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Sample records for assay cobas ampliscreen

  1. Analytical Performance Evaluation of a New Cobas Tacrolimus Assay on Cobas e411 Analyzer: Comparison of Values Obtained by the CMIA Tacrolimus Assay and a Liquid Chromatography Combined with Tandem Mass Spectrometric Method.

    Science.gov (United States)

    Dasgupta, Amitava; Khalil, Samir A; Johnson-Davis, Kamisha L

    2016-01-01

    Recently Roche Diagnostics (Indianapolis, IN) developed Cobas tacrolimus assay (currently for investigational use only in U.S) for application on multiple platforms including Cobas e 411 analyzer. We evaluated analytic performance of this new assay. Within run, between run and linearity of this new assay were evaluated. In addition, tacrolimus values in 40 specimens obtained by using this new method were compared with values obtained by using the CMIA assay (Abbott Laboratories). Moreover, 10 specimens where accurate tacrolimus values were determined by a reference method (LC-MS/MS), were further analyzed using Cobas tacrolimus assay and the CMIA assay. New Cobas tacrolimus assay showed excellent precision and accuracy. Comparing tacrolimus values obtained by using the CMIA tacrolimus assay (x-axis) with corresponding values obtained by using the Cobas tacrolimus assay (y-axis), the following regression equation was observed: y=0.922x+0.512 (n=40, r=0.99). For additional 10 specimens where tacrolimus values were determined by LC-MS/MS, tacrolimus values obtained by the Cobas tacrolimus assay as well as by the CMIA assay were higher than the corresponding LC-MS/MS values. The new Cobas tacrolimus assay is comparable to the FDA approved CMIA tacrolimus assay. Therefore, when this assay is approved by the FDA, it can be used for therapeutic drug monitoring of tacrolimus. © 2016 by the Association of Clinical Scientists, Inc.

  2. Comparison of the Roche Cobas® 4800 HPV assay to Roche Amplicor for detection of high-risk human papillomavirus.

    Science.gov (United States)

    Phillips, S; Cornall, A M; Machalek, D A; Garland, S M; Bateson, D; Garefalakis, M; Tabrizi, S N

    2016-08-01

    Roche Amplicor HPV (AMP) had previously been used for detection of high-risk human papillomavirus (HR-HPV) in epidemiological and clinical studies. As this assay is no longer available, we compared its performance using PreservCyt samples from women aged of 18-24 years attending for routine cervical cytology screening to Roche Cobas® 4800 (Cobas) to determine if subsequent studies could continue using the Cobas assay. Overall 507 samples were tested on Cobas and compared to previous AMP results, with discrepant samples tested on Roche Linear Array. Overall, agreement between the Cobas and AMP for the presence of HR HPV types was very high (κ = 0.81) (95 % CI: 0.76 - 0.87) with percentage agreement of 91.57 %. Cobas is comparable to AMP for the detection of HR-HPV types in a community recruited cohort of healthy women.

  3. Hybrid Capture 2 and cobas human papillomavirus assays perform similarly on SurePath samples from women with abnormalities

    DEFF Research Database (Denmark)

    Fornari, D; Rebolj, M; Bjerregaard, B

    2016-01-01

    OBJECTIVE: In two laboratories (Departments of Pathology, Copenhagen University Hospitals of Herlev and Hvidovre), we compared cobas and Hybrid Capture 2 (HC2) human papillomavirus (HPV) assays using SurePath® samples from women with atypical squamous cells of undetermined significance (ASCUS...

  4. Hybrid Capture 2 and cobas human papillomavirus assays perform similarly on SurePath samples from women with abnormalities.

    Science.gov (United States)

    Fornari, D; Rebolj, M; Bjerregård, B; Lidang, M; Christensen, I; Høgdall, E; Bonde, J

    2016-08-01

    In two laboratories (Departments of Pathology, Copenhagen University Hospitals of Herlev and Hvidovre), we compared cobas and Hybrid Capture 2 (HC2) human papillomavirus (HPV) assays using SurePath® samples from women with atypical squamous cells of undetermined significance (ASCUS) at ≥30 years and women after treatment of cervical intraepithelial neoplasia (CIN). Samples from 566 women with ASCUS and 411 women after treatment were routinely tested with HC2 and, thereafter, with cobas. Histological outcomes were retrieved from the Danish Pathology Data Base. We calculated the overall agreement between the assays, and compared their sensitivity and specificity for ≥CIN2. In women with ASCUS, HC2 and cobas testing results were similar in the two laboratories. The overall agreement was 91% (95% CI, 88-93). After CIN treatment, the overall agreement was 87% (95% CI, 82-91) at Herlev and 88% (95% CI, 82-92) at Hvidovre. There were no significant differences in the sensitivity for ≥CIN2 between the two tests [Herlev, 98% (95% CI, 89-100) for HC2 versus 94% (95% CI, 82-99) for cobas; Hvidovre, 97% (95% CI, 83-100) for HC2 versus 100% (95% CI, 88-100) for cobas]. The differences were also not significant for specificity. In women with the studied well-defined clinical indications for HPV testing, cobas and HC2 performed similarly in terms of the detection of HPV and ≥CIN2. © 2016 The Authors. Cytopathology Published by John Wiley & Sons Ltd.

  5. Performance of the Cobas® Influenza A/B Assay for Rapid Pcr-Based Detection of Influenza Compared to Prodesse ProFlu+ and Viral Culture

    Science.gov (United States)

    Chen, L.; Tian, Y.; Chen, S.; Liesenfeld, O.

    2015-01-01

    Rapid and accurate diagnosis of influenza is important for patient management and infection control. We determined the performance of the cobas® Influenza A/B assay, a rapid automated nucleic acid assay performed on the cobas® Liat System for qualitative detection of influenza A and influenza B from nasopharyngeal (NP) swab specimens. Retrospective frozen and prospectively collected NP swabs from patients with signs and symptoms of influenza collected in universal transport medium (UTM) were tested at multiple sites including CLIA-waived sites using the cobas® Influenza A/B assay. Results were compared to the Prodesse Pro-Flu+ assay and to viral culture. Compared to the Prodesse ProFlu+ Assay, sensitivities of the cobas® Influenza A/B assay for influenza A and B were 97.7 and 98.6%, respectively; specificity was 99.2 and 99.4%. Compared to viral culture, the cobas® Influenza A/B assay showed sensitivities of 97.5 and 96.9% for influenza virus A and B, respectively; specificities were 97.9% for both viruses. Polymerase chain reaction (PCR)/sequencing showed that the majority of viral culture negative but cobas® Influenza A/B positive results were true positive results, indicating that the cobas® Influenza A/B assay has higher sensitivity compared to viral culture. In conclusion, the excellent accuracy, rapid time to result, and remarkable ease of use make the cobas® Influenza A/B nucleic acid assay for use on the cobas® Liat System a highly suitable point-of-care solution for the management of patients with suspected influenza A and B infection. PMID:26716012

  6. Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

    Science.gov (United States)

    Mortier, Virginie; Vancoillie, Leen; Dauwe, Kenny; Staelens, Delfien; Demecheleer, Els; Schauvliege, Marlies; Dinakis, Sylvie; Van Maerken, Tom; Dessilly, Géraldine; Ruelle, Jean; Verhofstede, Chris

    2017-10-24

    Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA- containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of qPCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the Aids Reference Laboratory. An association between genomic DNA presence and spurious low-level viremia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viremia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cut-off. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viremia.

  7. Prevalence of human papillomavirus in 5,072 consecutive cervical SurePath samples evaluated with the Roche cobas HPV real-time PCR assay.

