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Analytical Sensitivity (analytical + sensitivity)

Distribution by Scientific Domains

Medical Sciences	52%
Chemistry	33%
Life Sciences	9%

Selected Abstracts

Evaluation of a new, fully automated immunoassay for detection of HTLV-I and HTLV-II antibodies

JOURNAL OF MEDICAL VIROLOGY, Issue 3 2008
Xiaoxing Qiu
Abstract Screening blood donations for human T-lymphotropic virus types I and II (HTLV-I/II) continues to be important in protecting the safety of blood products and controlling the global spread of these retroviruses. We have developed a fully automated, third generation chemiluminescent immunoassay, ARCHITECT rHTLV-I/II, for detection of antibodies to HTLV-I/II. The assay utilizes recombinant proteins and synthetic peptides and is configured in a double antigen sandwich assay format. Specificity of the assay was 99.98% (9,254/9,256, 95% CI,=,99.92,100%) with the negative specimens from the general population including blood donors, hospital patients and pregnant women from the US, Japan and Nicaragua. The assay demonstrated 100% sensitivity by detecting 498 specimens from individuals infected with HTLV-I (n,=,385) and HTLV-II (n,=,113). ARCHITECT rHTLV-I/II results were in complete agreement with the Murex HTLV-I/II reference assay and 99.7% agreement with the Genelabs HTLV Blot 2.4 confirmatory assay. Analytical sensitivity of the assay was equivalent to Murex HTLV-I/II assay based on end point dilutions. Furthermore, using a panel of 397 specimens from Japan, the ARCHITECT rHTLV-I/II assay exhibited distinct discrimination between the antibody negative (Delta Value,=,,7.6) and positive (Delta Value,=,7.6) populations. Based on the excellent specificity and sensitivity, the new ARCHITECT rHTLV-I/II assay should be an effective test for the diagnosis of HTLV-I/II infection and also for blood donor screening. J. Med. Virol. 80:484,493, 2008. © 2008 Wiley-Liss, Inc. [source]

Analytical sensitivity of arsenobetaine on atomic spectrometric analysis and the purity of synthetic arsenobetaine,

APPLIED ORGANOMETALLIC CHEMISTRY, Issue 9 2006
T. Narukawa
Abstract Arsenobetaine is one of the main organoarsenic compounds that exist in living organisms. Determination errors in total arsenic analyses for organoarsenic compounds occur because analytical sensitivities depend upon the chemical forms of the compounds. However, information on the analytical sensitivity of arsenobetaine by ICP-MS and ICP-AES and the purity of commercially available arsenobetaine standards is lacking. BCR CRM 626 (arsenobetaine solution) is a certified reference material from IRMM with a certified concentration of arsenobetaine. The sensitivity and behavior of arsenobetaine on ICP-MS and ICP-AES were investigated using the BCR arsenobetaine. The analytical sensitivity and behavior of arsenobetaine using ICP-MS and ICP-AES were also investigated using a commercially available synthetic arsenobetaine, and were compared with results for BCR-AB based on a Japan calibration service system (JCSS) arsenic standard solution. In the results, arsenic determined directly in arsenobetaine showed about 15% greater sensitivity in analysis by ICP-MS and ICP-AES than did inorganic (JCSS) arsenic. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Impedance Spectroscopy: A Powerful Tool for Rapid Biomolecular Screening and Cell Culture Monitoring

