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At least 181 records · Page 10Linked to original sources

PubMed2005

Bioresponsive hydrogel microlenses.

We report investigations of bioresponsive hydrogel microlenses as a new protein detection technology. Stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgels have been synthesized via free-radical precipitation polymerization. These hydrogel microparticles were then functionalized with biotin via EDC coupling. Hydrogel microlenses were prepared from the particles via Coulombic assembly onto a silane-modified glass substrate. Arrays containing both pNIPAm-co-AAc microgels (as an internal control) and biotinylated pNIPAm-co-AAc microgels were then used to detect multivalent binding of both avidin and polyclonal anti-biotin. Protein binding was determined by monitoring the optical properties of the microlenses using a brightfield optical microscopy technique. The microlens method is shown to be very specific for the target protein, with no detectable interference from nonspecific protein binding. Finally, the reversibility of the hydrogel microlens assay has been studied in the case of anti-biotin to determine the potential application of the microlens assay technology in a displacement-type assay. These results suggest that the microlens method may be an appropriate one for label-free detection of proteins or small molecules via displacement of tethered protein--ligand pairs.

Acrylamides↗

PubMed1986

Clinical considerations in the diagnosis of viral respiratory infections.

Recent advances are allowing the transfer of sensitive and precise rapid viral antigen detection technology from sophisticated research laboratories to standardly equipped clinical diagnostic facilities. It is now possible to identify many viral respiratory pathogens directly from clinical specimens in less than 1 hr. Rapid antigen detection promises to be of the most value in the identification of respiratory viruses 1) for which antiviral therapy is available, 2) which can be prevented by employing isolation precautions, chemoprophylaxis, and/or immunization, 3) whose presence usually is associated with acute respiratory disease, not just asymptomatic colonization, and 4) which ordinarily are not associated with concomitant bacterial infection, and thus, whose early detection may allow withholding or withdrawing antibiotics. Based on these considerations, the relative usefulness of rapid viral antigen detection of commonly encountered respiratory pathogens will be discussed. In addition, the role of rapid viral detection in diagnosis of respiratory infections in high risk versus otherwise healthy individuals will be explored.

Amantadine↗

PubMed2005

Infrared up-converting phosphors for bioassays.

The development of up-converting phosphor reporter particles has added a powerful tool to modern detection technologies. Carefully constructed phosphor reporters have core-shell structures with surface functional groups suitable for standard bio-conjugations. These reporters are chemically stable, possess the unique property of infrared up-conversion, and are readily detected. In contrast to conventional fluorescent reporters, up-converting phosphor particles do not bleach and allow permanent excitation with simultaneous signal integration. A large anti-Stokes shift (up to 500 nm) separates discrete emission peaks from the infrared excitation source. Along with the unmatched contrast in biological specimens due to the absence of autofluorescence upon infrared excitation, up-converting phosphor technology (UPT) has unique properties for highly-sensitive particle-based assays. The production and characteristics of UPT reporter particles as well as their application in various bioassays is reviewed.

Biological Assay↗

PubMed2002

A heteroduplex method for detection of targeted sub-populations of bacterial communities.

We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities. A small (ca. 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers. Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group. The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology. A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected. The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.

Bacteria↗

PubMed2005

High throughput P450 inhibition screens in early drug discovery.

This review of high throughput (HT) P450 inhibition technologies and their impact on early drug discovery finds the field at a mature stage. The relationship between P450 inhibition and drug-drug interactions is well understood. A wide variety of P450 inhibition detection technologies are readily available off-the-shelf, but what seems still to be missing is a general agreement on how much weight one should give to the various types of early discovery HT P450 inhibition data. Method-dependent potency differences are a cause of concern, and to resolve this issue the authors advocate calibration of the HT methods with a large set of marketed drugs.

Calibration↗

PubMed1994

Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting.

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.

Amino Acid Sequence↗

PubMed1996

Isolation of an in vitro transmissible agent with reverse transcriptase activity from a blood donor with a borderline-positive HIV-1 serology for more than five years.

