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PubMed1994

Nonradioactive labeling and high-sensitive detection of PCR products.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.

Biotechnology↗
PubMedDate not supplied

Newer methods for diagnosis of cytomegalovirus infection.

Recently developed techniques have greatly increased the sensitivity and speed of detection of CMV and of host antibody responses to it. Newer serologic assays such as enzyme immunoassay or latex agglutination assay are accurate and efficient for screening donors and recipients and for determining susceptibility to primary infection. Available IgM antibody assays have occasional utility in recognition of recent infection. The slow process of isolating CMV in cell culture has prompted development of effective rapid techniques that utilize CMV-specific monoclonal antibodies and DNA sequences. Immediate-early viral antigen can be detected in infected cell cultures within hours of specimen inoculation. CMV antigens can also be detected directly in cells within clinical specimens. DNA hybridization has been used for CMV analysis in dot-blot, Southern blot, and in situ hybridization assays; the use of the latter is increasing for the detection of virus in fixed, paraffin-embedded tissue sections. Antigen or nucleic acid detection procedures, when applied directly to relevant clinical specimens, aid in the recognition of tissue invasive disease for which antiviral therapy might be considered. DNA amplification, using the polymerase chain reaction, achieves new levels of sensitivity in viral detection and should be useful for clinical diagnosis and for investigation of CMV pathogenesis and latency.

Antibodies, Viral↗
PubMed1994

What direction of motion do we see if luminance but not colour contrast is reversed during displacement? Psychophysical evidence for a signed-colour input to motion detection.

To investigate the effects of colour upon motion detection, directional discrimination by human observers was determined using two-frame kinematograms in which the two classes of element composing the pattern could differ either in luminance alone (achromatic condition), or in both colour and luminance (chromatic condition). The elements in the second frame could either have the same colour/luminance as corresponding elements in the first frame, or they could be changed (swapped) in colour and/or luminance. The angular size of the elements was varied by changing the viewing distance. Changing colour between frames disrupted motion detection when the angular size of elements was large (0.9 deg) but not when they were small (0.225 deg), replicating a previous result. Detection of motion with chromatic patterns was generally superior to that with achromatic patterns, particularly with large element size. Luminance swap combined with colour swap produced the "reverse phi" phenomenon: however, when luminance was swapped with colour staying the same between frames, forward motion was seen, suggesting that forward motion based on colour dominated over reversed motion based on luminance. We conclude that signed chromatic information has an input to motion detection at low but less so at high spatial frequencies. Information across colour and luminance is combined in a final common pathway for motion detection, resulting either in enhancement if they are in agreement, or in disruption if they conflict.

Color Perception↗
PubMed2004

Analysis of glycerophosphoinositol by liquid chromatography-electrospray ionisation tandem mass spectrometry using a beta-cyclodextrin-bonded column.

Glycerophosphoinositol (GroPIns) has been demonstrated to have important roles in many intracellular regulatory processes. GroPIns has been analysed for many years by anion-exchange HPLC after radiolabelling of cells in culture, but no method has been developed, to our knowledge, for the direct detection and quantitation of the unlabelled compound in such biological samples. Here is reported a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct quantitative analysis of GroPIns that can indeed be applied to cell extracts. Analyses were performed on a beta-cyclodextrin-bonded HPLC column using a binary mobile phase of acetonitrile and 20 mM ammonium formate in water, which allowed direct on-line detection by tandem mass spectrometry in negative electrospray ionisation (ESI) mode. The method was applied to the quantitative analysis of GroPIns in selected rat cell lines after a two-phase acid extraction of cultured cells using external calibration. The potential matrix signal suppression effects were investigated by the parallel quantitation of GroPIns in extracts of selected cultured cell lines with both external calibration and the standard additions method. The accuracy data obtained demonstrated the feasibility of external calibration, so allowing a simpler and less time-consuming approach than that of the standard additions method.

Calibration↗
PubMed1995

Cell culture-PCR technique for detection of infectious cytomegalovirus in peripheral blood.

