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At least 217 records · Page 12Linked to original sources

PubMed2005

Detection of transferrin isoforms in human serum: comparison of UV and ICP-MS detection after CZE and HPLC separations.

Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L(-1) borate buffer (pH 8.4) containing 3 mmol L(-1) diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm x 50 microm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L(-1) in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP-MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP-MS instrument was used to minimize polyatomic interferences on the (56)Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02-0.04 micromol L(-1) Tf for HPLC-ICP (ORS)-MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S(2), S(3), S(4), S(5), and S(6)) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.

Chromatography, High Pressure Liquid↗

PubMed1996

HPLC analysis of carbohydrates on POLYSPHERCH(R)OH columns using pulsed amperometric detection (PAD) with sodium hydroxide as post column detection reagent.

Carbohydrates have been separated on POLYSPHER(R)CH OH columns using pulsed amperometric detection (PAD) and UV detection (lambda =196 nm) in series and pure water as mobile phase. Nearly baseline separations have been obtained for the glycoprotein carbohydrates of sialic acid ( N-acetylneuraminic acid, NANA), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). As carbohydrates dissolved and eluted with pure water are present in the neutral form they are not detectable with PAD in contrast to carbohydrate anions formed at high pH values. Therefore an additional NaOH post column reagent has been continuously pumped through a mixing chamber into the mobile phase to form carbohydrate anions resulting in improved detection limits. Monosaccharides as well as glycoprotein carbohydrates could be detected in the microg/ml-range. This method has been applied successfully to the analysis of sugars in fruit juice. With only 2 microl of juice per 50 ml water, the determination of the main constituents, sucrose, glucose and fructose, was possible in a few minutes without sample preparation.

Journal Article↗

PubMed1999

Detection of multiple fibronectin isoforms in fetal fibroblast monolayer culture: a novel method for the qualitative and quantitative detection of multiple antigens.

Analyzing the expression of multiple distinct antigens within a single monolayer culture involves cumbersome immunostaining techniques. We describe a simple and economical procedure for the detection and quantification of multiple antigens within a single monolayer. By generating an immunohistochemical grid which divides a monolayer in a standard tissue culture dish into 20 distinct areas, we were able to detect and quantify four individual fibronectin (FN) isoforms within a single fibroblast monolayer culture. Quantification of each isoform was performed using a modified enzyme-linked immunoassay. In addition, within the same monolayer, each FN isoform was detected using standard immunohistochemical detection with DAB visualization. Using this novel approach to immunohistochemical analysis we determined that within the first 4 days of culture, the quantity of all FN isoforms increases faster than the number of cells. However, upon reaching confluency, the quantity of FN/cell drops dramatically. After reaching confluency, the amount of FN/cell levels off and remains constant within the postconfluent monolayer. Statistical analysis of the quantity of FN/cell indicates that a significant reduction in the amount of FN/cell occurs in the 2 days prior to reaching confluency. The distribution of all the FN isoforms, with the exception of B-FN, was found along the length of the cell body. In contrast, the distribution of B-FN was altered in postconfluent monolayers where it was detected only in distinct locations within the monolayer.

Antigens↗

PubMed2005

Prognostic significance of intraperitoneal cancer cells in gastric carcinoma: detection of cytokeratin 20 mRNA in peritoneal washes, in addition to detection of carcinoembryonic antigen.

BACKGROUND: In patients with gastric cancer, the presence of gastric cancer cells in the peritoneal cavity detected by cytologic examination, is a significant prognostic factor. A more sensitive, reverse transcriptase-polymerase chain reaction (RT-PCR) technique, amplifying carcinoembryonic antigen (CEA), was introduced as a new detection system, but produced some false-positive results. A search for other molecular markers is ongoing. METHODS: Peritoneal washes were obtained from 195 patients with gastric carcinoma during surgery. Cytokeratin 20 (CK20) mRNA levels were quantified, in addition to those of CEA, using the LightCycler, and the feasibility of CK20 as a target was evaluated. RESULTS: CK20 was limited, in terms of sensitivity, for detecting disseminated cancer cells (sensitivity, 63%; specificity, 91%; positive predictive value, 70%; and negative predictive value, 88%). Multimarker analysis was performed, in which samples positive for either CK20 or CEA mRNA were considered to be positive for cancer cells. Multivariate analysis identified the multimarker analysis as a significant independent prognostic determinant. CONCLUSION: CK20 RT-PCR produced information that could add a significant impact to the knowledge obtained by CEA RT-PCR, although detection by CK20 alone was not sufficiently sensitive to replace CEA in the detection system.

