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At least 505 records · Page 28Linked to original sources
PubMed1983

A cell ELISA technique. The direct detection and semi-quantitation of immunoglobulin positive cells in 7 day lymphocyte cultures using the microtitre culture plates as the solid phase.

A sensitive ELISA is described which allows the detection and quantitation of immunoglobulin positive Peyer's patch and spleen lymphocytes in tissue culture microtitre plates. A methanol fixation step is used to fix the cells to the microtitre plate. The effects of various mitogens and an antigen on the number of immunoglobulin positive cells present after a 7 day culture were studied. Results were expressed either in optical densities or as percentages of the unstimulated control. The method proves a useful addition to existing techniques for studying the effects of mitogens and other agents on in vitro immunoglobulin synthesis by lymphocytes, and reflects immunoglobulin levels in the culture supernatants.

Animals↗
PubMed1981

Modified fluctuation test for the direct detection of mutagens in foods with Salmonella typhimurium TA98.

A modified fluctuation test (Green) with the Ames' tester strain Salmonella typhimurium TA98 has been examined for sensitivity to histidine feeding and for detection of minimal concentrations of daunomycin and S9 activated benzo[a]pyrene. The fluctuation test was found operable over a range of histidine concentrations between 0.25 and 1.25 microgram/ml using 48 tube assays and microtitre plates with 120 wells. The agar plate method yielded a comparable operational range for histidine concentration. With daunomycin, the microtiter fluctuation test was 48-fold greater in sensitivity than the macroscale fluctuation test. With benzo [a] pyrene, the microtiter fluctuation test was 4.8-fold greater in sensitivity than the macroscale test. The microtiter assay was 2.4 and 2.5-fold more sensitive than the plate and treat method with daunomycin and benzo [a] pyrene respectively.

Bacteriological Techniques↗
PubMed1988

The diagnosis of minor to moderate atherosclerotic lesions in the carotid artery bifurcation by means of spectral broadening combined with the direct detection of flow disturbances using a multi-gate pulsed Doppler system.

The diagnostic accuracy of noninvasive detection of carotid artery lesions with a diameter of less than 50% (minor to moderate) and greater than 50% reduction (severe) with the use of a multi-gate pulsed Doppler system (spectral analysis combined with the detection of flow disturbances) was studied in patients with focal neurological symptoms. Biplane arteriography was used as a reference. For all lesions the diagnostic accuracy (observed agreement), sensitivity and specificity was 88, 92, and 84%, respectively, and 84, 83, and 84%, respectively, when only minor to moderate lesions were considered. Kappa, a chance-corrected measure of agreement, which takes into account the marginal distribution of the data and, hence, may be considered to be a better parameter to describe diagnostic accuracy, was found to be 82.2%. The results in this study compare favorably with those obtained in studies employing duplex techniques (B-mode image combined with single-gate pulsed Doppler) and indicate that minor to moderate carotid artery lesions are detected rather accurately with the use of a multi-gate pulsed Doppler system.

Arteriosclerosis↗
PubMedDate not supplied

Direct detection and amplification of Helicobacter pylori ribosomal 16S gene segments from gastric endoscopic biopsies.

Helicobacter pylori is an organism thought to play an important causative role in gastritis and peptic ulcer diseases. We have designed an RNA dot blot assay for the detection of H. pylori, using as probe a synthetic oligonucleotide complementary to its 16S rRNA. We have also used oligonucleotide primers, complementary to conserved sequences within bacterial ribosomal 16S genes, to amplify a H. pylori ribosomal 16S DNA fragment via the polymerase chain reaction (PCR). After determining the DNA sequence of this amplified H. pylori fragment, primers were designed for specific PCR amplification of H. pylori ribosomal 16S DNA sequences. Samples from clinical endoscopic biopsies were PCR amplified with universal 16S ribosomal primers to detect the presence of bacteria and with H. pylori-specific primers to uniquely detect H. pylori. Finally, by comparing the H. pylori-specific PCR assay to commonly used diagnostic tests, we demonstrate that the molecular technique of PCR amplification shows promising applications for the clinical detection of H. pylori.

Base Sequence↗
PubMed1995

Direct detection and characterization of rotavirus into subgroups by dot blot hybridization and correlation with 'long' and 'short' electropherotypes.