    Directory of Open Access Journals (Sweden)

    Sarah Preisler

    Full Text Available New commercially available Human Papillomavirus (HPV assays need to be evaluated in a variety of cervical screening settings. Cobas HPV Test (cobas is a real-time PCR-based assay allowing for separate detection of HPV genotypes 16 and 18 and a bulk of 12 other high-risk genotypes. The aim of the present study, Horizon, was to assess the prevalence of high-risk HPV infections in an area with a high background risk of cervical cancer, where women aged 23-65 years are targeted for cervical screening. We collected 6,258 consecutive cervical samples from the largest cervical screening laboratory in Denmark serving the whole of Copenhagen. All samples were stored in SurePath media. In total, 5,072 samples were tested with cobas, Hybrid Capture 2 High Risk HPV DNA test (HC2 and liquid-based cytology. Of these, 27% tested positive on cobas. This proportion decreased by age, being 43% in women aged 23-29 years and 10% in women aged 60-65 years. HC2 assay was positive in 20% of samples, and cytology was abnormal (≥ atypical squamous cells of undetermined significance for 7% samples. When only samples without recent abnormalities were taken into account, 24% tested positive on cobas, 19% on HC2, and 5% had abnormal cytology. The proportion of positive cobas samples was higher than in the ATHENA trial. The age-standardized cobas positivity vs. cytology abnormality was 3.9 in our study and 1.7 in ATHENA. If in Copenhagen the presently used cytology would be replaced by cobas in women above age 30 years, an extra 11% of women would based on historical data be expected to have a positive cobas test without an underlying cervical intraepithelial lesion grade 3 or worse. Countries with a high prevalence of HPV infections should therefore proceed to primary HPV-based cervical screening with caution.

  8. Comparison of the Hologic Aptima HIV-1 Quant Dx Assay to the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 for the quantification of HIV-1 RNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Johansen, Kim; Landt, Bodil

    2017-01-01

    of the Aptima HIV-1 Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 (CAPCTMv2) for the quantification of HIV-1 RNA in plasma samples. Study design The performance of the two tests was compared on 216 clinical plasma samples, on dilutions series in seven replicates of five clinical......), but differed for the Low control (CV: 17.9% vs. 7.1%; Aptima assay vs. CAPCTMv2 test, respectively). However, this did not impact clinical categorization of clinical samples at neither the 50 cp/mL nor 200 cp/mL level. Conclusion The Aptima assay and the CAPCTMv2 test are highly correlated and are useful...

  9. Cross-reactivity profiles of hybrid capture II, cobas, and APTIMA human papillomavirus assays: split-sample study.

    Science.gov (United States)

    Preisler, Sarah; Rebolj, Matejka; Ejegod, Ditte Møller; Lynge, Elsebeth; Rygaard, Carsten; Bonde, Jesper

    2016-07-20

    High-risk Human Papillomavirus (HPV) testing is replacing cytology in cervical cancer screening as it is more sensitive for preinvasive cervical lesions. However, the bottleneck of HPV testing is the many false positive test results (positive tests without cervical lesions). Here, we evaluated to what extent these can be explained by cross-reactivity, i.e. positive test results without evidence of high-risk HPV genotypes. The patterns of cross-reactivity have been thoroughly studied for hybrid capture II (HC2) but not yet for newer HPV assays although the manufacturers claimed no or limited frequency of cross-reactivity. In this independent study we evaluated the frequency of cross-reactivity for HC2, cobas, and APTIMA assays. Consecutive routine cervical screening samples from 5022 Danish women, including 2859 from women attending primary screening, were tested with the three evaluated DNA and mRNA HPV assays. Genotyping was undertaken using CLART HPV2 assay, individually detecting 35 genotypes. The presence or absence of cervical lesions was determined with histological examinations; women with abnormal cytology were managed as per routine recommendations; those with normal cytology and positive high-risk HPV test results were invited for repeated testing in 18 months. Cross-reactivity to low-risk genotypes was detected in 109 (2.2 %) out of 5022 samples on HC2, 62 (1.2 %) on cobas, and 35 (0.7 %) on APTIMA with only 10 of the samples cross-reacting on all 3 assays. None of the 35 genotypes was detected in 49 (1.0 %), 162 (3.2 %), and 56 (1.1 %) samples, respectively. In primary screening at age 30 to 65 years (n = 2859), samples of 72 (25 %) out of 289 with high-risk infections on HC2 and < CIN2 histology were due to cross-reactivity. On cobas, this was 106 (26 %) out of 415, and on APTIMA 48 (21 %) out of 224. Despite manufacturer claims, all three assays showed cross-reactivity. In primary cervical screening at age ≥30 years, cross

  10. Comparison of clinical performances among Roche Cobas HPV, RFMP HPV PapilloTyper and Hybrid Capture 2 assays for detection of high-risk types of human papillomavirus.

    Science.gov (United States)

    Yu, Shinae; Kwon, Min-Jung; Lee, Eun Hee; Park, Hyosoon; Woo, Hee-Yeon

    2015-09-01

    The cervical cancer screening guidelines suggest that early detection of HPV16 and HPV18 is helpful for identifying women with cervical intraepithelial neoplasia (CIN) grade two or higher. We comparatively evaluated three HPV DNA assays, Roche Cobas HPV, RFMP HPV PapilloTyper, and Hybrid Capture 2 (HC2). A total of 861 cervical swab samples from women over 30 years of age were classified into two groups, that is, high grade squamous intraepithelial lesion (HSIL) and non-HSIL, according to cervical cytology results and analyzed by three assays. The results of direct sequencing or Linear array HPV genotyping test were considered true when the three assays presented discrepancies. The concordance rates between Roche Cobas HPV versus RFMP HPV PapilloTyper, RFMP HPV PapilloTyper versus HC2, and Roche Cobas versus HC2 were 94.5%, 94.3%, and 95.9%, respectively. For detection of HPV16 and HPV18, Roche Cobas HPV showed the concordance rates of 98.3% (κ = 0.73) and 99.4% (κ = 0.40) with the confirmation tests, respectively; and RFMP HPV PapilloTyper showed the concordance rates of 99.5% (κ = 0.92) and 100.0% (κ = 1.00), respectively. In conclusion, Roche Cobas HPV, RFMP HPV PapilloTyper, and HC2 showed high agreement rates. Roche Cobas HPV and RFMP HPV PapilloTyper are particularly useful, since both provide HPV specific genotypes, HPV16 and HPV18. © 2015 Wiley Periodicals, Inc.

  11. Performance of the NG OligoGen kit for the diagnosis of Neisseria gonorrhoeae: comparison with cobas 4800 assay.

    Science.gov (United States)

    Parra-Sánchez, M; García-Rey, S; Marcuello, A; Zakariya-Yousef, I; Bernal, S; Pueyo, I; Martín-Mazuelos, E; Palomares, J C

    2016-01-01

    PCR assays are nowadays between the most sensitive and reliable methods for screening and diagnosing sexually transmitted infections (STIs). The aim of this study was to analyze the reliability, accuracy, and usefulness of the new NG OligoGen kit in comparison with the cobas 4800 assay for the detection of Neisseria gonorrhoeae in clinical samples. A prospective study was designed for detection of N. gonorrhoeae including urine samples (n=152), rectal (n=80), endocervical (n=67), pharyngeal (n=41), and urethral swabs (n=5) that were sent from a regional STI clinic in Seville, Spain. Samples were collected from 255 (73.9%) men and 90 women. Sensitivity, specificity, positive and negative predicative values, and kappa value for N. gonorrhoeae detection using the NG OligoGen kit were 99.6%, 100%, 100%, 99.1%, and 0.99, respectively. Statistical data obtained in this study confirm the usefulness and reliable results of this new assay. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Prevalence of human papillomavirus in 5,072 consecutive cervical SurePath samples evaluated with the Roche cobas HPV real-time PCR assay

    DEFF Research Database (Denmark)

    Preisler, Sarah; Rebolj, Matejka; Untermann, Anette

    2013-01-01

    New commercially available Human Papillomavirus (HPV) assays need to be evaluated in a variety of cervical screening settings. Cobas HPV Test (cobas) is a real-time PCR-based assay allowing for separate detection of HPV genotypes 16 and 18 and a bulk of 12 other high-risk genotypes. The aim...... of the present study, Horizon, was to assess the prevalence of high-risk HPV infections in an area with a high background risk of cervical cancer, where women aged 23-65 years are targeted for cervical screening. We collected 6,258 consecutive cervical samples from the largest cervical screening laboratory...... in Denmark serving the whole of Copenhagen. All samples were stored in SurePath media. In total, 5,072 samples were tested with cobas, Hybrid Capture 2 High Risk HPV DNA test (HC2) and liquid-based cytology. Of these, 27% tested positive on cobas. This proportion decreased by age, being 43% in women aged 23...

