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At least 1,243 records · Page 69Linked to original sources
PubMed1993

Enzyme complex amplification--a signal amplification method for use in enzyme immunoassays.

An amplification system for enzyme immunoassays, in which complexes of streptavidin, biotinylated horseradish peroxidase, antibody are used to detect biotinylated target molecules was developed. In enzyme-linked immunosorbent assay (ELISA) experiments this enzyme complex enhancement (ECA) system gave up to a better than two orders of magnitude increase in sensitivity, compared to a horseradish peroxidase-conjugated streptavidin detection system. The ECA system was shown to be applicable to direct detection of biotinylated antigens, as well as in ELISA employing biotinylated antibodies and in sandwich ELISA. The ECA system was also applicable to immunoblots, giving a sensitivity comparable to that obtained using a radiochemical detection system.

Bacterial Proteins↗
PubMed1996

In situ detection of diamine oxidase activity using enhanced chemiluminescence.

In need of a simple and sensitive method for detection of diamine oxidase (EC 1.4.3.6) activity in connection with diamine oxidase purification from human placenta, we have developed an enhanced chemiluminescence method using putrescine as substrate and horseradish peroxidase and luminol for the detection of the H2O2 produced by diamine oxidase. The method allows direct detection of small amounts of diamine oxidase in serum samples after agarose gel electrophoresis and allows visualization of diamine oxidase activity in tissue sections. Employing this method we have detected diamine oxidase in sera from cow, horse, monkey, rabbit, and pregnant women. On tissue sections from term human placenta diamine oxidase activity was exclusively localized to the maternal side and was concentrated in vessels and fibrinoid areas.

Amine Oxidase (Copper-Containing)↗
PubMed2001

Screening for soluble expression of recombinant proteins in a 96-well format.

For future structural and functional genomics programs new tools will be required. The implementation of high-throughput (HTP) methods for protein production will be an essential element. Present HTP protein production developments in structural genomics are aimed at obtaining well-expressing and highly soluble proteins, which are preferred candidates for structure-function studies. Here, we describe a cheap and efficient procedure to identify well-expressing soluble proteins in Escherichia coli in a compact 96-well format. Reproducible lysis on filter plates, followed by a filtration step on 96-well filter plates, allows the efficient separation of inclusion bodies from the soluble fraction. In the following step a dot blot procedure using anti-RGS-His(4) antibody (Qiagen) to detect expression of recombinant His-tagged protein is applied allowing direct detection of the target protein in the soluble fraction. The method is well suited for automation and should be applicable to expression screening of most proteins and fusion domains to which specific antibodies are available.

Biotechnology↗
PubMed2005

Analysis of alkyl polyglucosides in industrial products by capillary electrophoresis with pulsed amperometric detection.

Pulsed amperometric detection following micellar electrokinetic chromatography has been applied successfully to the direct detection of alkyl polyglucosides (APGs) in shampoos and other industrial products without prior conversion to highly absorbing or fluorescing derivatives. For electrochemical detection, it is necessary to dissociate the hydroxyl groups of the APGs. Thus, we used 0.1 M NaOH in the outlet vial to dissociate the APGs. The main problems associated with the combination of electrochemical detection and capillary electrophoresis are the need to isolate the detector from the electric field used in the capillary electrophoresis separation and the difficulty of aligning the working electrode with the end of the capillary. To overcome these problems, a simple capillary-electrode holder was constructed. This holder automatically aligns the capillary and the electrode in a wall-jet configuration without the aid of micropositioners and facilitates the replacement of electrodes and capillaries without reconstruction of the entire capillary/electrode setup. Special microcylindrical gold electrodes have been produced by sealing 300-microm-diameter gold wire into borosilicate-glass capillaries.

Alkylation↗
PubMed1994

Adenovirus myocarditis: retrospective diagnosis by gene amplification from formalin-fixed, paraffin-embedded tissues.