ELECTROANALYSIS, Issue 23 2005
Isaac
Abstract Dielectric spectroscopy or Electrochemical impedance spectroscopy (EIS) is traditionally used in corrosion monitoring, coatings evaluation, batteries, and electrodeposition and semiconductor characterization. However, in recent years, it is gaining widespread application in biotechnology, tissue engineering, and characterization of biological cells, disease diagnosis and cell culture monitoring. This article discusses the principles and implementation of dielectric spectroscopy in these bioanalytical applications. It provides examples of EIS as label-free, mediator-free strategies for rapid screening of biocompatible surfaces, monitoring pathogenic bacteria, as well as the analysis of heterogeneous systems, especially biological cells and tissues. Descriptions are given of the application of nanoparticles to improve the analytical sensitivities in EIS. Specific examples are given of the detection of base pair mismatches in the DNA sequence of Hepatitis B disease, TaySach's disease and Microcystis spp. Others include the EIS detection of viable pathogenic bacteria and the influence of nanomaterials in enhancing biosensor performance. Expanding applications in tissue engineering such as adsorption of proteins onto thiolated hexa(ethylene glycol)-terminated (EG6) self-assembled monolayer (SAM) are discussed. [source]

Functional Nanostructured Plasmonic Materials

ADVANCED MATERIALS, Issue 10 2010
Jimin Yao
Abstract Plasmonic crystals fabricated with precisely controlled arrays of subwavelength metal nanostructures provide a promising platform for sensing and imaging of surface binding events with micrometer spatial resolution over large areas. Soft nanoimprint lithography provides a robust, cost-effective method for producing highly uniform plasmonic crystals of this type with predictable optical properties. The tunable multimode plasmonic resonances of these crystals and their ability for integration into lab-on-a-chip microfluidic systems can both be harnessed to achieve exceptionally high analytical sensitivities down to submonolayer levels using even a common optical microscope, circumventing numerous technical limitations of more conventional surface plasmon resonance techniques. In this article, we highlight some recent advances in this field with an emphasis on the fabrication and characterization of these integrated devices and their demonstrated applications. [source]

Analytical sensitivity of arsenobetaine on atomic spectrometric analysis and the purity of synthetic arsenobetaine,

Accelerating drug development: methodology to support first-in-man pharmacokinetic studies by the use of drug candidate microdosing

DRUG DEVELOPMENT RESEARCH, Issue 1 2007
Matthew A. McLean
Abstract Microdosing of experimental therapeutics in humans offers a number of benefits to the drug development process. Microdosing, conducted under an exploratory Investigational New Drug (IND) application, entails administration of a sub-pharmacological dose of a new chemical entity (NCE) that allows for early evaluation of human pharmacokinetics. Such information can be pivotal for: (1) selecting a compound for full drug development from a small group of candidates; (2) defining the amount of material needed for early development; and (3) setting the initial Phase I dose regimen in humans. Appropriate safety studies must be conducted to support microdosing in humans, but the requirements are generally less extensive than those needed to support a traditional IND. To date, microdosing has not been broadly applied by the pharmaceutical industry due to concerns about analytical sensitivity and the possibility of non-linear pharmacokinetics at extremely low doses. The primary method for detecting analytes following microdosing until now has been accelerator mass spectrometry, which is expensive, not generally available, and requires test agents to be radiolabeled. Presented in this report is an example of pharmacokinetics analysis using LC/MS/MS following microdosing of an experimental agent in cynomolgus monkeys. The results show good linearity in plasma pharmacokinetics for oral doses of 10,mg/kg (therapeutic dose) and 0.0005,mg/kg (microdose) of the test agent. The results also demonstrate the feasibility of applying standard laboratory analytics to support microdosing in humans and raise the possibility of establishing an animal model to screen for compounds having non-linear pharmacokinetics at low dose levels. Drug Dev. Res. 68:14,22, 2007. © 2007 Wiley-Liss, Inc. [source]

Evaluating and expressing the propagation of uncertainty in chemical fate and bioaccumulation models