BACKGROUND: Reverse transcriptase (RT) is present in all infectious retrovirus particles. Sensitive RT tests should thus detect all such particles. A family of ultrasensitive RT tests, product-enhanced reverse transcriptase (PERT) assays, have been designed. Accumulated results show that (i) a first version of the PERT assay that uses microtiter/ELISA technology detects RT of only 3-11 retroviral particles, (ii) very different human and animal lenti- and oncoviruses are detected very sensitively, (iii) HIV-1 is detected as sensitively as with polymerase chain reaction (PCR) for viral RNA, and (iv) prevalence of elevated particle-associated RT in plasma of unselected Swiss blood donors was 1.9%. OBJECTIVE: To investigate whether the RT activity detected in one unselected donor with a chronically elevated level of the liver enzyme alanine aminotransferase and two selected donors with chronically indeterminate or borderline-positive HIV-1 serology was due to human immunodeficiency virus (HIV) or human T-cell leukemia virus (HTLV) infection. STUDY DESIGN: Blood samples were tested by PCR for viral DNA and/or RNA of HIV-1, HIV-2, HTLV-1, HTLV-2, hepatitis B and C virus. Serological tests and virus cultures were also employed. RESULTS: Infection with any of the above agents could not be demonstrated. Virus cultivation in one case of borderline-positive HIV-1 Western blot for more than 5 years yielded a peak of RT production that was repeatedly transmissible to fresh cells. CONCLUSIONS: The findings suggest the presence of an infectious RT-positive agent, probably different from HIV-1/2 or HTLV-1/2, in a healthy individual with chronically borderline-positive HIV-1 serology. The PERT assay may detect retroviruses currently undetectable by other tests. The use of more stringent Western blot interpretation guidelines other than those of CDC or WHO is strongly recommended.

Journal Article↗

PubMed2000

A reduced estimate of the number of kilometre-sized near-Earth asteroids.

Near-Earth asteroids are small (diameters < 10 km), rocky bodies with orbits that approach that of the Earth (they come within 1.3 AU of the Sun). Most have a chance of approximately 0.5% of colliding with the Earth in the next million years. The total number of such bodies with diameters > 1 km has been estimated to be in the range 1,000-2,000, which translates to an approximately 1% chance of a catastrophic collision with the Earth in the next millennium. These numbers are, however, poorly constrained because of the limitations of previous searches using photographic plates. (One kilometre is below the size of a body whose impact on the Earth would produce global effects.) Here we report an analysis of our survey for near-Earth asteroids that uses improved detection technologies. We find that the total number of asteroids with diameters > 1 km is about half the earlier estimates. At the current rate of discovery of near-Earth asteroids, 90% will probably have been detected within the next 20 years.

Earth, Planet↗

PubMed2003

Contrast M-mode power Doppler ultrasound in the detection of right-to-left shunts: utility of submandibular internal carotid artery recording.

Cardiac right-to-left shunts (RLSs) can be detected by echocardiography and transcranial Doppler ultrasound (TCD). In patients without adequate transtemporal bone windows, results may be obtained by insonating extracranial arteries; however, the sensitivity and practicality of this approach is unknown. In 34 patients evaluated with echocardiography for RLSs, 73 studies were performed with unilateral, simultaneous contrast TCD (cTCD) of the middle cerebral artery (MCA) and anterior cerebral artery (ACA) and submandibular power M-mode Doppler (PMD) ultrasound of the extracranial internal carotid artery (ecICA). The number of microbubble (MB) signals and their times of first appearance were determined. RLS volume was graded on 6 levels (I = trace, II = small, III = medium, IVa = large, IVb = shower, IVc = curtain) and compared between MCA and ecICA recordings. In 2 of 24 cTCD studies in 15 patients without evidence of RLSs on single-gated MCA monitoring, low-volume RLSs (grades I and II) were detected via ecICA insonation; in both, MB signatures were tracked in the ecICA, passing into the ipsilateral ACA. In 40 of 49 studies (26 patients) in which RLSs were demonstrated with single-gated MCA monitoring, more MBs were detected in the ecICA than the MCA, with either single-gated or M-mode images, with increases of 76.9% and 66.1%, respectively (P = .027). Compared to single-gated studies, M-mode technology detected nonsignificant increases in MB number in both the MCA and the ecICA (by 20.2% and 14.0%, respectively). Contrast PMD with cervical ICA recording is at least as sensitive and specific as the traditional MCA method in detecting RLSs; furthermore, this method seems to be more sensitive for low-volume RLSs (grades I-III) because of air MB decay (9.2%) and entry into the ipsilateral ACA (34.2%). This is in concordance with the increase of detected RLS grades observed in 32.7% of patients with echocardiography-documented RLSs. The authors therefore suggest the incorporation of ecICA PMD not only in patients with poor ultrasonic bone windows but also in every patient being evaluated for suspected RLSs.