We determined the efficacy of PCR for the direct detection of infectious cytomegalovirus (CMV) in specimen-inoculated MRC-5 tube cultures (cell culture-PCR [CC-PCR]). Parallel testing was performed by a shell vial assay-indirect immunofluorescence assay (SVA-IFA) and isolation (conventional MRC tube cultures [TC-CPE]. The sensitivity, specificity, and positive and negative predictive values for CC-PCR, SVA-IFA, and TC-CPE were 81, 99, 94, and 94%, 86, 100, 100, and 96%, and 77, 100, 100, and 93%, respectively. Future application of CC-PCR may be directed toward its use as a screening tool for cytomegalovirus genotypic variants.

Blood↗
PubMed2002

A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration.

Protein phosphatase inhibition assays currently used for the detection of cyanobacterial peptide hepatotoxins in drinking water require an enrichment step using C18 cartridges to achieve lower the detection limit. This paper describes a colorimetric and fluorometric protein phosphatase inhibition method for the direct detection of microcystin-LR (MCYST-LR) in drinking water without complex clean-up steps and preconcentration procedures. In this assay three different substrates, p-nitrophenyl phosphate (p-NPP) and two fluorogenic compounds, 4-methylumbelliferyl phosphate (MUP) and 6,8-difluoro-4-methylumbelliferyl phosphate DiFMUP), were tested. The detection limits of the assay are 0.25 and 0.1 microg/l using colorimetric and fluorometric methods, respectively. These levels are well below the provisional guideline value for MCYST-LR of 1 microg/l of drinking water. The detection limit of the fluorometric method is comparable to that of the classical ELISA test. Although both the latter tests allow the detection of MCYST-LR in drinking water directly without pretreatment, the protein phosphatase inhibition assay remain less expensive and therefore more attractive for use in the routine assessment of drinking water contamination by microcystins.

Bacterial Toxins↗
PubMed1994

A novel kit for rapid detection of Vibrio cholerae O1.

We report on the development and testing of a novel, rapid, colorimetric immunodiagnostic kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae O1 in clinical specimens. Unlike conventional culture methods requiring several days to complete, the Cholera SMART kit can be used directly in the field by untrained or minimally skilled personnel to detect V. cholerae O1 in less than 15 min, without cumbersome laboratory equipment. A total of 120 clinical and environmental bacterial strains, including both O1 and non-O1 serotypes of V. cholerae isolated from samples collected from a variety of geographical regions, were tested, and positive reactions were observed only with V. cholerae O1. Also, results of a field trial in Bangladesh, employing Cholera SMART, showed 100% specificity and 96% sensitivity compared with conventional culture methods. Another field trial, in Mexico, showed that Cholera SMART was 100% in agreement with a recently described coagglutination test when 108 stool specimens were tested.

Bangladesh↗
PubMed1994

Detection of lectin activity on western blots using erythrocytes.

A method is described for the direct detection of lectins, agglutinating erythrocytes, on nitrocellulose membranes after Western blotting, thus avoiding protein extraction from specific bands in the gel, followed by agglutination assays. The methodology essentially involves exposing the lectin band on a nitrocellulose strip to trypsinized rabbit erythrocytes (2%, in 0.15 M NaCl) for 30 min at 37 degrees C and then carefully transferring the membrane to saline (4 degrees C) for a few gentle washes and then fixing it in a solution (0.2% glutaraldehyde in 0.15 M NaCl) for 30 min. Later, the membrane is gently washed several times in 0.15 M NaCl containing 10 mM beta-alanine. The lectin band is visualized as a red agglutinated patch. The method is specific for lectins that can agglutinate red blood cells and virtually has no cross reactivity with the various nonlectin proteins tested. Binding of erythrocytes to the lectin band on the nitrocellulose strip can be prevented by specific competing sugars. The method can be applied to screen for the presence of lectins in natural materials and to monitor lectin fractions during purification.

Animals↗
PubMed1991

Electrochemical detection of proteins in high-performance liquid chromatography using on-line, postcolumn photolysis.

Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective.

Chromatography, High Pressure Liquid↗
PubMed1988

Comparison of Visuwell enzyme immunoassay to culture for detection of group A Streptococcus in throat swab specimens.