Understanding the pathogenesis of viral myocarditis is linked to the availability of sensitive assays to detect viruses in clinical material. Recent advances in molecular techniques permit direct detection of viral-specific nucleic acid in tissue samples. This report describes a protocol for DNA extraction and amplification of adenovirus genome from formalin-fixed, paraffin-embedded human tissues that detects as little as 10 copies of viral genome in a background of 0.5 micrograms of human DNA. This sensitive assay permitted the examination of archived tissues to establish a retrospective diagnosis of adenoviral myocarditis in two pediatric patients.

Adenovirus Infections, Human↗
PubMed1992

Convergent and overlapping transcripts of the Chlamydia trachomatis 7.5-kb plasmid.

Transcription of the 7.5-kb cryptic plasmid of Chlamydia trachomatis serovar L2 was investigated. Faint, diffuse transcripts of about 1.6 and 2.2 kb and intense short transcripts of about 250 and 430 bases were identified by Northern blot analysis. The short transcripts were found to have a common 5' end corresponding to bp 501 relative to the unique BamHI site of the plasmid and to terminate at different downstream sites. Putative promoter sequences of TTGCCA and TATATT, which closely resemble the consensus recognition site of Escherichia coli sigma 70, were identified at the -35 and -10 positions upstream from the 5' end of the short transcripts made in chlamydia. Transcripts of similar sizes were also expressed from this promoter in E. coli harboring a recombinant plasmid encoding the short transcripts. The short transcripts encode a common open reading frame (ORF) of 34 codons; however, a strong ribosome binding site was not found in the vicinity of the initiator codon, and it is not known whether the transcripts are translated in vivo. A large ORF of 330 codons, which has been shown to encode a hypothetical protein containing conserved domains of recombinase-like proteins, is antisense to the short transcripts. Transcripts encoding the large ORF could not be detected directly by Northern blot or primer extension analysis. However, transcripts were detected by polymerase chain reaction amplification of the large ORF cDNA and when Southern blots of single-stranded antisense DNA for the large ORF were probed with radiolabeled RNA synthesized by host-free chlamydial reticulate bodies. Thus, both strands of the chlamydial plasmid are transcribed in the region encoding the short transcripts. We propose that the short transcripts play a regulatory role as antisense RNAs.

Amino Acid Sequence↗
PubMed2000

Temporal and anatomical distribution of nitric oxide synthase mRNA expression and nitric oxide production during central nervous system inflammation.

Nitric oxide (NO) has important roles in inflammatory processes. It was the aim of this study to ascertain whether changes in nitric oxide synthase (NOS) mRNA expression lead to similar temporal and anatomical changes in NO production in an experimental model of CNS inflammation. NOS-II (inducible NOS) mRNA expression was analyzed 2, 4, 6 and 24 h after intracerebroventricular (i.c.v.) injection of interleukin-1beta (IL-1beta) or vehicle. Increased expression of NOS-II mRNA was observed surrounding the microinjection site and meninges. The changes were significantly higher than controls at 4 and 6 h, returning to baseline at 24 h. Using the novel fluorometric NO detection system, 4,5-Diaminofluorescein diacetate (DAF-2/DA), for the direct detection of NO production, we observed a significant increase in NO production after 4 and 6 h. NO production was observed in areas surrounding the injection site, meninges surrounding the brain and perivascular cells and neuron-like cells throughout the cortex. However, increases in NO production in these areas remained significantly higher than controls at 24 h. These findings demonstrate for the first time that, in fresh frozen tissue, that the anatomical distribution of NOS-II mRNA is consistent with the distribution of NO production. We conclude that increases in NOS-II mRNA following i.c.v. administration of IL-1beta lead to increases in NO production. While the mRNA is degraded by 24 h post treatment, the enzyme remains active. We propose that the DAF-2/DA method can be used as a potential marker in the diagnosis of CNS inflammation.

Animals↗
PubMed1989

Separation and detection of bis(alkylthio)selenides of penicillamine and glutathione by liquid chromatography with electrochemical detection.

