Lastly, the measuring vial was placed into a magnetic permeability detector, which measured the enrichment of superparamagnetic tracer in the sediment due to complex formation with human albumin. Total analysis time was 6.5 min. A linear response was obtained for 0-400 mg/L albumin with a detection limit of 5 mg/L. The total coefficient of variation (CV) was 11% calculated from four consecutive runs on a urine sample containing 11.1 mg/L human albumin during 3 consecutive days. Human urinary albumin analysis was performed on 149 patient samples using the MIA technique and the obtained results showed good correlation with the hospital immunoturbidimetric reference method (y = 1.004x + 4.01, R2 = 0.978, N = 149) and a commercially available point of care albumin analysis provided by HemoCue Inc. (y = 0.98x + 5.8, R2 = 0.833, N = 90).

Albumins↗

PubMed2004

Single-step quantitation of DNA in microchip electrophoresis with linear imaging UV detection and fluorescence detection through comigration with a digest.

We demonstrate a convenient single-step quantitation technique for double-stranded DNA (dsDNA) fragments in polymerase chain reaction (PCR) products based on microchip capillary electrophoresis (micro-CE)/UV or fluorescence detection. PCR products of polymorphisms on the human Y-chromosome related to spermatogenic failure did not need purification. They were premixed and comigrated with a DNA digest whose concentration was known. Hydroxyethyl cellulose (HEC) dissolved in 5x Tris-borate-EDTA (5x TBE, pH 8.3) was used as a separation matrix in a linear polyacrylamide-coated quartz microchip, while mixed poly(ethyl oxides) (PEOs) of different molar-masses dissolved in 1 x TBE (pH 8.3) containing 1 ng/microl ethidium bromide was used as a separation matrix in an uncoated poly(methyl methacrylate) (PMMA) microchip. Elution profiles were monitored under either real-time linear imaging UV detection in the snapshot mode where the total separation time is fixed, or light-emitting diode (LED) confocal fluorescence detection in the finishline mode where solutes migrate over the same separation length. It is found that, in both modes, a linear relation exists between the peak areas (A) and the multiplication of the digest concentrations (C) and the fragment sizes (L) in a DNA restrictive digest. Using the comigration electropherogram of a single-step experiment, the concentrations of PCR products were directly determined using the A versus LC linear relationship. The sole condition to obey is that the chosen digest has different fragment sizes with the PCR products of interest. This condition is easy to obey, because micro-CE owns high separation ability, and many digests are commercially available. The recovery of the technique was between 98 and 105%. The R.S.D. for chip-to-chip concentration measurements was less than 6.0% (n = 6). Hence, the technique was accurate and reliable for DNA assays.

DNA↗

PubMed2005

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of seleno amino acids.

This article describes an effective microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related seleno amino acids, including Se-methionine (Se-Met), Se-ethionine (Se-Eth), Se-methyl cysteine (Se-Cys), utilizing o-phtaldialdeyde/2-mercaptoethanol (OPA/2-ME) derivatization. Relevant parameters of the chip separation and amperometric detection are examined and optimized using a response surface methodology (RSM). Under optimum conditions, the analytes could be separated and detected in a 30 mM borate buffer (pH 9.3, with 28 mM sodium dodecul sulfate) within 300 s using a separation voltage of 2000V and a detection voltage of +0.9 V. Linear calibration plots are observed for micromolar concentrations of the Se-amino acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of selenoamino-acid compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption.

Amino Acids↗

PubMed2006

Determination of gamma- and alpha-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV-vis detection and comparison with evaporative light scattering detection.

A rapid direct method (Method I) for measuring gamma- and alpha-tocopherols in human milk was developed and validated using reversed-phase high-performance liquid chromatography with ultraviolet/visible (UV-vis) detection. Human milk, with an internal standard (alpha-tocopherol acetate) added, was diluted in hexane. The chromatographic system consisted of a short column (50 mm x 2.1mm I.D., 3 microm particle size) that allowed the separation of the gamma- and alpha-tocopherols in less than 6 min. The new direct method (Method I) was compared with other methods. Method II (saponification with ultraviolet/visible detection) determined 24% and 22% less gamma- and alpha-tocopherols, respectively. Method III (saponification with evaporative light scattering detection) gave the same values for alpha-tocopherol content as Method II. However, the amount of sample used in the application of Method III was higher than that used in Method II. Furthermore, Method I uses smaller amounts of solvents, and it is simpler and faster than Methods II or III. Only a small volume of sample is needed, which is an additional advantage for biological assays.

Chromatography, High Pressure Liquid↗

PubMed2006

Trends in detection of warfare agents. Detection methods for ricin, staphylococcal enterotoxin B and T-2 toxin.

An overview of the different detection methods available for ricin, staphylococcal enterotoxin B (SEB) and T-2 toxin is presented here. These toxins are potential biological warfare agents (BWA). The aim of this review is not to cover all the papers that had been published but rather to give an overall picture of the trend in the detection methodologies for potential biological warfare agents as we do see the emerging threats from these three toxins. The advantages and disadvantages of each methodology as well as the detection limit will be reviewed. It seems that mass spectrometry has created a niche for analysis of proteinaceous toxins, ricin and SEB as well as molecular toxin, T-2 toxin given its high sensitivity, high selectivity, high specificity and capability to identify and quantify unknown agents simultaneously in a short time frame. But its main drawbacks are its sophisticated instrumentation and its high cost. Improvised immunoassay may be an alternative.