  13. Multi-center evaluation of the cobas(®) Liat(®) Influenza A/B & RSV assay for rapid point of care diagnosis.

    Science.gov (United States)

    Gibson, Jane; Schechter-Perkins, Elissa M; Mitchell, Patricia; Mace, Sharon; Tian, Yu; Williams, Kemi; Luo, Robert; Yen-Lieberman, Belinda

    2017-10-01

    Point of Care Testing (POCT) provides the capability for rapid laboratory test results in patient care environments where a traditional clinical laboratory is not available. POCTs have shorter turn-around times (TATs), they may be performed by non-laboratory personnel, and the need for transport time is eliminated. The Food and Drug Administration (FDA) recently granted Clinical Laboratory Improvements Amendment (CLIA) waiver status to the cobas(®) Influenza A/B & RSV assay, a rapid, accurate point-of-care test for Influenza and respiratory syncytial virus (RSV) performed on the Liat(®) System. The performance characteristics of this test were determined though a multi-site study consisting of different point of care testing environments. Prospectively collected Nasopharyngeal (NP) swabs from 1361 patients seen at 8 primary care clinics and 4 emergency departments (EDs) and 295 retrospectively identified specimens were tested for Influenza A/B and RSV on the cobas(®) Liat(®) platform. Performance characteristics were determined through comparison to ProFlu+, a laboratory-based PCR test for Influenza A/B and RSV (reference test). Discordant specimens were adjudicated following bi-directional sequencing. The cobas(®) Influenza A/B and RSV assay showed sensitivities of 99.6%, 99.3%, and 96.8% for Influenza A, Influenza B, and RSV, respectively as determined from percent positive agreement (PPA) following comparison to the reference test. Sequencing confirmed cobas(®) Influenza A/B and RSV results in 49.2% of reference test discordant specimens, while crossing threshold data suggest increased sensitivity compared to the reference test. The cobas(®) Influenza A/B and RSV assay was found to be a rapid, sensitive POCT for the detection of these viruses, and provides laboratory-quality PCR-based diagnostic results in point of care settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Cross-reactivity profiles of hybrid capture II, cobas, and APTIMA human papillomavirus assays

    DEFF Research Database (Denmark)

    Preisler, Sarah Nørgaard; Rebolj, Matejka; Ejegod, Ditte Møller

    2016-01-01

    Background High-risk Human Papillomavirus (HPV) testing is replacing cytology in cervical cancer screening as it is more sensitive for preinvasive cervical lesions. However, the bottleneck of HPV testing is the many false positive test results (positive tests without cervical lesions). Here, we...... evaluated to what extent these can be explained by cross-reactivity, i.e. positive test results without evidence of high-risk HPV genotypes. The patterns of cross-reactivity have been thoroughly studied for hybrid capture II (HC2) but not yet for newer HPV assays although the manufacturers claimed...

  15. Comparison of the Roche Cobas(®) 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia.

    Science.gov (United States)

    Phillips, Samuel; Garland, Suzanne M; Tan, Jeffery H; Quinn, Michael A; Tabrizi, Sepehr N

    2015-01-01

    The recently FDA (U.S. food and drug administration) approved Roche Cobas(®) 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. Evaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. A selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. Overall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. The Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens.

    NARCIS (Netherlands)

    W.H.F. Goessens (Wil); P. de Man (Peter); J.G. Koeleman; A. Luijendijk (Ad); R. te Witt (René); H.P. Endtz (Hubert); A.F. van Belkum (Alex)

    2005-01-01

    textabstractThe performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference

  17. Comparison of the Analytical Performance Between cobas EGFR Assay and PCR-Clamp Method in the Detection of EGFR Mutations in Japanese Non-Small Cell Lung Cancer Patients.

    Science.gov (United States)

    Ai, Tomohiko; Yuri, Maiko; Tabe, Yoko; Kakimoto, Atsushi; Morishita, Soji; Tsuchiya, Koji; Takamochi, Kazuya; Kodama, Yuzo; Takahashi, Fumiyuki; Shigeki, Misawa; Horii, Takashi; Suzuki, Kenji; Takahashi, Kazuhisa; Miida, Takashi; Ohsaka, Akimichi

    2017-05-01

    EGFR, a tyrosine-kinase, plays an important role in the progression of lung cancer. Since genetic abnormality of EGFR alters the effects of tyrosine-kinase inhibitors targeting EGFR, molecular analyses of EGFR have recently gained more attention in the treatment of lung cancer. However, several different techniques are available and which method is superior has not been determined. In this study, we compared two recently developed PCR-based techniques, PCR-clamp method and cobas EGFR assay. Ninety-four surgical samples and 58 biopsy samples from patients suffering from non-small cell lung cancers (NSCLCs) were included in the study. Samples with positive and negative genetic abnormalities, 66 and 28 respectively, were chosen for PCR-Clamp methods. Those same samples were reanalyzed with cobas EGFR assay. The concordance between PCR-Clamp and cobas EGFR methods was 95.7%. PCR-Clamp failed to detect four mutations that were detected with cobas EGFR assay. These two methods were further tested by analyzing 58 random biopsy samples. The concordance for the biopsy samples was 93.1%, and PCR-Clamp, again, failed to detect three mutations that were detected with cobas EGFR assay. Our results showed both methods detected most of the known EGFR mutations and the concordance was similar to those previously reported in different ethnicities. However, in our study, PCR-Clamp method failed to detect a total of seven mutations that were detected with cobas EGFR assay. Thus, we concluded that cobas EGFR assay is an easier and more accurate screening assay than PCR-Clamp method in detecting EGFR genetic abnormalities.

  18. Performance of cobas® 4800 and m2000 real-time™ assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and self-collected vaginal specimen

    NARCIS (Netherlands)

    Geelen, Tanja H; Rossen, John W; Beerens, Antoine M; Poort, Linda; Morré, Servaas A; Ritmeester, Wilma S; van Kruchten, Harry E; van de Pas, Masja M; Savelkoul, Paul H M

    2013-01-01

    A prospective, multicenter trial was designed to compare the performance characteristics of the cobas® 4800 (Roche Diagnostics, Indianapolis, IN, USA) and m2000 real-time™ (Abbott Molecular Inc., Des Plaines, IL, USA) assays for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)

  19. Comparison of the Abbott RealTime High-Risk Human Papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 assays to direct sequencing and genotyping of HPV DNA.

    Science.gov (United States)

    Park, Yongjung; Lee, Eunhee; Choi, Jonghyeon; Jeong, Seri; Kim, Hyon-Suk

    2012-07-01

    Infection with high-risk (HR) human papillomavirus (HPV) genotypes is an important risk factor for cervical cancers. We evaluated the clinical performances of two new real-time PCR assays for detecting HR HPVs compared to that of the Hybrid Capture 2 test (HC2). A total of 356 cervical swab specimens, which had been examined for cervical cytology, were assayed by Abbott RealTime HR and Roche Cobas HPV as well as HC2. Sensitivities and specificities of these assays were determined based on the criteria that concordant results among the three assays were regarded as true-positive or -negative and that the results of genotyping and sequencing were considered true findings when the HPV assays presented discrepant results. The overall concordance rate among the results for the three assays was 82.6%, and RealTime HR and Cobas HPV assays agreed with HC2 in 86.1% and 89.9% of cases, respectively. The two real-time PCR assays agreed with each other for 89.6% of the samples, and the concordance rate between them was equal to or greater than 98.0% for detecting HPV type 16 or 18. HC2 demonstrated a sensitivity of 96.6% with a specificity of 89.1% for detecting HR HPVs, while RealTime HR presented a sensitivity of 78.3% with a specificity of 99.2%. The sensitivity and specificity of Cobas HPV for detecting HR HPVs were 91.7% and 97.0%. The new real-time PCR assays exhibited lower sensitivities for detecting HR HPVs than that of HC2. Nevertheless, the newly introduced assays have an advantage of simultaneously identifying HPV types 16 and 18 from clinical samples.