Carotid Artery, Internal↗

PubMed2004

High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase.

A number of assays have been developed for high-throughput screening (HTS) of potentially bioactive compounds. To screen millions of chemical compounds efficiently, the best detection technology prior to initiating HTS must be chosen. Ideally, a non-radioisotope (non-RI), homogeneous method, equivalent to the most reliable assay for a particular target, should be selected as an HTS method. Protein kinases are among the most important classes for drug discovery because they participate in various signaling pathways. Several HTS technologies are available for kinase activity: SPA (Amersham, Piscataway, NJ, U.S.A.), HTRF (CIS-US, Inc., Bedford, MA, U.S.A.), IMAP (Molecular Devices, Sunnyvale, CA, U.S.A.), and Z'-LYTE (Invitrogen, Carlsbad, CA, U.S.A.). The amount of phosphorylated product is detected by different methods in these assays. Recently, Kinase-Glo Luminescent Kinase Assay, a non-RI, homogeneous, adenosine triphosphate (ATP) quantitative kit useful for kinase activity detection, has become available from Promega (Madison, WI, U.S.A.). ATP is a universal substrate for kinases. Thus, the Kinase-Glo assay shows promise for becoming the primary method of determining kinase activity in HTS. We have developed a Kinase-Glo system for cyclin-dependent kinase 4 (Cdk4), and compare its results with those of the filtration method, the most reliable assay for in vitro Cdk4 activity. In addition, the reliability and sensitivity of the Kinase-Glo are discussed.

Adenosine Triphosphate↗

PubMed2001

Detection of West Nile virus in mosquitoes by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay employing < > detection technology was developed to identify West Nile virus in experimentally infected mosquitoes. The specificity of the assay was evaluated with the following viruses: eastern equine encephalitis, Ilheus, West Nile and yellow fever viruses. The limits of detection were determined using West Nile viral RNA extracted from serial dilutions of virus culture in infected mosquitoes. Limit of detection was 5 PFU from extracted mosquitoes. We were able to detect the presence of one infected mosquito in a pool of 50 repeatedly. When the RT-PCR was used with coded samples of intrathoracically-infected and uninfected mosquitoes, the assay detected the virus in all infected mosquitoes. Analytic sensitivity and specificity were 100%. This assay offers an efficient and rapid method of identifying West Nile virus in infected mosquitoes or cell culture.

Animals↗

PubMedDate not supplied

[Evaluation of a new technology for the detection of enterovirus in sewage].

This paper reported the appraisal of a novel technology for the detection of enterovirus in sewage based on a viral recovery method using polyethylenglycol as a concentrating agent and on the combined use of viral isolation and polymerase chain reaction as viral detection and identification techniques. It was also confirmed that the viral recovery method is highly efficient since it allows to recover all viruses present in sewage and to preserve their infective capacity. It was proved that the polymerase chain reaction (PCR) is less sensitive than cell culture for detecting enteroviruses in sewage. It is also possible to use guadinine isothiocyanate, whose components can be prepared in the lab, as an alternative method for taking out and purifying nucleic acids instead of using the conventional TRIZOL method which is the one recommended in these cases by the World Health Organization.

Enterovirus↗

PubMedDate not supplied

Estimating diagnostic accuracy from multiple conflicting reports: a new meta-analytic method.

Reports of diagnostic accuracy often differ. The authors present a method to summarize disparate reports that uses a logistic transformation and linear regression to produce a summary receiver operating characteristic curve. The curve is useful for summarizing a body of diagnostic accuracy literature, comparing technologies, detecting outliers, and finding the optimum operating point of the test. Examples from clinical chemistry and diagnostic radiology are provided. By extending the logic of meta-analysis to diagnostic testing, the method provides a new tool for technology assessment.

Data Interpretation, Statistical↗

PubMed1997

Do HPV-negative cervical carcinomas exist?