Bacterial Toxins↗

PubMed2006

Selective ultratrace detection of Al(III) and Ga(III) complexed with a calcein isomer by capillary zone electrophoresis with laser-induced fluorescence detection. Comparison of emissive polyaminocarboxylates as derivatizing ligands.

A highly sensitive and selective capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) for Al(3+) and Ga(3+) was developed using a calcein 4',5'-isomer as a pre-capillary complexing agent. Direct fluorescent detection without an agent added to the carrier buffer provided the notable detection limits of 3.9x10(-10) and 4.6x10(-10)moldm(-3) for Al and Ga, respectively. By applying this system successfully to a river water sample, its potential for practical applications was ascertained. A comparison of three aromatic polyaminocarboxylate ligands related to the coordination structure and the distance between the fluorophore and the chelating moiety, revealed some interesting factors regarding their selectivity.

Aluminum↗

PubMed2004

High sensitivity detection of protein molecules picked up on a probe of atomic force microscope based on the fluorescence detection by a total internal reflection fluorescence microscope.

We developed a method to detect and identify proteins on a probe of the atomic force microscope (AFM) with a high sensitivity. Due to a low background noise of the total internal reflection fluorescence microscope employed as a detecting system, we were able to achieve a high enough sensitivity to detect zeptomole orders of protein molecules immobilized on the tip. Several different methods to immobilize protein molecules to AFM-probes were tested, meant for a wide range of applications of this method. Furthermore, we demonstrated that different proteins were clearly distinguished by immunofluorescence microscopy on the probe using their specific antibodies.

Antibodies↗

PubMed2005

The detection of salmochelin and yersiniabactin in uropathogenic Escherichia coli strains by a novel hydrolysis-fluorescence-detection (HFD) method.

Escherichia coli strains produce a variety of structurally different siderophores of which enterobactin, aerobactin and yersiniabactin have been reported earlier to occur in strains of extraintestinal infections. In uropathogenic E. coli (UPEC) strains novel siderophores, named salmochelins, have recently been identified which contain C-glucosylated 2,3-dihydroxybenzoyl-L-serine (glucosyl-DHB-serine) residues connected in a linear (mono-, di- , trimeric) or cyclic form. We report here on a fast and simple hydrolysis-fluorescence-detection (HFD) method, based on identification of C-glucosylated dihydroxybenzoic acid (glucosyl-DHB). Salmochelin containing culture filtrates were bound to DEAE cellulose spin columns, hydrolyzed and the breakdown products were subsequently identified by HPLC or thin layer chromatography (TLC). The hydrolysis products can be easily detected by their fluorescence, either during HPLC separation connected to a fluorescence detector or after TLC on cellulose plates viewed under a UV254 or UV365 lamp. While DHB originates from the hydrolysis of enterobactin and salmochelin, glucosyl-DHB is only found as a characteristic hydrolysis product of salmochelins (S1, S2, S4). The HFD method allows detection of salmochelin in the presence of other siderophores, such as enterobactin, aerobactin and yersiniabactin. Several clinical UPEC isolates containing the iroN gene cluster were analyzed by this procedure, showing that all isolates were glucosyl-DHB positive indicating salmochelin production, while a collection of other pathogenic E. coli strains (EHEC, EIEC, ETEC, EAggEC and EPEC) were glucosyl-DHB negative. In addition, the HFD method allowed the identification of yersiniabactin due to a fluorescent salicylate-containing degradation product.

Chromatography, High Pressure Liquid↗

PubMed2005

Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in Plasma Preparation Tubes (PPT).

BACKGROUND: The accurate and reliable quantification of HIV RNA is an essential part of the management of HIV infected individuals, and elucidation of factors that may affect HIV RNA measurements, such as the use of Vacutainer Plasma Preparation Tubes (PPT), is crucial. OBJECTIVES: The objective of this study was to determine if plasma samples with viral loads close to the lower limit of the dynamic range of the assay collected in PPT tubes had increased levels of HIV RNA as compared to samples collected in standard EDTA tubes. STUDY DESIGN: HIV RNA levels were compared in 112 paired plasma samples collected in PPT and standard EDTA tubes. All samples had been frozen prior to testing. RESULTS: Discrepancies between PPT and EDTA tubes did not occur for samples with high viral loads. However, in samples with viral loads close to the lower limit of the dynamic range, levels of HIV RNA detected were higher in a large proportion of PPT as compared to the corresponding EDTA plasma samples. Forty percent of plasma pairs had no detectable HIV RNA in the EDTA aliquot, but had low levels of HIV RNA in the corresponding PPT aliquot. CONCLUSIONS: This prospective study underlines the need for cautious interpretation of small transient viral load changes in samples with values close to the detection limit.

Acquired Immunodeficiency Syndrome↗