  20. Comparison of the Abbott RealTime High-Risk Human Papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 Assays to Direct Sequencing and Genotyping of HPV DNA

    OpenAIRE

    Park, Yongjung; Lee, Eunhee; Choi, Jonghyeon; Jeong, Seri; Kim, Hyon-Suk

    2012-01-01

    Infection with high-risk (HR) human papillomavirus (HPV) genotypes is an important risk factor for cervical cancers. We evaluated the clinical performances of two new real-time PCR assays for detecting HR HPVs compared to that of the Hybrid Capture 2 test (HC2). A total of 356 cervical swab specimens, which had been examined for cervical cytology, were assayed by Abbott RealTime HR and Roche Cobas HPV as well as HC2. Sensitivities and specificities of these assays were determined based on the...

  1. A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS(®) TAQMAN(®) HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-10-01

    Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System.(1) OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS(®) TaqMan(®) HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT(®) HBV Assay (Versant), and 74 specimens tested with Veris and artus(®) HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log10 IU/mL between Veris and Cobas, -0.46 log10IU/mL between Veris and RealTime, -0.36 log10IU/mL between Veris and Versant, and -0.12 log10IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in an STI population: performances of the Presto CT-NG assay, the Lightmix Kit 480 HT CT/NG and the COBAS Amplicor with urine specimens and urethral/cervicovaginal samples

    Science.gov (United States)

    Schuurs, T A; Verweij, S P; Weel, J F L; Ouburg, S; Morré, S A

    2013-01-01

    Objectives This study assessed the performances of the Presto CT-NG assay, the Lightmix Kit 480 HT CT/NG and the COBAS Amplicor for Chlamydia trachomatis and Neisseria gonorrhoeae detection. Design A cross-sectional study design. Setting Izore, Centre for Diagnosing Infectious Diseases in Friesland, the Netherlands, tested samples sent from regional sexually transmitted infection (STI) outpatient clinics and regional hospitals from the province Friesland, the Netherlands. Participants Samples were collected from 292 men and 835 women. These samples included 560 urine samples and 567 urethral/cervicovaginal samples. Primary and secondary outcome measures The primary outcome measure is C trachomatis infection. No secondary outcome measures are available. Results The sensitivity, specificity, positive predicative value (PPV) and negative predictive value (NPV) for C trachomatis detection in urine samples using the Presto CT-NG assay were 100%, 99.8%, 98.1% and 100%, respectively; for the Lightmix Kit 480 HT CT/NG: 94.2%, 99.8%, 96.1% and 99.4%, respectively; for the COBAS Amplicor: 92.3%, 99.6%, 96% and 99.2%, respectively. The sensitivity, specificity, PPV and NPV for C trachomatis detection in urethral/cervicovaginal swabs using the Presto CT-NG assay and the COBAS Amplicor were 100%, 99.8%, 97.7% and 100%, respectively; for the Lightmix Kit 480 HT CT/NG: 100%, 99.6%, 97.7% and 100%, respectively. Calculations for N gonorrhoeae could not be made due to a low prevalence. Conclusions All three assays had a high sensitivity, specificity, PPV and NPV for C trachomatis, with best performance for the Presto CT-NG assay. PMID:24381252

  3. Accurate Detection of Streptococcus pyogenes at the Point of Care Using the cobas Liat Strep A Nucleic Acid Test.

    Science.gov (United States)

    Wang, Fangnian; Tian, Yu; Chen, Lingjun; Luo, Robert; Sickler, Joanna; Liesenfeld, Oliver; Chen, Shuqi

    2017-10-01

    The performance of a polymerase chain reaction-based point-of-care assay, the cobas Strep A Nucleic Acid Test for use on the cobas Liat System (cobas Liat Strep A assay), for the detection of group A Streptococcus bacteria was evaluated in primary care settings. Throat swab specimens from 427 patients were tested with the cobas Liat Strep A assay and a rapid antigen detection test (RADT) by existing medical staff at 5 primary care clinics, and results were compared with bacterial culture. The cobas Liat Strep A assay demonstrated equivalent sensitivity (97.7%) and specificity (93.3%) to reference culture with a 15-minute turnaround time. In comparison to RADTs, the cobas Liat Strep A assay showed improved sensitivity (97.7% Liat vs 84.5% RADT). The Clinical Laboratory Improvement Amendments-waived cobas Liat Strep A assay demonstrated the ease of use and improved turnaround time of RADTs along with the sensitivity of culture.

  4. Direct detection of markers associated with Neisseria gonorrhoeae antimicrobial resistance in New Zealand using residual DNA from the Cobas 4800 CT/NG NAAT assay.

    Science.gov (United States)

    Nicol, Mackenzie; Whiley, David; Nulsen, Mary; Bromhead, Collette

    2015-03-01

    To use nucleic acid amplification techniques (NAAT) for detection of markers associated with gonococcal antimicrobial resistance (AMR) in non-cultured clinical samples to enhance surveillance of Neisseria gonorrhoeae AMR in New Zealand. A total of 198 clinical samples from patients living in two cities, Wellington and Auckland and the more rural region of Gisborne, New Zealand, which were positive for N. gonorrhoeae by the Cobas 4800 were tested for three markers that predict reduced susceptibility or resistance to three antibiotics. Residual DNA extracts from the Cobas 4800 NG/CT test were tested for a single-nucleotide polymorphism in the gyrA gene at codon 91 associated with quinolone resistance; a sequence on the plasmid in penicillinase-producing N. gonorrhoeae (PPNG) which confers resistance to penicillin and the mosaic penA sequence associated with reduced susceptibility to extended-spectrum cephalosporins in N. gonorrhoeae. A total of 186/198 (94%) of the samples provided a valid result on gyrA genotyping, confirming the utility of N. gonorrhoeae DNA extracted by the Roche Cobas 4800 CT/NG test for subsequent detection of AMR markers. The NAAT results for Wellington, Auckland and Gisborne, respectively, showed that 77%, 33% and 32% of samples had the marker associated with quinolone resistance, while 4%, 15% and 0% were positive for the PPNG plasmid marker, and 9%, 5% and 0% samples were positive for mosaic penA sequence. The use of residual clinical DNA samples from the Cobas 4800 CT/NG test proved an efficient and effective method for performing AMR genotyping. These data also show for the first time the presence of gonococci with a mosaic penA sequence in New Zealand. Overall, the results further highlight the potential of molecular methods to aid N. gonorrhoeae AMR surveillance, particularly for regions where gonococcal culture is no longer performed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a

  5. Diagnostic accuracy of the real-time PCR cobas(®) Liat(®) Influenza A/B assay and the Alere i Influenza A&B NEAR isothermal nucleic acid amplification assay for the detection of influenza using adult nasopharyngeal specimens.