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2002
Matthew MacLeod
Abstract First-order analytical sensitivity and uncertainty analysis for environmental chemical fate models is described and applied to a regional contaminant fate model and a food web bioaccumulation model. By assuming linear relationships between inputs and outputs, independence, and log-normal distributions of input variables, a relationship between uncertainty in input parameters and uncertainty in output parameters can be derived, yielding results that are consistent with a Monte Carlo analysis with similar input assumptions. A graphical technique is devised for interpreting and communicating uncertainty propagation as a function of variance in input parameters and model sensitivity. The suggested approach is less calculationally intensive than Monte Carlo analysis and is appropriate for preliminary assessment of uncertainty when models are applied to generic environments or to large geographic areas or when detailed parameterization of input uncertainties is unwarranted or impossible. This approach is particularly useful as a starting point for identification of sensitive model inputs at the early stages of applying a generic contaminant fate model to a specific environmental scenario, as a tool to support refinements of the model and the uncertainty analysis for site-specific scenarios, or for examining defined end points. The analysis identifies those input parameters that contribute significantly to uncertainty in outputs, enabling attention to be focused on defining median values and more appropriate distributions to describe these variables. [source]

WDX Studies on Ceramic Diffusion Barrier Layers of Metal Supported SOECs

FUEL CELLS, Issue 6 2009
D. Wiedenmann
Abstract Solid oxide electrolyser cells (SOECs) have great potential for efficient and economical production of hydrogen fuel. Element diffusion between the Ni-cermet electrode and the metal substrate of metal supported cells (MSC) is a known problem in fuel cell and electrolysis technology. In order to hinder this unintentional mass transport, different ceramic diffusion barrier layers (DBLs) are included in recent cell design concepts. This paper is based on wavelength dispersive X-ray fluorescence investigations of different SOEC and focuses on Fe, Cr and Ni diffusion between the metal grains of the cathode and the metal substrate. Due to the low detection limits and therefore high analytical sensitivity, wavelength dispersive electron probe microanalysis (EPMA) provides a precise method to determine element distribution, absolute element concentration and changes between the reference material and aged cells on a microstructural level by element mappings and concentration profiles. The results of this work show considerable concentration gradients in the metal grains caused by mass exchange during cell operation. Diffusion can be inhibited significantly by integrating different ceramic DBLs of doped LaCrO3 -type or doped LaMnO3 -type perovskite, either by vacuum plasma spraying (VPS) or physical vapour deposition technique (PVD). [source]

Improving blood donor screening by nucleic acid technology (NAT)

ISBT SCIENCE SERIES: THE INTERNATIONAL JOURNAL OF INTRACELLULAR TRANSPORT, Issue n1 2010
M. Schmidt
The description of the ABO blood group system by Landsteiner and coworkers marked a sea change in making blood transfusions feasible and safe for a broad range of indications. Nevertheless, with an increase in blood transfusions, side-effects such as transfusion-transmitted infections (TTIs) became more and more important. A major challenge in transfusion medicine was (and is) to develop screening assays with maximum analytical sensitivity and analytical specificity to reduce the diagnostic window period as much as possible. Until the late 1990s, blood screening for TTIs depended entirely on serological assays. Except for HBV, where the virus can be detected using HBs-antigen assays, tests for the detection of other TTIs relied almost exclusively on antibody detection. These tests, however, are associated with a relatively long diagnostic window period because they detect the response of the immune system to an infection. [source]