Biomarkers, Tumor↗

PubMed1987

Detection of influenza viruses through selective adsorption and detection of the M-protein antigen.

A model system has been developed which permits rapid detection of influenza viruses through targeting of the M (membrane or matrix)-protein; a type-specific antigen, in an enzyme-linked immunosorbent assay system. This technique exploits the hydrophobic properties of M-protein; the M-protein is selectively and rapidly adsorbed to polystyrene surfaces even in the presence of a 5000-fold excess of bovine serum albumin. Hyperimmune antiserum prepared to purified M-protein is used as the detecting reagent. All type A influenza viruses could be detected by this technique, type B influenza viruses reacted to a slight extent and Sendai virus (parainfluenza virus, type 1) did not react. Virus could be detected to levels as low as 3 ng. Purification of M-protein and preparation of hyperimmune sera from other related virus groups, such as type B influenza viruses, paramyxoviruses and rhabdoviruses should permit detection of these agents by a similar technique.

Adsorption↗

PubMed1996

Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: optimization, validation, and comparative analysis with other detection systems.

An ultra-sensitive assay for reverse transcriptase (RT) activity called Amp-RT has been developed. An in vitro transcribed heteropolymeric RNA sequence was used as a template, and polymerase chain reaction (PCR) amplification with Southern-blot hybridization served as a detection system for the cDNA product of the reaction. Titration of Mg2+ and Mn2+ concentrations using the human immunodeficiency virus type 1 (HIV-1) and the human T lymphotropic virus type 1 (HTLV-I), respectively, showed optimal assay reactivity for both viruses at 2-20 mM of Mg2+. Analysis of density banded HIV-1 showed that the peak RT activity of the assay was associated with the fractions consistent with retrovirus particles. The sensitivity of Amp-RT was also compared with that of three conventional RT assays by using seven different retroviruses including HIV-1, simian immunodeficiency virus (SIV), caprine arthritis-encephalitis virus (CAEV), HTLV-I and HTLV-II, simian retrovirus type 2 (SRV-2), and gibbon ape leukemia virus (GALV). HTLV-I, HTLV-II, and GALV could not be detected by the three conventional RT assays. Amp-RT was able to detect all these viruses in 10(1)-10(3)-fold dilutions. Similarly, Amp-RT was found to be 10(3)-10(6)-fold more sensitive than the other RT assays in detecting HIV-1, SIV< or CAEV. Culture supernatants from uninfected cell lines were all Amp-RT negative. A quantitative Amp-RT assay was also developed by using recombinant HIV-1 RT and signal quantitation. The assay was found to have a 5 log linear range, and therefore, provides a useful tool for quantitating RT and retroviruses. Amp-RT offers a sensitive generic tool for the qualitative and quantitative detection of known and unknown retroviruses.

Animals↗

PubMed1994

Rapid and sensitive detection of the femA gene in staphylococci by enzymatic detection of polymerase chain reaction (ED-PCR): comparison with standard PCR analysis.

Despite its recent cloning and characterization, the physiological role of FemA remains unclear. To easily and reliably identify femA in staphylococci, we analysed 45 blood isolates of staphylococci using enzymatic detection of polymerase chain reaction (ED-PCR). ED-PCR is a new method that detects amplified PCR products using biotin-streptavidin affinity and an enzyme-linked antibody. Of 45 samples, 34 strains contained the femA gene as detected by ED-PCR. Phenotyping analysis showed that these 34 strains (femA-positive) were Staphylococcus aureus and that the other 11 (femA-negative) were S. epidermidis. These results were completely consistent with the results of femA detection using standard PCR and subsequent Southern blot hybridization. The ED-PCR procedure was complete within 4 h and could be done in one tube. We conclude that ED-PCR is a rapid, simple and reliable method for detecting the femA gene and that it provides an important means of classifying staphylococcal strains in the clinical field.

Base Sequence↗

PubMed2004

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system.

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.

Bacterial Proteins↗

PubMed2005

SPR-based immunosensor for the CRP detection--a new method to detect a well known protein.