BACKGROUND: Enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE) of viral RNA are well-established methods for detection of rotavirus in stool samples. Dot-blot hybridization has also been found to be a sensitive and specific technique for detection and characterization of rotaviruses. OBJECTIVES: To compare the performance of dot blot hybridization with ELISA and PAGE for detection of rotavirus in stool samples. To assess the use of dot blot hybridization for characterization of rotaviruses into subgroups. STUDY DESIGN: Stool samples were collected from 214 children presenting to the hospital with acute diarrhoea. These were assayed for rotavirus by ELISA and PAGE. Dot-blot hybridization was done with full length cloned radiolabelled c-DNA probes of gene segment 6 of SA-11 (subgroup I) and Wa (subgroup II) rotaviruses. RESULTS: Out of 214 stool samples 134 were found to be positive for rotavirus by one of the three methods. Among these 134 positive specimens 114 were positive by dot blot hybridization, this included 18 specimens which were positive only by dot blot assay. One-hundred-and-twelve of these 114 specimens could be subgrouped. Fifteen of these were classified as subgroup I, 97 as subgroup II and two had a dual subgroup specificity. Three subgroup 1 strains had a 'long' RNA pattern, whereas one subgroup II strain had a 'short' RNA pattern which has not been reported earlier for human rotaviruses. CONCLUSION: Dot blot hybridization as described here is a sensitive and specific assay for detection and subgrouping of rotaviruses. However, as there is a considerable genomic diversity among rotaviruses, the panel should include probes from all the genotypes of gene segment 6.

Journal Article↗
PubMed2004

Molecularly imprinted polypyrrole-based synthetic receptor for direct detection of bovine leukemia virus glycoproteins.

Preparation and basic characterization of polypyrrole-based molecularly imprinted polymer (MIP) for label-free detection of bovine leukemia virus (BLV) glycoprotein gp51 (gp51) is firstly described. Polypyrrole (Ppy) was selected as a matrix for preparation of MIP. Polypyrrole doped by gp51 (gp51/Ppy) was prepared by electrochemical deposition of this polymer on the surface of platinum-black electrode. Then, molecules of gp51 were removed from polymeric backbone and molecularly imprinted polypyrrole (mPpy) was ready for recognition of gp51 in the aqueous solution. Pulsed amperometric detection (PAD) was applied for label-free detection of gp51 in the samples. Anti-gp51 antibodies and secondary antibodies labeled with horseradish peroxidase (HRP) were involved as markers for the control of mPpy preparation procedures. Control experiments were also simultaneously performed by spectrophotometrical detection of HRP activity. Application of anti-gp51 and HRP labelled secondary antibodies confirmed that generation of analytical signal was based on redoping of mPpy by gp51. During our experiments, only few mPpy redoping/dedoping cycles were effective, but generally this method seems to be very effective for the future development of mPpy-based MIPs. Preparation, electrochemical investigation and control procedures are described in the current paper.

Biomimetic Materials↗
PubMed2004

Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA).

A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.

Bacteriological Techniques↗
PubMed2006

Blood and urine samples as useful sources for the direct detection of tuberculosis by polymerase chain reaction.

The aim of the study was to assess the utility of the polymerase chain reaction (PCR) assay in blood and urine for the diagnosis of tuberculosis (TB). We prospectively evaluated the usefulness of PCR performed in blood and urine samples from patients with proved or probable TB compared with a control group of patients. The PCR technique was performed using IS6110 primers. We included in the study 57 patients (43 with definite TB and 14 with probable TB) and 26 controls. Blood and urine samples were drawn at the time of microbiologic diagnosis and 3, 6, 9, and 12 months later. Cultures were positive in the early period (<1 month after treatment) in 11 of 57 patients (19%) with probable or definite TB, in comparison with 42% of patients (24/57) who yielded a positive PCR (P = 0.02). Urine samples increased the sensitivity of PCR determination in blood samples by 10%. The PCR in blood and/or urine was positive in 41% of patients with pulmonary TB, in 36% of patients with extrapulmonary TB, and in 50% of patients with disseminated TB. Mycobacterium tuberculosis was still detectable by PCR in 5 of 13 patients with cured TB after 1 or more months of antituberculous treatment. The PCR detection of M. tuberculosis in blood and urine samples is useful for the diagnosis of different clinical forms of TB, mostly in those patients in which sample extraction is difficult or requires aggressive techniques. The sensitivity of this technique could be improved studying more than 1 sample in each patient, even after initiating an antituberculous treatment.

Antitubercular Agents↗
PubMed2004

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry.

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.

Animals↗
PubMed2006

13C direct detected COCO-TOCSY: a tool for sequence specific assignment and structure determination in protonless NMR experiments.