A microwell enzyme immunoassay (Visuwell) for direct detection of Group A streptococcal antigen from throat swab specimens has been developed. It incorporates urease conjugated antibody as the detector and is easily interpreted by a yellow to purple color change. Throat specimens obtained on rayon-tipped swabs were transported moist in modified Stuarts medium and cultured before being tested in Visuwell (n = 585, prevalence 17.1%, sensitivity 88%, specificity 92.4%, predictive value positive 70.4%, predictive value negative 97.4%, and accuracy 91.6%). In instances of discrepancy between culture and Visuwell, throat swab extracts were tested in a latex agglutination test. In 21 of 37 instances of Visuwell-positive, culture-negative specimens, latex agglutination was positive. Throat specimens obtained using double rayon swabs and transported to the laboratory dry had one swab cultured and the other tested in Visuwell (n = 280, prevalence 20.4%, sensitivity 75.4%, specificity 88.3%, predictive value positive 62.3%, predictive value negative 93.4%, and accuracy 85.7%). When 1+ culture positive specimens were considered negative, a sensitivity of 97.6% was obtained. In 14 of 26 instances of Visuwell-positive, culture-negative specimens, latex agglutination was positive. Cross-reaction with organisms other than Group A Streptococcus found in the oropharynx was negligible in Visuwell. Limit of detection of Group A streptococcal antigen was equivalent for Visuwell and latex agglutination.

Adolescent↗
PubMed1985

Immunostaining free oligosaccharides directly on thin-layer chromatograms.

Oligosaccharides are chromatographed on amino-bonded high-performance thin-layer chromatography silica gel plates and after chromatography the aldehydes on the reducing ends of the oligosaccharides react with the amino groups on the silica gel. Bound oligosaccharides are immunostained directly on the chromatograms by monoclonal antibodies. The binding of antibodies is detected by autoradiography after a second incubation with 125I-labeled goat anti-mouse immunoglobulin. Using this method, 10 pmol of lacto-N-difucopentaose I (Leb hapten) and lacto-N-fucopentaose III (Lex hapten) are detected directly on the thin-layer chromatograms by monoclonal antibodies 10c17 and 534F8, respectively. Previously undescribed larger oligosaccharides containing these epitopes are also detected in human milk. This method may be used to identify and characterize antibody-binding oligosaccharides liberated from glycoconjugates by hydrazinolysis, by trifluoroacetolysis, by ozonolysis, or by treatment with endoglycosidases. This technique may also be used to determine the structural specificity of other carbohydrate-binding proteins such as lectins, toxins, and hormones or of bacteria and viruses that bind to cell surface glycoproteins.

Antibodies, Monoclonal↗
PubMed2005

[Rapid prenatal detection of Down syndrome by homologous gene quantitative PCR].

OBJECTIVE: To evaluate the use of homologous gene quantitative PCR (HGQ-PCR) as a method for non-invasive diagnosis of Down syndrome and for prevention of the birth of Down syndrome children. METHODS: HGQ-PCR, which can directly detect the additional copy of chromosome 21 by comparing simultaneously amplified two highly homologous genes, i.e. the human liver-type phosphofructokinase located on chromosome 21 critical region of Down syndrome (PFKL-CH21) and the human muscle-type phosphofructokinase located on chromosome 1 (PFKM-CH1), was performed in 38 clinically diagnosed Down syndrome patients and 178 normal controls. RESULTS: The ratios of PFKM-CH1/PFKL-CH21 products were 1.40 +/- 0.367 (mean +/- SD) and 0.46 +/- 0.21 (mean +/- SD) for disomy 21 and trisomy 21, respectively. The difference between these two groups was statistically significant (P<0.001). CONCLUSION: This approach has proven to be a practical and direct method for the detection of trisomy 21 and may also be applied to the detection of the extra piece of 21q involved in translocation-type of Down syndrome.

Chromosomes, Human, Pair 1↗
PubMed2001

Reaction of human myoglobin and peroxynitrite: characterizing biomarkers for myoglobin-derived oxidative stress.

Mixtures of human myoglobin (Mb) (or the Y103F variant of human Mb), authentic peroxynitrite (ONOO(-), ONOO(-):protein 2 mol/mol), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave radicals adducts at cysteine-110 (DMPO-C110) that are detected directly by electron paramagnetic magnetic spectroscopy (EPR). DMPO-C110 was detected exclusively over a range of DMPO concentrations (DMPO:protein ratios 25-100 mol/mol). Treatment of human Mb (or Y103F Mb) with the ONOO(-) generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) chloride (ONOO(-):protein 5 mol/mol) yielded a cross-linked Mb dimer as judged by SDS-PAGE analyses. Addition of DMPO or carbonate effectively eliminated the cross-linked product. Mass analyses of samples containing human Mb (or Y103F Mb), carbonate, and ONOO(-) indicated that nitration occurs exclusively at Y103. Thus, reaction of human Mb and ONOO(-) yields specific products that depend on the presence or absence of physiological concentrations of carbonate. These products may serve as biomarkers for the participation of Mb-derived radicals in the oxidative damage associated with myocardial reperfusion injury.