The c-reactive protein (CRP) is one of the significant human blood serum markers for inflammatory processes. The serum presence of this hepatic approximately 115 kDa protein of five identical subunits accompanies several diseases (e.g. CVD, inflammatory bowel diseases) and is nowadays detected by high-sensitivity ELISA assays in blood serum. To enable CRP detection in other matrices, an SPR-based (surface plasmon resonance) immunosensor for the CRP detection has been established. A linear detection range of 2-5 microg CRP per ml was found, using two different antiCRP antibodies (monoclonal, IgG) for CRP trapment and detection. Furthermore, the kinetic antibody association and dissociation constants of one antibody (antiCRP, clone C2) could be determined.

Antibodies↗

PubMed2005

Computer-aided detection (CAD) of cancers detected on double reading by one reader only.

We evaluated the role of computer-aided detection (CAD) in cancers undergoing double reading and detected by one reader only. A series of 33 cancers, originally missed by the first reader and detected by the second reader, and 75 negative controls were processed to assess CAD sensitivity, and was read by the six radiologists who originally missed the cancers with the help of CAD printouts. CAD case-based sensitivity, specificity and positive predictive value were 51.5%, 18.6% and 21.7%, respectively. Average sensitivity of all radiologists in all cancers in the series was 74.7%, being higher for CAD+ (86.2%) than for CAD- (62.5%) cancers (P<0.01). When reading cancer cases that they had originally missed, radiologists had a sensitivity of 75.8%, which was higher for CAD+ (100.0%) than for CAD- (58.3%) cancers. The average recall rate was 14.2%, the majority of recalls (45 out of 64) occurring for lesions marked by CAD. CAD may help in detecting at most half of cancers missed at a single reading but detected by a second reader.

Breast Neoplasms↗

PubMed2006

Ion chromatographic determination of trace iodate, chlorite, chlorate, bromide, bromate and nitrite in drinking water using suppressed conductivity detection and visible detection.

An ion chromatography method for the simultaneous determination of trace iodate, chlorite, chlorate, bromide, bromate and nitrite in drinking water has been developed using an anion-exchange column and the suppressed conductivity detector, followed by post-column addition of reagent to enhance visible absorbance detection of ions. A high capacity anion exchange Ion Pac9-HC column (250 mm x 4 mm I.D.) was used. Eight millimole per liter sodium carbonate was used as eluent, an auto-suppression external water mode was selected, 0.5 g/l o-dianisidine.2HCl (ODA)+4.5 g/l KBr+25% methanel+5.6% nitric acid was used as post-column reagent. The post-column reaction (PCR) temperature was at 60 degrees C, and the visible absorbance detected wavelength at 450 nm. The sample's pH and coexist anions had no influence on determination. The method enjoyed a wide linear range and a good linear correlation coefficient (r>0.999). The method detection limits were between 0.023 and 2.0 microg/l. The average recoveries ranged from 87.5 to 110.0%, and the relative standard deviations (RSD) were in the range of 1.1-4.6%. The analytical results by the method of post-column addition of reagent to enhance visible absorbance detection of anions was compared with that of the suppressed conductivity detection, and the former was proved to be better in sensitivity and selectivity. The results showed that this method was accurate, sensitive and might be good for application and suitable for trace analysis at the level of mug/l.

Anions↗

PubMed2006

Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry.

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.

Caseins↗

PubMed2006

Ion-trap tandem mass spectrometry-based analytical methodology for the determination of polychlorinated biphenyls in fish and shellfish. Performance comparison against electron-capture detection and high-resolution mass spectrometry detection.

Optimization of the Varian Saturn 2200 ion-trap tandem mass spectrometry (IT-MS/MS) system and comparison of its data quality with two other detection methods [electron-capture detection (ECD) and high-resolution mass spectrometry (HRMS)] was pursued by measuring polychlorinated biphenyls (PCBs) levels in fish and shellfish samples. IT-MS/MS methodology provided limits of detection (LOD) comparable to those obtained by ECD but superior specificity for the detection of a selected number of 39 PCB native congeners and 9 (13)C-labelled PCB standards. The method detection limits (MDLs) established for IT-MS/MS ranged between 1.0 and 5.0 pg/g on a wet weight basis while those obtained by ECD and HRMS were 1.0-4.0 pg/g and 0.1-2.0 pg/g, respectively. Overall, the results obtained in the study demonstrate that gas chromatography (GC) combined with IT-MS/MS provide higher data quality than those achievable by GC-ECD. For this particular set of target analytes the specificity achievable with IT-MS/MS was comparable to that obtained by HRMS and both techniques provided comparable data in terms of accuracy and precision.