    Science.gov (United States)

    Young, Stephen; Illescas, Patrick; Nicasio, Joclin; Sickler, Joanna Jackson

    2017-09-01

    Accurate detection of influenza requires diagnostic testing; however, methods such as RADTs and central laboratory-based tests are limited by low sensitivity and time constraints, respectively. To compare the performances of the cobas(®) Liat(®) Influenza A/B and Alere™ i Influenza A&B point-of-care (POC) assays for detecting influenza A and B viruses using fresh nasopharyngeal specimens with the GenMark Dx(®) Respiratory Viral Panel as the reference method, a FDA cleared IVD PCR test. A total of 87 samples collected in viral transport medium from adults ≥18 years of age were re-tested on both POC assays (based on the reference PCR method, 29 were influenza A and 18 were influenza B virus positive). The overall sensitivity and specificity of the cobas Influenza A/B for the detection of influenza A and B relative to reference PCR was 97.9% (95% confidence interval [CI] 88.9%, 99.6%) and 97.5% (95% CI: 87.1%, 99.6%), respectively, while the sensitivity of the Alere i Influenza A&B assay relative to the reference PCR method was 63.8% (95% CI: 49.5%, 76.0%) and the specificity was 97.5% (95% CI: 87.1%, 99.6%). The individual sensitivities and specificities of the cobas Influenza A/B assay for influenza A alone and influenza B alone were comparable to those of the reference PCR method (influenza A: sensitivity of 100% [95% CI: 88.3%, 100.0%] and specificity of 98.3% [95% CI: 90.9%, 99.7%]; influenza B: sensitivity of 94.4% [95% CI: 74.2%, 99.0%] and specificity of 100% [95% CI: 94.7%, 100.0%]). For the Alere i Influenza A&B assay, the individual specificities for influenza A and B were comparable to those of the reference PCR method (98.3% [95% CI: 90.9%, 99.7%] and 97.1% [95% CI: 90.0%, 99.2%], respectively), while the individual sensitivities were low relative to reference PCR (55.2% [95% CI: 37.5%, 71.6%] and 72.2% [95% CI: 49.1%, 87.5%], respectively). The cobas Influenza A/B assay demonstrated performance equivalent to laboratory-based PCR, and could replace

  6. Lab-in-a-tube: Real-time molecular point-of-care diagnostics for influenza A and B using the cobas(R) Liat(R) system

    NARCIS (Netherlands)

    Melchers, W.J.G.; Kuijpers, J; Sickler, J.J.; Rahamat-Langendoen, J.C.

    2017-01-01

    Rapid diagnosis of influenza A and B is important for direct treatment decisions in patient care and for the reduction of in-hospital transmissions. The new real-time PCR based molecular point-of-care (POC) assay, the cobas(R) Influenza A/B test on the cobas(R) Liat(R) System (cobas(R) Liat(R)

  7. Analytical performance evaluation of the Elecsys® Cyclosporine and Elecsys® Tacrolimus assays on the cobas e411 analyzer

    Directory of Open Access Journals (Sweden)

    Maki Sasano

    2017-08-01

    Full Text Available Background: Cyclosporine (CsA and tacrolimus (TAC are immunosuppressant drugs that are often used to treat autoimmune diseases and as transplantation therapy; therefore, their concentrations need to be monitored carefully. We herein evaluated the analytical performance of the Elecsys® Cyclosporine and Elecsys® Tacrolimus assay kits, which have been newly developed to measure CsA and TAC concentrations in the whole blood. Methods: We used residual whole blood samples from autoimmune disease and transplantation patients who were being treated with CsA or TAC. CsA concentrations were measured using an affinity chrome-mediated immunoassay (ACMIA and an electrochemiluminescence immunoassay (ECLIA. TAC concentrations were measured using a chemiluminescence immunoassay (CLIA and ECLIA. We investigated assay precision, linearity, lower limit of quantitation (LOQ, stability of calibration, influence of interference substances and the hematocrit, correlation of ACMIA with ECLIA, and correlation of CLIA with ECLIA. Results: Within-assay coefficients of variation were 1.8−3.6% (CsA: 94−1238 ng/mL and 2.9−3.9% (TAC: 2.1−17.8 ng/mL, whereas day-to-day coefficients of variation ranged between 3.0−4.1% (CsA and 2.8−3.9% (TAC. The limits of quantitation were defined as the concentration at which the CV was approximately 10%. Each lower LOQ obtained was 16 ng/mL (CsA, and 0.95 ng/mL (TAC. CsA and TAC calibrations were stable for at least 21 days. Neither the presence of conjugated bilirubin, unconjugated bilirubin, chyle, and rheumatoid factor nor the hematocrit affected these assays. A method comparison using a standardized major axis regression analysis of ACMIA and ECLIA was r=0.995, y=0.924x −1.175, n=200 (CsA, while that of CLIA and ECLIA was r=0.994, y=1.080x −0.197, n=200 (TAC. Conclusions: The analytical performances of the Elecsys® Cyclosporine and Elecsys®Tacrolimus assays were acceptable

  8. TSH Comparison Between Chemiluminescence (Architect) and Electrochemiluminescence (Cobas) Immunoassays: An Indian Population Perspective.

    Science.gov (United States)

    Sarkar, Rajarshi

    2014-04-01

    Although 3rd generation TSH assays are the most widely used immunoassays, credible comparison studies, specially involving Indian sub-populations are practically non-existent. To compare the TSH measurements between chemiluminescence (Architect) and electrochemiluminescence (Cobas) inmmunoassays in an urban ambulatory Indian population. 1,615 subjects were selected randomly from the usual laboratory workflow, their TSH measured in Architect and Cobas and the paired data thus generated were statistically analysed. TSH values of Cobas were observed to be higher than the Architect values by 28.7 %, with a significant proportional difference between the two, but majority of the Cobas values (above 90 %) were within the limits of agreement with Architect values. In situations where both the instruments are in use simultaneously, a standardization of the methods is imperative, in larger interest of the patient populace.

  9. Lab-in-a-tube: Real-time molecular point-of-care diagnostics for influenza A and B using the cobas® Liat® system.

    Science.gov (United States)

    Melchers, Willem J G; Kuijpers, Judith; Sickler, Joanna Jackson; Rahamat-Langendoen, Janette

    2017-08-01

    Rapid diagnosis of influenza A and B is important for direct treatment decisions in patient care and for the reduction of in-hospital transmissions. The new real-time PCR based molecular point-of-care (POC) assay, the cobas(®) Influenza A/B test on the cobas(®) Liat(®) System (cobas(®) Liat(®) Influenza A/B assay), generated a PCR result in less than 20 min, was evaluated for the detection of influenza A and B. One hundred twenty-one retrospectively collected respiratory specimens, previously analyzed with a routine influenza A/B test (Diagenode) were tested using the cobas(®) Liat(®) Influenza A/B assay. The cobas(®) Liat(®) Influenza A/B assay allows influenza A and B testing by RT-PCR within 20 min. This assay detected influenza A in 51 of 56 samples positive by the Diagenode test. The five discrepant results were retested with the Cepheid Influenza A/B test, confirming two positive cases. All 30 influenza B Diagenode positive samples were found positive by the cobas(®) Liat(®) Influenza A/B assay. Control samples (viral negative and non-influenza pathogens) were all negative by the cobas(®) Liat(®) Influenza A/B assay. The cobas(®) Liat(®) Influenza A/B assay showed a sensitivity for influenza A/B of 96% and 100%, respectively, and 100% specificity for both targets. The cobas(®) Liat(®) Influenza A/B assay is a useful tool for accurate, rapid, and sensitive detection of influenza A and B, offering timely and personalized patient management and infection control when implemented at the point-of-care. © 2017 Wiley Periodicals, Inc.

  10. Analytic and clinical performance of cobas HPV testing in anal specimens from HIV-positive men who have sex with men.