Sensitive and selective methodology is described for the simultaneous determination of thiols, disulfides and bis(alkylthio)selenides in mixtures formed by reaction of selenious acid with thiols. The methods are based on separation of the various compounds by reversed-phase ion pairing chromatography followed by detection with an electrochemical detector equipped with dual mercury/gold amalgam electrodes in series in the eluent stream. Thiols are detected directly at the downstream electrode. Bis(alkylthio)selenides and disulfides are detected indirectly by first reducing them at the upstream electrode followed by detection of the reduction products at the downstream electrode. Bis(alkylthio)selenides are reduced at less negative potentials than disulfides, making it possible to selectively detect them in mixtures with disulfides. Detector response to bis(penicillamine)selenides is linear from 1.1.10(-7) M up to at least 1.2.10(-4) M; the detection limit is estimated to be less than 2 pmol bis(penicillamine)selenide.

Chromatography, High Pressure Liquid↗
PubMed1997

Pulsed electrochemical detection of thiols and disulfides following capillary electrophoresis.

Pulsed electrochemical detection (PED) following capillary electrophoresis (CE) has been applied to the direct detection of thiocompounds. Both reduced and oxidized thiol moieties are detected without the need of derivatization. Thiocompounds can be detected over a wide range of pH conditions (i.e., pH 0-14), and except for pH, their response is relatively unperturbed by buffer composition. Integrated pulsed amperometric detection (IPAD) results in more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection than other PED waveforms. Mass detection limits using optimized IPAD waveforms are typically 2 pg (5 fmol) or less. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler electropherograms of complex samples containing these biologically significant compounds.

Cysteine↗
PubMed1984

N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen.

N-acetyl-p-benzoquinone imine (NAPQI) has been proposed as the toxic metabolite of acetaminophen for the past 10 years, although it has never been detected as an enzymatic oxidation product of acetaminophen. We report (i) direct detection of NAPQI formed as an oxidation product of acetaminophen by cytochrome P-450 and cumene hydroperoxide and (ii) indirect evidence that is compelling for NAPQI formation from acetaminophen by cytochrome P-450, NADPH, and NADPH-cytochrome P-450 reductase. Evidence is provided for the rapid reduction of NAPQI back to acetaminophen by NADPH and NADPH-cytochrome P-450 reductase such that steady-state levels of NAPQI were below our detection limits of 6.7 X 10(-8) M. In mouse liver microsomal incubations, radiolabeled analogs of both NAPQI and acetaminophen bound covalently to microsomal protein with the loss of approximately equal to 20% of the acetyl group as acetamide. The binding in each case was decreased by glutathione with concomitant formation of 3-S-glutathionylacetaminophen. The binding also was decreased by L-ascorbic acid, NADPH, and NADH with reduction of NAPQI to acetaminophen. Results of partitioning experiments indicate that NAPQI is a major metabolite of acetaminophen, a considerable fraction of which is rapidly reduced back to acetaminophen.

Acetaminophen↗
PubMed1995

Detection of Yersinia enterocolitica O:3 in faecal samples and tonsil swabs from pigs using IMS and PCR.

Dilutions of faecal samples spiked with Yersinia enterocolitica O:3 were analysed using immunomagnetic separation (IMS) followed by PCR. In 10% faecal dilutions with added Y. enterocolitica cells, the limit of detection was 200 cells g-1 faeces. Faecal samples from 38 pigs were analysed by IMS-PCR in parallel with detection and quantification of Y. enterocolitica O:3 using cold pre-enrichment culturing. Of the 15 culture-positive samples, only two were detected with IMS-PCR. These two samples contained 40-400 Y. enterocolitica O:3 cells g-1 faeces; the highest level found in the investigation. This indicated that the low sensitivity of IMS-PCR was due to low amounts of cells in the faecal samples. Swab samples from 195 pig tonsils, taken on a slaughterline were examined using IMS-PCR and culture detection. Of 164 culture-positive samples, 60 were positive with IMS-PCR. In addition, IMS-PCR was positive for three culture-negative samples. Forty-five of the samples were further examined by IMS-PCR after 7-10 d of cold pre-enrichment. All 31 culture-positive samples as well as five culture-negative samples were detected by IMS-PCR. From these data it can be concluded that IMS-PCR can be used to detect Y. enterocolitica O:3 cells after pre-enrichment, but direct detection needs further optimization of the sample preparation procedures.