Comparison of different methods of bacterial detection in blood components

ISBT SCIENCE SERIES: THE INTERNATIONAL JOURNAL OF INTRACELLULAR TRANSPORT, Issue 1 2009
M. Schmidt
Background, Over the last two decades, the residual risk of acquiring a transfusion-transmitted viral infection has been reduced to less than 1 : 1 000 000 via improvements in different techniques (e.g. donor selection, leuco-depletion, introduction of 3rd or 4th generation enzyme-linked immunosorbent assays and mini-pool nucleic acid testing (MP-NAT). In contrast, the risk for transfusion-associated bacterial infections has remained fairly stable, and is estimated to be in a range between 1 : 2000 and 1 : 3000. Platelets are at an especially higher risk for bacterial contamination, because they are stored at room temperature, which provides good culture conditions for a broad range of bacterial strains. To improve bacterial safety of blood products, different detection systems have been developed that can be divided into culture systems like BacT/ALERT or Pall eBDS, rapid detection systems like NAT systems, immunoassays and systems based on the FACS technique. Culture systems are used for routine bacterial screening of platelets in many countries, whereas rapid detection systems so far are mainly used in experimental spiking studies. Nevertheless, pathogen-reduction systems are currently available for platelet concentrates and plasma, and are under investigation for erythrocytes. Methods, In this review, the functional principles of the different assays are described and discussed with regard to their analytical sensitivity, analytical specificity, diagnostic sensitivity, diagnostic specificity and clinical efficiency. The detection methods were clustered into three groups: (i) detection systems currently used for routine screening of blood products, (ii) experimental detection systems ready to use for routine screening of blood products, and (iii) new experimental detection systems that need to be investigated in additional spiking studies and clinical trials. Results, A recent International Society of Blood Transfusion international forum reported on bacterial detection methods in 12 countries. Eight countries have implemented BacT/ALERT into blood donor screening, whereas in three countries only quality controls were done by culture methods. In one country, shelf-life was reduced to 3 days, so no bacterial screening was implemented. Screening data with culture methods can be used to investigate the prevalence of bacterial contamination in platelets. Differing results between the countries could be explained by different test definitions and different test strategies. Nevertheless, false-negative results causing severe transfusion-related septic reactions have been reported all over the world due to a residual risk of sample errors. Rapid screening systems NAT and FACS assays have improved over the last few years and are now ready to be implemented in routine screening. Non-specific amplification in NAT can be prevented by pre-treatment with Sau3AI, filtration of NAT reagents, or reduction of the number of polymerase chain reaction cycles. FACS systems offer easy fully automated handling and a handling time of only 5 min, which could be an option for re-testing day-5 platelets. New screening approaches like immunoassays, detection of bacterial adenosine triphosphate, or detection of esterase activity need to be investigated in additional studies. Conclusion, Bacterial screening of blood products, especially platelets, can be done with a broad range of technologies. The ideal system should be able to detect one colony-forming unit per blood bag without a delay in the release process. Currently, we are far away from such an ideal screening system. Nevertheless, pathogen-inactivation systems are available, but a system for all blood components will not be expected in the next few years. Therefore, existing culture systems should be complemented by rapid systems like NAT or FACS especially for day-5 platelets. [source]

Development of a Novel Immunoradiometric Assay Exclusively for Biologically Active Whole Parathyroid Hormone 1,84: Implications for Improvement of Accurate Assessment of Parathyroid Function

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2001
Ping Gao
Abstract We developed a novel immunoradiometric assay (IRMA; whole parathyroid hormone [PTH] IRMA) for PTH, which specifically measures biologically active whole PTH(1,84). The assay is based on a solid phase coated with anti-PTH(39,84) antibody, a tracer of125I-labeled antibody with a unique specificity to the first N-terminal amino acid of PTH(1,84), and calibrators of diluted synthetic PTH(1,84). In contrast to the Nichols intact PTH IRMA, this new assay does not detect PTH(7,84) fragments and only detects one immunoreactive peak in chromatographically fractionated patient samples. The assay was shown to have an analytical sensitivity of 1.0 pg/ml with a linear measurement range up to 2300 pg/ml. With this assay, we further identified that the previously described non-(1,84)PTH fragments are aminoterminally truncated with similar hydrophobicity as PTH(7,84), and these PTH fragments are present not only in patients with secondary hyperparathyroidism (2° -HPT) of uremia, but also in patients with primary hyperparathyroidism (1° -HPT) and normal persons. The plasma normal range of the whole PTH(1,84) was 7,36 pg/ml (mean ± SD: 22.7 ± 7.2 pg/ml, n = 135), whereas over 93.9% (155/165) of patients with 1° -HPT had whole PTH(1,84) values above the normal cut-off. The percentage of biologically active whole PTH(1,84) (pB%) in the pool of total immunoreactive "intact" PTH is higher in the normal population (median: 67.3%; SD: 15.8%; n = 56) than in uremic patients (median:53.8%; SD: 15.5%; n = 318; p < 0.001), although the whole PTH(1,84) values from uremic patients displayed a more significant heterogeneous distribution when compared with that of 1° -HPT patients and normals. Moreover, the pB% displayed a nearly Gaussian distribution pattern from 20% to over 90% in patients with either 1° -HPT or uremia. The specificity of this newly developed whole PTH(1,84) IRMA is the assurance, for the first time, of being able to measure only the biologically active whole PTH(1,84) without cross-reaction to the high concentrations of the aminoterminally truncated PTH fragments found in both normal subjects and patients. Because of the significant variations of pB% in patients, it is necessary to use the whole PTH assay to determine biologically active PTH levels clinically and, thus, to avoid overestimating the concentration of the true biologically active hormone. This new assay could provide a more meaningful standardization of future PTH measurements with improved accuracy in the clinical assessment of parathyroid function. [source]