Animals↗

PubMed2004

Detection and differentiation of Plasmodium species by polymerase chain reaction and colorimetric detection in blood samples of patients with suspected malaria.

Polymerase chain reaction (PCR) is now recognized as a sensitive and specific method for detecting Plasmodium species in blood. In this study, we tested 279 blood samples, from patients with suspected malaria, by a PCR assay utilizing species-specific colorimetric detection, and compared the results to light microscopy. Overall, both assays were in agreement for 270 of the 279 specimens. P. vivax was detected in 131 (47.0%) specimens, P. falciparum in 64 (22.9%) specimens, P. ovale in 6 (2.1%) specimens, and P. malariae in 5 (1.8%) specimens. Both P. falciparum and P. vivax were detected in a further 10 (3.6%) specimens, and 54 (19.3%) specimens were negative by both assays. In the remaining nine specimens, microscopy either failed to detect the parasite or incorrectly identified the species present. In summary, the sensitivity, specificity and simplicity of the PCR assay makes it particularly suitable for use in a diagnostic laboratory.

Animals↗

PubMed2006

A novel method based on ligase detection reaction for low abundant YIDD mutants detection in hepatitis B virus.

A novel method based on ligase detection reaction (LDR) coupled with polymerase chain reaction (PCR) was used in this study to detect low abundant YIDD mutants in hepatitis B virus (HBV), which was able to detect 10(2) mutants from 10(8) wild type copies in plasmids and 10(3) from 10(7) in clinical serum samples. Direct sequencing and microarray hybridization were selected for sensitivity comparison together with this assay. By direct sequencing, 6 of 50 clinical specimens infected with HBV were found to be YIDD and the others to be YMDD, while by microarray hybridization, 28 specimens were detected to contain both YMDD and YIDD, and the others contained YMDD only, and with this assay, 31 specimens containing both YMDD and YIDD were detected with the others containing YMDD only, which indicated that the assay was relatively more sensitive.

Journal Article↗

PubMed2004

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.

BACKGROUND: The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. OBJECTIVES: Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. STUDY DESIGN: The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. RESULTS AND CONCLUSION: Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.

Base Sequence↗

PubMed2004

Event-related potential correlates of judgment categories and detection sensitivity in a visual detection task.

Event-related potentials were recorded from human subjects performing a visual detection task to find correlates of detection performance (d' and beta) in accordance with the theory of signal detection. Two spatial frequencies of square waves shifted horizontally with three shifting-step varieties were presented to the subjects who reported whether or not the waves were perceived to shift. Although three components of N1, P2, and P3 were observed for all of four response categories of hit, miss, false alarm, and correct rejection, only the amplitudes of the P3 component at vertex and parietal sites highly correlated to the detection sensitivity of d'. It was also found that coefficients of correlation between the P3 amplitudes at these sites and observed hit and false alarm rates were highly significant and a d'-extrapolation value reproduced by the normalized P3 amplitudes and the usual d' indicated a highly linear trend. Results suggest that generation of the P3 component is associated with "threshold-modulating" mechanisms which determine detection sensitivity of a task for each perceptual event.

Adult↗

PubMed2001

Derivatization, stabilization and detection of biogenic amines by cyclodextrin-modified capillary electrophoresis-laser-induced fluorescence detection.

o-Phthalaldehyde (OPA) derivatives of eight biogenic amines were stabilized at 5 degrees C by forming inclusion complexes with methyl-beta-cyclodextrin (MBCD). The derivatives were separated and detected by cyclodextrin-modified capillary electrophoresis (CE) with UV or laser-induced fluorescence (LIF) detection. Using a borate buffer, pH 9.0 consisting of ethanol and a mixture of negatively charged sulfobutylether-beta-cyclodextrin and neutral MBCD, baseline separation of the eight OPA derivatives was achieved within 25 min with high separation efficiencies. The detection limits (S/N=3) obtained by UV and LIF detection were determined to be 10 microM and 0.250 microM, respectively. Glutamic acid was added after the initial derivatization step to neutralize residual OPA which otherwise caused a significant interference, particularly when analysis was performed around the detection limit of the OPA derivatives. Important biogenic amines in fish, wine and urine were then derivatized and determined by CE-LIF. In the case of sole and rainbow trout, the results obtained were validated by an enzymatic assay using putrescine oxidase.

Biogenic Amines↗