Biomarkers↗
PubMed2000

Free radicals in licorice-flavored sweets can Be detected noninvasively using low frequency electron paramagnetic resonance after oral administration to mice.

The observation of the fate of free radicals coming from food after oral administration could be important in evaluating their reactivity in vivo. The aim of this study was to demonstrate that it is feasible to detect directly in vivo free radicals coming from food with the use of low frequency electron paramagnetic resonance (EPR) spectroscopy. Because polyphenols are easily oxidized into stable radicals, we assumed that these radicals could be detected in food. We chose licorice, which contains several types of polyphenols. The presence of free radicals was demonstrated in licorice-flavored sweets. Using low frequency EPR spectroscopy, we detected these free radicals directly and noninvasively after oral administration to mice. These radicals were rather stable in the guts of the mice. This study is the first report demonstrating noninvasively the presence of free radicals in vivo coming from food.

Administration, Oral↗
PubMed2007

Hepatitis C viral infection is not associated with Waldenström's macroglobulinemia.

While a familial predisposition may exist in up to 20% of patients with Waldenström's Macroglobulinemia (WM), the precipitating cause of this B-cell malignancy remains unknown in most patients. In previous studies, an association between hepatitis C virus (HCV) infection and WM has been suggested as etiological. This relationship has been the subject of debate, however, with some studies demonstrating increased incidence of HCV infection among WM patients and other studies showing no such association exists. This discordance might be attributable to the analytical method used, HCV antibody detection, which might be ineffective in patients with immunosuppression. We therefore analyzed the prevalence of HCV in a large population of WM patients utilizing both an HCV antibody detection immunoassay as well as qualitative polymerase chain reaction assay to directly detect HCV presence in serum samples. None of 100 randomly tested WM patients in this study tested positive for HCV by either analytical method. Our results therefore demonstrate a lack of association between HCV and WM.

Adult↗
PubMed1991

High performance liquid chromatography of glucose using a post-column reactor of immobilized enzyme followed by electrochemical detection (glucose-LCEC).

A sensitive and selective, reasonably fast method for the determination of glucose content has been developed. A glucose oxidase immobilized column was coupled to a small-size anion exchange column/borate buffer chromatograph. The hydrogen peroxide produced in the enzyme reaction was detected directly by an amperometric detector using a platinum working electrode. The detection limit was 0.03 ppm (1.5 x 10(-7) M, 3 pmol/injection). The linear dynamic range was three orders of magnitude at least. The system was stable and reproducible both in short- and long-term operation. The proposed method is suitable for analysis of complicated matrices of biological samples because of its good selectivity and sensitivity.

Chromatography, High Pressure Liquid↗
PubMed1996

Pulsed electrochemical detection of nonchromophoric compounds following capillary electrophoresis.

Pulsed electrochemical detection (PED) following capillary electrophoresis (CE) has been applied to the direct detection of polar aliphatic compounds. These compounds typically have poor optical detection properties and are considered to be electroinactive under constant applied potentials. The voltammetric responses at microelectrodes of model compounds were studied under various buffers and pH conditions. The detection of unsubstituted carbohydrates requires highly alkaline conditions, whereas amine-containing compounds (e.g., glycopeptides, peptides, and amino acids) and thiocompounds can best be detected at mildly alkaline (i.e., pH 9.0) and mildly acidic (i.e., pH 5.5) conditions, respectively. In a comparative study of pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (IPAD), IPAD is determined to be better suited to manage the large oxide-formation backgrounds, which accompany oxide-catalyzed detections of amine- and sulfur-containing compounds. IPAD results in more stable baselines, eliminates oxide-induced artifacts, and often yields lower limits of detection. Mass detection limits under optimal conditions are typically 10 fmole or less. This paper delineates PED waveform parameters for the optimal detection of underivatized nonchromophoric compounds of biological significance in various operating buffers and electrolytes.

Cysteine↗