Novel rapid immunochromatographic test based on an enzyme immunoassay for detecting nucleocapsid antigen in SARS-associated coronavirus

JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2005
Hiroyuki Kogaki
Abstract A novel severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been discovered. The detection of both antigens and antibodies in SARS-CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all-in-one device for detecting the native nucleocapsid antigen (N-Ag) of SARS-CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N-Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N-Ag as well as viral N-Ag of SARS-CoV. rSN122 and rSN21-2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N-Ag at 31 pg/mL for recombinant N-Ag, and at 1.99×102 TCID50/mL for SARS-CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21-2 pair is a sensitive, specific, and reliable rapid assay for detecting N-Ag in SARS-CoV treated with either heat or Triton X-100. J. Clin. Lab. Anal. 19:150,159, 2005. © 2005 Wiley-Liss, Inc. [source]

Cardiac Troponin I in Pastured and Race-Training Thoroughbred Horses

JOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 4 2003
Wade Phillips
Cardiac troponin I (cTnI), a myocardial polypeptide, is a highly sensitive and specific biomarker of myocardial injury in people and dogs. The structure of cTnI is highly conserved across species, and equine myocardium has high reactivity with human immunoassays. The purpose of this study was to describe cTnI concentrations in normal pastured and race-training Thoroughbred horses. Ten horses on pasture and 10 horses in race training were studied. Horses were considered normal on the basis of physical examination, training performance, electrocardiography (ECG), and echocardiography. Serum cTnI concentrations were determined with a colorimetric immunoassay. The assay has an analytical sensitivity of 0.04 ng/mL. Serum cTnI concentrations in race-training horses were not significantly different from those of pastured horses. When groups were combined, mean cTnI concentration (±SD) was 0.047 ± 0.085 ng/mL, and the median was 0 (range, 0-0.35 ng/mL). The 90th percentile for both groups combined was 0.11 ng/mL. This study establishes a preliminary reference range for serum cTnI in normal Thoroughbred horses. Key words: Cardiac disease; Cardiac markers; Creatine kinase; Myocarditis. [source]

Liquid chromatography,tandem mass spectrometry applications in endocrinology

MASS SPECTROMETRY REVIEWS, Issue 3 2010
Mark M. Kushnir
Abstract Liquid chromatography,tandem mass spectrometry (LC,MS/MS) has been recognized as a primary methodology for the accurate analysis of endogenous steroid hormones in biological samples. This review focuses on the use of LC,MS/MS in clinical laboratories to assist with the diagnosis of diverse groups of endocrine and metabolic diseases. Described analytical methods use on-line and off-line sample preparation and analytical derivatization to enhance analytical sensitivity, specificity, and clinical utility. Advantages of LC,MS/MS as an analytical technique include high specificity, possibility to simultaneously measure multiple analytes, and the ability to assess the specificity of the analysis in every sample. All described analytical methods were extensively validated, utilized in routine diagnostic practice, and were applied in a number of clinical and epidemiological studies, including a study of the steroidogenesis in ovarian follicles. © 2009 Wiley Periodicals, Inc. Mass Spec Rev 29:480-502, 2010 [source]