Animals↗
PubMed2002

Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA.

The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

Bacteria↗
PubMed1996

A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs.

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.

Animals↗
PubMed1994

Reactive oxygen species produced from chromate pigments and ascorbate.

The reactions of various chromate pigments and ascorbate were investigated by an ESR spin trapping technique. Production of Cr(V) was detected directly and productions of very electrophilic reactive oxygen species (ROS) was detected via the oxidation of formate. We demonstrated previously that both dissolved oxygen and Cr (V) were essential in the production of ROS in this system, and that ROS production was inhibited by catalase. We studied here the effect of solubility of different chromate pigments: sodium, calcium, strontium, basic zinc, basic lead supported on silica, and lead and barium chromates on the production of ROS in buffered medium and cell culture medium (Dublecco's Modified Eagle medium + fetal calf serum). Sodium, calcium, basic zinc, and basic lead chromates were active in the production of ROS in presence of cell culture medium, whereas lead and barium chromates were inactive.

Ascorbic Acid↗
PubMedDate not supplied

Molecular beacon probes of photodamage in thymine and uracil oligonucleotides.

Molecular beacons (MB) are becoming more common as sequence-selective detectors of nucleic acids. Although they can easily detect single-base mismatches, they have never been used to directly detect DNA or RNA damage. To measure the degree of ultraviolet (UV) light damage in oligonucleotides, we report a novel MB approach for general detection of photoproducts in UV-irradiated rU17 and dT17 oligonucleotides. With monochromatic UV light irradiation at ca 280 nm under anoxic conditions, the oligonucleotide absorption decays with a single-exponential time constant of 123+/-1 min for rU17 and with double-exponential time constants of 78+/-0.5 min (99%) and 180+/-5 min (0.05%) for dT17 oligonucleotides. Under the same conditions, the MB fluorescence decays more quickly, with single-exponential time constants of 19+/-2 and 26+/-3 min for rU17 and dT17, respectively. Similar kinetics were observed with broadband UV light irradiation of oligonucleotides. The differences in the UV damage kinetics of dT17 and rU17 and their detection by absorption and fluorescence techniques will be discussed in the context of differential instabilities introduced in the nucleic acid-MB duplex by the different photoproducts formed.

Kinetics↗
PubMed2000

Depletion of tumor oxygenation during photodynamic therapy: detection by the hypoxia marker EF3 [2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl)acetamide ].