    Science.gov (United States)

    Wentzensen, Nicolas; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Sahasrabuddhe, Vikrant V; Chen, Jie; Lorey, Thomas S; Gage, Julia C; Fetterman, Barbara; Boyle, Sean; Sadorra, Mark; Tang, Scott Dahai; Darragh, Teresa M; Castle, Philip E

    2014-08-01

    Anal human papillomavirus (HPV) infections are common, and the incidence of anal cancer is high in HIV-infected men who have sex with men (MSM). To evaluate the performance of HPV assays in anal samples, we compared the cobas HPV test (cobas) to the Roche Linear Array HPV genotyping assay (LA) and cytology in HIV-infected MSM. Cytology and cobas and LA HPV testing were conducted for 342 subjects. We calculated agreement between the HPV assays and the clinical performance of HPV testing and HPV genotyping alone and in combination with anal cytology. We observed high agreement between cobas and LA, with cobas more likely than LA to show positive results for HPV16, HPV18, and other carcinogenic types. Specimens testing positive in cobas but not in LA were more likely to be positive for other markers of HPV-related disease compared to those testing negative in both assays, suggesting that at least some of these were true positives for HPV. cobas and LA showed high sensitivities but low specificities for the detection of anal intraepithelial neoplasia grade 2/3 (AIN2/3) in this population (100% sensitivity and 26% specificity for cobas versus 98.4% sensitivity and 28.9% specificity for LA). A combination of anal cytology and HPV genotyping provided the highest accuracy for detecting anal precancer. A higher HPV load was associated with a higher risk of AIN2/3 with HPV16 (P(trend) < 0.001), HPV18 (P(trend) = 0.07), and other carcinogenic types (P(trend) < 0.001). We demonstrate that cobas can be used for HPV detection in anal cytology specimens. Additional tests are necessary to identify men at the highest risk of anal cancer among those infected with high-risk HPV. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Validation of Cobas AmpliPrep/Cobas TaqMan HIV-1 Test on dried blood spots

    Directory of Open Access Journals (Sweden)

    N Ruiz

    2012-11-01

    Full Text Available The plasma specimen is the gold standard for viral load monitoring, the key method to assess the effect of antiviral chemotherapy and to monitor progression of the disease toward AIDS. Nevertheless, several works endorse the use of dried blood spots (DBS on filter paper for the reliable quantification of the levels needed to take therapeutic decisions, detect of treatment failure and monitor the occurrence of drug resistance. The purpose of this study was to validate the use of Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0, with DBS. To evaluate the performance of the above mentioned kit, three stages were involved: 1- Standardization of DBS working conditions, 2- Stability studies at three temperature conditions and 3- Performance evaluation of the kit using this alternative specimen. Additionally, the viral load was quantified in parallel (plasma and DBS to 43 genetically characterized samples, with different levels of viral load. The Pearson correlation coefficient was calculated and the prediction of the value of RNA in plasma starting from the obtained value in DBS was made. Linear regression analysis was performed and coefficients of variation in precision assays were calculated. The best conditions pickups to the work with DBS were: 100 µL of blood (2 spots/50 µl, dried time between 16 and 18 hours at room temperature and, elution of the blood, 2 hours, between 2 and 8°C; in TRIS-EDTA buffer. The samples on DBS proved to be stable during the study periods. A strong correlation was attained between the measurements of viral load in plasma and DBS samples (r=0.96. The detection rate was 90.7 and the coefficient of variation between the values obtained in plasma-DBS sample pairs averaged 3.42%. The CAP/CTM HIV-1 test provided a linear response in DBS, from 330 copies/mL to 420 000 copies/mL. Overall, coefficients of variation in precision tests were below 10%. Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 had a good

  12. A Comparison of the Roche Cobas HPV Test With the Hybrid Capture 2 Test for the Detection of High-Risk Human Papillomavirus Genotypes.

    Science.gov (United States)

    Levi, Angelique W; Bernstein, Jane I; Hui, Pei; Duch, Kara; Schofield, Kevin; Chhieng, David C

    2016-02-01

    All Food and Drug Administration-approved methods in the United States for human papillomavirus testing including the Hybrid Capture 2 human papillomavirus assay and the Roche cobas human papillomavirus test are approved for cytology specimens collected into ThinPrep media but not for specimens collected into SurePath solution. To compare the performance of the Roche cobas and Hybrid Capture 2 tests for the detection of high-risk human papillomavirus using both ThinPrep and SurePath preparations as part of a validation study. One thousand three hundred seventy-one liquid-based cytology samples, including 1122 SurePath and 249 ThinPrep specimens, were tested for high-risk human papillomavirus DNA using the Roche cobas human papillomavirus test and the Hybrid Capture 2 human papillomavirus assay. For cases with discrepant results, confirmatory testing was performed using Linear Array human papillomavirus testing. One hundred and fifty-six (11.38%) and 184 (13.42%) of the 1371 specimens tested positive for high-risk human papillomavirus DNA using the Hybrid Capture 2 human papillomavirus assay and Roche cobas human papillomavirus assay, respectively. In addition, 1289 (94.0%) of 1371 specimens demonstrated concordant high-risk human papillomavirus results with a κ value of 0.72 (95% confidence interval, 065-0.78). There was no statistically significant difference in the percentage of positive high-risk human papillomavirus results between the 2 liquid-based preparations with either assay. Discordant results between the 2 assays were noted in 82 of 1371 cases (6%). Twenty-seven of 82 cases (32.9%) were Hybrid Capture 2 positive/Roche cobas negative and 55 of 82 cases (67.1%) were Roche cobas positive/Hybrid Capture 2 negative. Two of 20 Hybrid Capture 2-positive/Roche cobas-negative cases (10%) and 26 of 37 Roche cobas-positive/Hybrid Capture 2-negative cases (70%) tested positive for high-risk human papillomavirus by Linear Array. Both assays showed good agreement

  13. Comparison of the performance of Anyplex II HPV HR, the Cobas 4800 human papillomavirus test and Hybrid Capture 2.

    Science.gov (United States)

    Lee, Do-Hoon; Hwang, Na Rae; Lim, Myong Cheol; Yoo, Chong Woo; Joo, Jungnam; Kim, Joo-Young; Park, Sang-Yoon; Hwang, Sang-Hyun

    2016-09-01

    Anyplex II HPV HR (Anyplex_HR; Seegene, Seoul, Korea) is a new multiplex real-time polymerase chain reaction assay for screening cervical cancer, and it is designed to detect 14 high-risk human papillomaviruses along with all the genotype information in a single tube. The aim of this study was to evaluate the performance of the Anyplex_HR in comparison to that of the Cobas 4800 HPV (Cobas_4800; Roche Molecular Diagnostics, Pleasanton, CA, USA) and the Hybrid capture 2 (HC2; Qiagen GmbH, Hilden, Germany). The performance of the Anyplex_HR for high-risk human papillomavirus genotype detection was prospectively evaluated against that of the HC2 and the Cobas_4800 at the National Cancer Center using 400 cervical samples. All discrepant samples were confirmed by polymerase chain reaction with type-specific primers followed by sequencing. The overall agreement and kappa value of the Anyplex_HR with the Cobas_4800 were 98.0% and 0.96, respectively. The level of agreement between the two assays and the corresponding kappa values for human papillomavirus16, human papillomavirus18 and other high-risk human papillomaviruses were 99.5%, 99.8% and 98.8%, and 0.98, 0.96 and 0.97, respectively. The agreement and kappa value of the HC2 with the Cobas_4800 were 95.3% and 0.91. The human papillomavirus positivity of the Anyplex_HR and the Cobas_4800 in low-grade squamous intraepithelial lesion/high-grade squamous intraepithelial lesion samples demonstrated 100% concordance. Both the Anyplex_HR and the Cobas_4800 showed excellent results in the precision test. The Anyplex_HR is comparable with the Cobas_4800 and the HC2 for human papillomavirus DNA testing, and it may prove more useful for follow-up testing and patient management by providing genotyping information additional to human papillomavirus16 and human papillomavirus18. © The Author(s) 2016.

  14. Comparison of AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR for Detection of Mycobacterium tuberculosis Complex in Routine Clinical Practice.

    Science.gov (United States)

    Cho, Won-Hyung; Won, Eun-Jeong; Choi, Hyun-Jung; Kee, Seung-Jung; Shin, Jong-Hee; Ryang, Dong-Wook; Suh, Soon-Pal

    2015-05-01

    The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.

  15. Evaluation of NGAL TestTM on Cobas 6000

    DEFF Research Database (Denmark)

    Hansen, Young B L; Damgaard, Anette; Poulsen, Jørgen H

    2014-01-01

    BACKGROUND: Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a promising biomarker for acute kidney injury (AKI). Our objectives were to evaluate the NGAL Test(TM) from Bioporto for both urine NGAL and plasma NGAL on the Cobas 6000 c501 (Roche Diagnostics, Rotkreuz, Switzerland) with matched...