Based on improvements in HPV detection technology, it appears that an almost 100% HPV association is reached for cervical cancer, as demonstrated in an accompanying paper in this issue of the Journal. Factors which may explain the rare cases in which no HPV DNA is detectable include: improper sampling; disruption of HPV by integration events; the existence of still unidentified HPVs; sensitivity of the method; and the mechanism of transformation. Finally, epidemiological studies identifying HPV independent risk factors are necessary to answer the question of whether HPV independent pathways exist for cervical carcinogenesis.

DNA, Viral↗

PubMed2006

Gas-based detectors for synchrotron radiation.

Gas-filled detectors have played an important role in experiments at synchrotron sources for many years. Although other X-ray detection technologies are gaining wider use, particularly with the higher flux of new sources, gas-filled detectors are still well suited for specific experiments. This article describes some fundamental characteristics of gas-filled detectors for X-ray detection, with emphasis on position encoding, position resolution and linearity. Recently developed micropattern structures for achieving electron multiplication are described. Some applications of conventional wire chambers and micro-pattern detectors are given.

Crystallography, X-Ray↗

PubMed2005

Bacillus anthracis: toxicology, epidemiology and current rapid-detection methods.

B. anthracis, the causative agent for anthrax, has been well studied for over 150 years. Due to the genetic similarities among various Bacillus species, as well as its existence in both a spore form and a vegetative state, the detection and specific identification of B. anthracis have been proven to require complex techniques and/or laborious methods. With the heightened interest in the organism as a potential biological threat agent, a large number of interesting detection technologies have recently been developed, including methods involving immunological and nucleic acid-based assay formats. The technologies range from culture-based methods to portable Total Analysis Systems based on real-time PCR. This review with 170 references provides a brief background on the toxicology and epidemiology of B. anthracis, discusses challenges associated with its detection related to genetic similarities to other species, and reviews immunological and, with greater emphasis, nucleic acid-based detection systems.

Animals↗

PubMed1997

Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique.

The continuing threat posed by viral diseases in cultured shrimp calls for the development of detection technologies for monitoring the animals, especially broodstock. Two of the most highly pathogenic viruses of penaeid shrimp are the yellow-head virus (YHV) and Chinese baculovirus (CBV, also called white spot baculovirus). A Western blot (WB) protocol capable of detecting YHV and CBV in the hemolymph of infected shrimp was developed. The use of the hemolymph as material for virus detection allowed for sample collection without sacrificing the animals. This protocol was highly specific, rapid, and sensitive enough to detect the presence of the viruses before the appearance of overt symptoms. It was also useful for demonstrating the growth of both viruses in primary shrimp lymphoid cell cultures.

Animals↗

PubMed2006

Improved marker combination for detection of de novo genetic variation and aberrant DNA in colorectal neoplasia.

BACKGROUND: The genetic heterogeneity of sporadic colorectal cancer (CRC) makes the choice of genetic markers and sequence variation-detection technologies critical to the performance of screening assays. We have previously described the effectiveness of a CRC assay composed of 22 known variants in KRAS, APC, TP53, and BAT-26 (V1). We introduce a new marker formulation (V2) that includes detection of de novo variation in APC, PIK3CA, and CTNNB1, hypermethylated sequences within SMARCA3 and VIM, and a single-base variation within BRAF. We compared the abilities of the V1 and V2 markers to detect aberrant DNA in colorectal neoplasias. METHODS: V1 and V2 marker formulations were used to analyze 144 colorectal tissue samples comprising 50 precancerous adenomas, 94 carcinomas, and 11 nonpathologic tissues. V1 analysis consisted of single-base extension analysis of the 22 V1 variants. V2 analysis consisted of DNA scanning of the APC mutation cluster region, PIK3CA exons 9 and 20, CTNNB1 exon 3, analysis for the BRAF Val600Glu substitution, and methylation-specific PCR analysis of VIM and SMARCA3. RESULTS: The V2 marker formulation had significantly higher sensitivity than the V1 markers for carcinomas (93.6% and 72.3%, respectively; P = 0.0002) and adenomas (92.0% and 62.0%, respectively; P = 0.0006). None of the nonpathologic samples were positive for any marker. CONCLUSIONS: We demonstrate improved sensitivity of a new marker formulation (V2) to detect aberrant DNA in CRC and precancerous adenoma tumor tissues.

Adenomatous Polyps↗