Photodynamic therapy (PDT) of tumors can create hypoxia when oxygen is depleted by photochemical consumption or the oxygen supply is compromised by microvascular damage. However, oxygen is a requirement for PDT, and hypoxia during illumination can lead to poorer tumor response. As such, sensitive methods of quantifying tumor oxygen and evaluating its distribution may help in the development and optimization of treatment protocols. In this study, the hypoxia marker EF3 [2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl)acetam ide] was used to evaluate the oxygenation of PDT-treated radiation-induced fibrosarcoma tumors. Tumor-bearing mice were administered Photofrin (5 mg/kg) 24 h before PDT illumination at 75 mW/cm2, 135 J/cm2 (30 min). EF3 (52 mg/kg) was injected either within 3 min before PDT illumination, with tumor excision at the conclusion of illumination, or within 3 min after illumination, with tumor excision 30 min later. Control animals received EF3 alone, EF3 plus Photofrin, or EF3 plus illumination. After tumor disaggregation, staining with a fluorochrome-conjugated monoclonal antibody, and flow cytometric analysis, control tumors demonstrated an averaged median fluorescence intensity (+/- SE) of 17.1 +/- 2.8. EF3 binding significantly (P = 0.007) increased during PDT to a median fluorescence intensity of 48.9 +/- 8.3. In the 30 min after PDT, EF3 binding returned to control levels (median, 18.3 +/- 3.3). To evaluate the oxygen concentrations corresponding to these fluorescence intensities, an in vitro standard curve was created based on the in vivo exposure conditions. From this curve, the oxygen tensions of tumors exposed to EF3 under control conditions, during PDT, or after PDT were calculated to be 3.1-5.3, 1.2-2.4, and 3.0-5.2 mm Hg, respectively. Detection of EF3 binding using a monoclonal antibody correlated well with direct detection of binding using a radioactive assay. EF3 binding was linear with drug incubation for times from 1.5 to 60 min. Overall, this work demonstrates that hypoxia during PDT illumination of radiation-induced fibrosarcoma tumors can be detected by the hypoxia marker EF3. Hypoxia during illumination can be labeled separately from that found before or after PDT. Tissue oxygen tensions corresponding to EF3 binding levels can be calculated.

Animals↗
PubMed2000

Current understanding of cytomegalovirus infection in immunocompetent individuals.

Human cytomegalovirus (CMV) is a member of the herpes family of viruses. After primary infection, it undergoes latency/persistence. Significant progress has been made in the last few years in detecting CMV. The most available approach to the diagnosis of CMV infection is the direct detection of CMV antigen in nuclei of peripheral blood leukocytes, an assay known as pp65 direct antigenemia test. CMV infection is well controlled in the immunocompetent hosts; however, there are various immunological changes in immune function during and after recovery from CMV infections. Characteristic changes in lymphocyte subsets occur during CMV infection, mainly involving expansion and activation of CD8+ T lymphocytes and NK cells. On the other hand, CMV has an array of immune escape strategies for establishing a life long latent state: CMV inhibits major histocompatibility complex (MHC) class I expression within infected cells and impairs IFN-gamma-induced MHC class II-dependent antigen presentation by macrophages; it can also encode proteins that can interfere with the presentation of viral peptide antigens to T cells. While cutaneous manifestations of CMV seen in immunocompromised patients have been extensively reported, those in adult immunocompetent individuals have received relatively little attention: in this setting the primary CMV infection appears as CMV mononucleosis. At the time of occurrence of the mononucleosis syndrome, a variety of extracutaneous and cutaneous manifestations occur. These clinical symptoms are not the direct consequence of proliferation of CMV in given tissues but indicative of the immunological response toward CMV. The incidence of the appearance of eruptions in CMV mononucleosis is variable. Certain drugs given in the early stage of this disease play an important role in the development of eruption, just as with the ampicillin rashes in the Epstein-Barr virus mononucleosis. Although the mechanism by which drugs trigger the development of rashes in patients with CMV mononucleosis is unknown, it is assumed that CMV is likely to be a potential amplifier of drug rashes induced by activation of drug-specific T cells. By improving methods for detection of CMV, we can recognize that many types of eruptions other than CMV mononucleosis could be induced by primary infection or reactivation of CMV.

Acute Disease↗
PubMed1985

Enzyme immunoassay for the detection of Chlamydia trachomatis antigen in urethral and endocervical swabs.

An enzyme immunoassay technique based on the direct detection of Chlamydia trachomatis antigen in urethral or cervical swabs was used for the rapid diagnosis of chlamydial genital infection. Urethral and cervical samples from 140 patients were tested in parallel by enzyme immunoassay and cell culture using iodine staining. The direct test had a sensitivity of 92.5% and specificity of 97.2% when compared with the cell culture system. The enzyme immunoassay technique provides a rapid and simple method for diagnosing chlamydial genital infection and may be performed on a large number of samples in laboratories which do not have tissue culture facilities or a trained microscopist.

Antigens, Bacterial↗