  16. Comparison of Xpert MTB/RIF with ProbeTec ET DTB and COBAS TaqMan MTB for direct detection of M. tuberculosis complex in respiratory specimens

    OpenAIRE

    Antonenka, Uladzimir; Hofmann-Thiel, Sabine; Turaev, Laziz; Esenalieva, Ainura; Abdulloeva, Mokhonim; Sahalchyk, Evgeni; Alnour, Tarig; Hoffmann, Harald

    2013-01-01

    Background Nucleic acid amplification assays allow for the rapid and accurate detection of Mycobacterium tuberculosis (MTB) directly in clinical specimens thereby facilitating diagnosis of tuberculosis (TB). With the fully automated Xpert MTB/RIF system (Cepheid) an innovative solution of TB diagnostics has been launched. We performed a direct head-to-head comparison of Xpert MTB/RIF with two widely used commercial assays, ProbeTec ET DTB (DTB) (Becton-Dickinson) and COBAS TaqMan MTB (CTM-MTB...

  17. Comparison of human papillomavirus detection by Aptima HPV and cobas HPV tests in a population of women referred for colposcopy following detection of atypical squamous cells of undetermined significance by Pap cytology.

    Science.gov (United States)

    Castle, Philip E; Eaton, Barbara; Reid, Jennifer; Getman, Damon; Dockter, Janel

    2015-04-01

    Few studies have compared the cobas HPV test to the Aptima HPV assay (AHPV) and the Aptima HPV 16 18/45 genotype assay (AHPV GT) for high-risk human papillomavirus (hrHPV) detection, clinical performance in detecting cervical intraepithelial neoplasia grade 2 (CIN2) or more severe (CIN2+) diagnoses, and risk stratification by partial HPV genotyping. The cobas HPV test is a DNA test that separately and concurrently detects HPV16, HPV18, and a pool of 12 other hrHPV types. AHPV is an RNA test for a pool of 14 hrHPV genotypes, and AHPV GT is an RNA test run on AHPV-positive results to detect HPV16 separately from HPV18 and HPV45, which are detected together. In a population of patients (n=988) referred for colposcopy because of a cervical Pap cytology result of atypical squamous cells of undetermined significance (ASC-US), a cervical scrape specimen was taken, placed into a ThinPrep Pap test vial containing PreservCyt liquid cytology medium, and tested in a blinded fashion with cobas and AHPV and with AHPV GT for AHPV-positive results. The final diagnoses were based on a consensus panel review of the biopsy specimen histology. AHPV and cobas were equally sensitive for CIN2+ diagnoses (89.4% each; P=1.000), and AHPV was more specific than cobas (63.1% versus 59.3%; P≤0.001). The percent total agreement, percent positive agreement, and kappa value were 90.9%, 81.1%, and 0.815, respectively. Risk stratification using partial HPV genotyping was similar for the two assays. AHPV and AHPV GT had similar sensitivity and risk stratification to cobas HPV, but they were more specific than cobas HPV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS(®) Ampliprep/COBAS(®)TaqMan HBV test v2.0.

    Science.gov (United States)

    Salpini, Romina; Piermatteo, Lorenzo; Gill, Upkar; Battisti, Arianna; Stazi, Francesca; Guenci, Tania; Giannella, Sara; Serafini, Valentina; Kennedy, Patrick T F; Perno, Carlo Federico; Svicher, Valentina; Ciotti, Marco

    2017-08-01

    Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBAS(®)Ampliprep/COBAS(®)TaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R (2) = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R (2) = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBAS(®)Ampliprep/COBAS(®)TaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

  19. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, Martin; Peters, D; Back, N K T; Beld, M; Beuselinck, K; Foulongne, V; Geretti, A-M; Pandiani, L; Tiemann, C; Niesters, H G M

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS

  20. Evaluation of the fully automated Cobas Core enzyme immunoassay for the quantitation of antibodies against hepatitis B virus surface antigen.

    Science.gov (United States)

    Doche, C; Thomé, M; Dimet, I; Bienvenu, J

    1996-04-01

    The Roche Cobas Core automated enzyme immunoassay of antibodies against hepatitis B virus surface antigen (anti-HBs) was evaluated using a protocol complying with the Société Française de Biologie Clinique guidelines. Results showed good precision (CV below 6 and 9% for intra- and inter-assay precision) and accuracy (according to the Paul-Ehrlich-Institute standard); the detection limit of the test was 2 IU/l. The manufacturer's specifications were confirmed and a satisfactory correlation (r = 0.901) was found with an automated method (IMx, Abbott) used for comparison.

  1. An Evaluation of the Cobas4800 HPV Test on Cervico-Vaginal Specimens in Liquid versus Solid Transport Media.

    Directory of Open Access Journals (Sweden)

    Hongxue Luo

    Full Text Available Determine the ability of the Cobas 4800 assay to detect high-risk human papillomavirus (HrHPV and high-grade cervical lesions when using cervico-vaginal samples applied to liquid medium and solid media cards compared to a direct cervical sample.Two cervico-vaginal specimens (pseudo self-collected were obtained from 319 women. One was applied to an iFTA Card (FTA then the brush placed in liquid-based medium (LSELF; the other was applied to a new solid media: POI card (POI. The clinical performance of Cobas4800 assay using the three aforementioned specimens was compared to direct collected endocervical specimens in liquid media (LDOC.The overall agreements of HrHPV detection were 84.2% (LSELF vs. LDOC, 81.0% (FTA vs. LDOC, and 82.3% (POI vs. LDOC. LSELF, FTA and POI identified 98.0%, 79.6%, and 97.5% positive cases of LDOC. Sensitivity to identify CIN2+ were 98.4% (LSELF, 73.8% (FTA, 95.1% (POI, and 93.4% (LDOC respectively. FTA had 78.1% and 90.4% agreement with the LSELF samples for all HrHPV and HPV16/18 detection respectively, while POI had 91.6% for both.Cobas4800 HPV test combined with cervico-vaginal specimens applied to both liquid media and POI solid card are accurate to detect HrHPV infection and high-grade cervical lesions as compared with direct endocervical samples in liquid media.

  2. Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma.

    Science.gov (United States)

    Pettus, Jason R; Wilson, Terri L; Steinmetz, Heather B; Lefferts, Joel A; Tafe, Laura J

    2017-02-01

    Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-07-01

    Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Tekno Ekonomi Uji Coba Alat Pembuat Kancing Tempurung

    Directory of Open Access Journals (Sweden)

    Endang Pristiwati

    2016-04-01

    Full Text Available Tempurung kelapa mempunyai sifat fisik yang menarik dan cocok dimanfaatkan menjadi produk kerajinan khususnya kerajinan perhiasan atau perabot busana. Salah satu barang kerajinan yang dapat dibuat dari tempurung kelapa adalah kancing baju untuk busana ataupun kancing untuk asesoris tas. Pembuatan kancing tempurung dengan menggunakan alat hasil rekayasa Balai Besar Industri Kerajinan dan Batik yang terdiri dari 3 unit alat, yaitu alat pengeplong, alat pelubang dan alat bubut. Untuk mengetahui kelayakan operasional dari alat tersebut maka perlu dilakukan uji coba dan analisa secara tekno ekonominya.Hasil pengujian alat menunjukkan bahwa 1 jam dapat membuat kancing tempurung sebanyak 12,43 set. Kapasitas produksi perhari sebanyak 87 set kancing tempurung rangkap dengan tenaga kerja 5 orang. Alat hasil rekayasa tersebut akan mencapai efektifitas dan efisiensi dengan spesialisasi dalam proses produksinya, yaitu 1 orang spesialisasi dalam proses plong dan bur dan 4 orang spesialisasi dalam pembubutan. Perhitungan ekonomi uji coba a/at tersebut sebagai berikut : - Total modal Rp. 4.752.150,- - Biaya produksi Rp. 6.426.050,-- Jumlah produksi 6.525 set per 3 bulan - Harga jual perset sekitar Rp. 1.350 .-- Hasil penjualan per 3 bulan Rp. 8.808. 750,- - Keuntungan sebelum pajak Rp. 2.382. 700,- - Prosentase keuntungan 50,14 % - Waktu pengembalian modal 5,6 bulan - Prosentase batas rugi laba 65,2 %- Harga jual perset sekitar Rp.1.350 ,-Tempurung kelapa mempunyai sifat fisik yang menarik dan cocok dimanfaatkan menjadi produk kerajinan khususnya kerajinan perhiasan atau perabot busana. Salah satu barang kerajinan yang dapat dibuat dari tempurung kelapa adalah kancing baju untuk busana ataupun kancing untuk asesoris tas. Pembuatan kancing tempurung dengan menggunakan alat hasil rekayasa Balai Besar Industri Kerajinan dan Batik yang terdiri dari 3 unit alat, yaitu alat pengeplong, alat pelubang dan alat bubut. Untuk mengetahui kelayakan operasional dari alat

  5. Performance of the cobas MRSA/SA Test for Simultaneous Detection of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus From Nasal Swabs.