A novel experiment is proposed to provide inter-residue sequential correlations among carbonyl spins in (13)C detected, protonless NMR experiments. The COCO-TOCSY experiment connects, in proteins, two carbonyls separated from each other by three, four or even five bonds. The quantitative analysis provides structural information on backbone dihedral angles phi as well as on the side chain dihedral angles of Asx and Glx residues. This is the first dihedral angle constraint that can be obtained via a protonless approach. About 75% of backbone carbonyls in Calbindin D(9K), a 75 amino acid dicalcium protein, could be sequentially connected via a COCO-TOCSY spectrum. 49(3)J(C')(C') values were measured and related to backbone phi angles. Structural information can be extended to the side chain orientation of aminoacids containing carbonyl groups. Additionally, long range homonuclear coupling constants, (4)J(CC) and (5)J(CC), could be measured. This constitutes an unprecedented case for proteins of medium and small size.

Calbindins↗
PubMed2006

A novel strategy for quantitative isoform detection directly performed from culture supernatant.

Currently, one of the most used techniques for the determination of isoform pattern analysis is isoelectric focusing. Routinely, this is performed by immunoblotting. Blotting of proteins after isoelectric focusing on IPG gels may cause several problems, such as protein loss by the blotting itself and band broadening, in some cases the immunostaining with antibodies might be problematic. In the present study, an alternative isoform prestaining method with CyDye fluors is presented. For this approach, a highly glycosylated fusion protein, Epo-Fc, was used consisting of two recombinant human erythropoietin attached to the Fc part of a human IgG(1) molecule. By using CyDye fluors, up to three samples can be focused on the same lane under identical electrophoretic conditions. A fundamental benefit of this technique is the ability to perform quantitative isoform pattern analysis directly from serum-free culture supernatant.

Animals↗
PubMed2004

Solid-state 17O nuclear magnetic resonance spectroscopy without isotopic enrichment: direct detection of bridging oxygen in radiation damaged zircon.

Protocols are presented for obtaining natural abundance (17)O magic angle spinning and static NMR spectra in the solid state. Rotor-assisted population transfer (RAPT), Carr-Purcell-Meiboom-Gill (CPMG) echo trains and cross-polarisation (CP) are all used to obtain spectra of sites with large as well as small electric field gradients in proton and non-proton containing inorganic materials. Spectra are of sufficient quality to obtain the typical NMR parameters by standard fitting of the spectra. The protocol is then applied to identifying the changes that accompany radioactive decay in zircon (ZrSiO(4)) where enrichment is impossible. The (17)O NMR spectra of a partially metamict zircon sample clearly show evidence of bridging oxygens being produced as a consequence of radiation damage. The spectra have been acquired at moderate magnetic fields over periods typically of 60 h (1 weekend) and it is concluded that a 'routine' overnight (17)O experiment of 15 h at high field (e.g. 21 T) may well be possible.

Binding Sites↗
PubMed1991

Slow motions in lipid bilayers. Direct detection by two-dimensional solid-state deuterium nuclear magnetic resonance.

Two-dimensional solid-state 2H NMR spectroscopy of specifically deuteriated lipids is used to detect and to characterize the rate and mode of slow motions in two lipid bilayer systems. Lateral diffusion of lipid molecules over the curved surface of dipalmitoylphosphatidylcholine liposomes can be detected by two-dimensional exchange 2H NMR and it is shown that molecular orientational exchange is complete on the timescale of 100 ms. In contrast, it is shown that for the glycolipid 1,2-di-O-tetradecyl-3-O-Beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL), there is no evidence of a corresponding orientational exchange in the liquid-crystalline phase suggesting that this lipid forms relatively flat bilayers. In the gel phase of hydrated multibilayers of beta-DTGL, a slow (10(3) s(-1)) whole molecule axial motion is demonstrated at 40 degrees C. Comparison of the experimental and simulated 2D-NMR ridge patterns suggests that large angle jumps about the long molecular axis, rather than small step Brownian diffusion, can best account for the 2D-exchange spectra of beta-DTGL in the gel phase. The significance of this technique for the study of dynamics in other biological systems is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

A modified citrulline assay of NOS activity in rat brain homogenates does not detect direct effects of halothane on the kinetics of NOS activity.

An improved citrulline radioassay of nitric oxide synthase (NOS) activity was developed to study the direct effects of the volatile anesthetic (VA) halothane on the enzyme kinetics of neuronal NOS derived from different regions of the rat central nervous system (CNS). The Vmax of NOS in both soluble cytosolic and membrane bound particulate fractions varied across regions with greatest activity in the cerebellum and least in the spinal cord. In contrast, the Km was not different across regions or in the cytosolic and particulate fractions. Halothane at 0.5, 1, 2 or 3% delivered concentration had no effect on either kinetic parameter of NOS in any of the regions studied indicating that the VAs have no direct effects on NOS activity.

Animals↗