Matrix vapor deposition/recrystallization and dedicated spray preparation for high-resolution scanning microprobe matrix-assisted laser desorption/ionization imaging mass spectrometry (SMALDI-MS) of tissue and single cells

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2010
Werner Bouschen
Matrix preparation techniques such as air spraying or vapor deposition were investigated with respect to lateral migration, integration of analyte into matrix crystals and achievable lateral resolution for the purpose of high-resolution biological imaging. The accessible mass range was found to be beyond 5000 u with sufficient analytical sensitivity. Gas-assisted spraying methods (using oxygen-free gases) provide a good compromise between crystal integration of analyte and analyte migration within the sample. Controlling preparational parameters with this method, however, is difficult. Separation of the preparation procedure into two steps, instead, leads to an improved control of migration and incorporation. The first step is a dry vapor deposition of matrix onto the investigated sample. In a second step, incorporation of analyte into the matrix crystal is enhanced by a controlled recrystallization of matrix in a saturated water atmosphere. With this latter method an effective analytical resolution of 2,µm in the x and y direction was achieved for scanning microprobe matrix-assisted laser desorption/ionization imaging mass spectrometry (SMALDI-MS). Cultured A-498 cells of human renal carcinoma were successfully investigated by high-resolution MALDI imaging using the new preparation techniques. Copyright © 2010 John Wiley & Sons, Ltd. [source]

The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity

THE JOURNAL OF PATHOLOGY, Issue 1 2003
Peter JF Snijders
Abstract Given the fact that infection with high-risk human papillomavirus (HPV) is causally involved in cervical cancer, addition of high-risk HPV testing to a cervical smear may improve the efficacy of cervical cancer screening programmes, the triage of women with equivocal or borderline Pap smears, and the monitoring of women who have been treated for cervical intraepithelial neoplasia grade 3 (CIN 3). Compared to a cervical smear HPV tests revealed a superior sensitivity (ie clinical sensitivity) for lesions , CIN 3, and a negative predictive value approaching 100%. However, a potential complication is the availability of several HPV testing methods, all displaying a different sensitivity and specificity to detect HPV-positive women (ie analytical sensitivity and specificity). There is now compelling evidence that the clinical sensitivity and specificity of HPV tests are not simply synonymous to their analytical sensitivity and specificity, respectively. In fact, a distinction between so-called clinically relevant and irrelevant high-risk HPV infections should be made when considering HPV tests for primary screening, triage policies, or post-treatment monitoring. Here, we discuss the potential importance of HPV load in the context of currently widely applied HPV detection methods, to distinguish clinically relevant from irrelevant HPV infections. From this it can be concluded that it is of utmost importance to define criteria, involving viral load threshold and the type of HPV detection method that should be fulfilled by an HPV test before implementation of such a test in clinical practice and population-based cervical cancer screening programmes. Copyright © 2003 John Wiley & Sons, Ltd. [source]

Analytical sensitivity of arsenobetaine on atomic spectrometric analysis and the purity of synthetic arsenobetaine,

RNA helicase encoded by melanoma differentiation,associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease,

ARTHRITIS & RHEUMATISM, Issue 7 2009
Shinji Sato
Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti,CADM-140 antibody,positive prototype serum sample was used to screen a HeLa cell,derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro,transcribed and in vitro,translated products using anti,CADM-140 antibody,positive and anti-CADM-140 antibody,negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti,CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro,transcribed and in vitro,translated products, we obtained a cDNA clone encoding melanoma differentiation,associated gene 5 (MDA-5). The anti,CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti,CADM-140 antibody routinely available. [source]

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples

AUSTRALIAN VETERINARY JOURNAL, Issue 7 2006
AJ FOORD
Objective To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. Design A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. Results The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per µl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. Conclusion The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis. [source]

Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

ACADEMIC EMERGENCY MEDICINE, Issue 4 2008
Samuel Yang MD
Abstract Objectives:, To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. Methods:, The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. Results:, The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. Conclusions:, A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents. [source]