    Science.gov (United States)

    Peterson, Lance R; Woods, Christopher W; Davis, Thomas E; Wang, Zi-Xuam; Young, Stephen A; Osiecki, John C; Lewinski, Michael A; Liesenfeld, Oliver

    2017-08-01

    Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) infections are continuing problems. Rapidly determining the MRSA colonization status of a patient facilitates practice to reduce spread of MRSA clinical disease. Sensitive detection of all SA prior to surgery, followed by decolonization, can significantly reduce postoperative infection from this pathogen. Our goal was to validate a new automated assay for this testing. We compared performance of the cobas MRSA/SA Test on the cobas 4800 System to direct and enriched chromogenic culture using nasal swabs collected from patients at six United States sites. Compared to direct and enriched culture, the sensitivity for MRSA and SA was 93.1% and 93.9%, and the specificity was 97.5% and 94.2%, respectively. After discrepancy analysis, the sensitivity for MRSA and SA was 97.1% and 98.6%, and the specificity was 98.3% and 95.5%, respectively. Compared to direct culture, sensitivity for detecting any SA was 99.6%. The cobas MRSA/SA Test is an effective tool to simultaneously perform surveillance testing for nasal colonization of both MRSA and MSSA.

  6. Comparative study of the serum measurement of PTH on Roche Cobas e411® versus the Abbott Architect ci8200®.

    Science.gov (United States)

    Bensalah, Mohammed; Bouayadi, Ouardia; Rahmani, Nawal; Lyagoubi, Amina; Lamrabat, Somiya; Choukri, Mohammed

    2017-12-15

    Parathormone (PTH) is the main hormone of phosphocalcic homeostasis. It is synthesized and secreted by the parathyroid glands. PTH has become a routine test in the medical biology laboratory. However, its measurement presents analytical difficulties with the various marketed kits. The aim of this work is to present the results of a comparative study between the PTH measurment on Abbott architect ci8200 and on Roche's Cobas e411 automaton. It is a prospective study carried out for 252 hospitalized patients in the various departments of the University Hospital Center Mohammed VI of Oujda. The "intact" PTH tests were performed on two automata: Abbott Architect ci8200 and Roche Cobas e411. The first uses chemiluminescent microparticle immunoassay. The second uses electrochemiluminiscence sandwich enzyme immunoassay. The agreement of the results between the different techniques was evaluated using the Bland-Altman difference diagram and the Passing-Bablok and Deming regression line (MedCalc software version 14.8.1.0®). By analyzing the diagram of Bland-Altman, we note that the average bias between both methods is of the order of 193.9 pg/mL. As for the equation of the right of Passing-Bablok, it is: Y(Architect) = 3.11 X (Cobas) - 12.26. In conclusion, our study shows a great discrepancy between the results of the PTH assay on the Architect ci8200 versus the Cobas e411, hence the biologist's indisputable role in the control and evaluation of the kits marketed through the various validation tests.

  7. Prevalence of high-risk human papillomavirus by cobas 4800 HPV test in urban Peru

    OpenAIRE

    Iwasaki, Ricardo; Galvez-Philpott, Felipe; Arias-Stella Jr.,Javier; Arias-Stella, Javier

    2014-01-01

    Background: Molecular tests allow the detection of high-risk human papillomavirus in cervical samples, playing an important role in the prevention of cervical cancer. Objectives: We performed a study to determine the prevalence of HPV 16, HPV 18 and other high-risk human papillomavirus (pool 12 genotypes) in Peruvian females from diverse urban areas using the cobas 4800 HPV test. Methods: Routine cervical samples collected in our laboratory were analyzed by cobas 4800 HPV test. Results:...

  8. Performance of the Xpert HPV assay in women attending for cervical screening

    Directory of Open Access Journals (Sweden)

    Jack Cuzick

    2015-12-01

    Full Text Available Objectives: This study evaluated the Xpert HPV Assay in women attending screening in general practice by comparing Xpert with two established HPV tests, cytology and histology. Methods: A prospective study in women aged 20–60 years attending screening in Bristol, Edinburgh and London using residual Preservcyt cytology samples. Sample order was randomised between Roche cobas4800 and Cepheid Xpert assays with Qiagen hc2 third. Results: 3408 cases were included in the primary analysis. Positivity for Xpert was 19.6%, cobas 19.2% and hc2 19.9% with high concordance (kappa=86.8% vs cobas, 81.55 vs hc2. Xpert, cobas and hc2 showed similar sensitivity (98.7%, 97.5%, 98.7% for CIN2+. All pairwise comparisons had high concordance (Kappa ≥0.78 with any abnormal cytology. Xpert and hc2 were positive for all cases of ≥moderate dyskaryosis (N=63, cobas was negative in two. Histology was available for 172 participants. 79 reported CIN2+, 47 CIN3+. All CIN3+ was positive on Xpert and hc2 and one case negative for cobas. One case of CIN2 was negative for all assays. Conclusions: The performance of Xpert HPV Assay in a general screening population is comparable to established HPV tests. It offers simplicity of testing, flexibility with non-batching of individual samples and rapid turnaround time. Keywords: Human papillomavirus, Xpert, Cervical screening, HPV testing

  9. Human Papillomavirus Assays and Cytology in Primary Cervical Screening of Women Aged 30 Years and Above

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah

    2016-01-01

    In women aged ≥30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30-65 years in Copenhagen, Denmark....... Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected...... more ≥CIN3 than cytology (HC2: 35, cobas, CLART: 37, APTIMA: 34, cytology: 31), although statistically the differences were not significant. Cobas and CLART detected significantly more ≥CIN2 than cytology (cobas, CLART: 49, cytology: 39). The proportion of women with false-positive test results...

  10. Performance Evaluation and Comparison of the Fully Automated Urinalysis Analyzers UX-2000 and Cobas 6500.

    Science.gov (United States)

    Wesarachkitti, Bongkot; Khejonnit, Varanya; Pratumvinit, Busadee; Reesukumal, Kanit; Meepanya, Suriya; Pattanavin, Chanutchaya; Wongkrajang, Preechaya

    2016-05-01

    To evaluate and compare the performances of the automated urinalysis devices UX-2000 and Cobas 6500. A total of 258 urine specimens were collected from the routine specimen workload. We analyzed all specimens on both automated instruments and recorded the turnaround time from each method. Physical, chemical, and sedimentary urine components were compared between the automated and the manual method for each analyzer. The correlation of urine physical/chemical properties between the 2 instruments was excellent. The Cobas 6500 instrument demonstrated a higher level of agreement for red blood cells (Cobas 6500:R= 0.94; UX-2000:R= 0.78) and white blood cells (Cobas 6500:R= 0.95; UX-2000:R= 0.85). The UX-2000 demonstrated higher sensitivity for small round cells, hyaline casts, pathological casts, and bacteria. The median turnaround time was 1.5 minutes and 8.5 minutes for the Cobas 6500 and UX-2000, respectively. The 2 devices showed similar performance in technical evaluation; they each reduce workload and increase time saving. However, manual examination by technicians is recommended for pathological specimens. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Performance evaluation of the new Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 for quantification of human immunodeficiency virus type 1 RNA

    NARCIS (Netherlands)

    Pas, Suzan; Rossen, John W. A.; Schoener, Daniel; Thamke, Diana; Pettersson, Annika; Babiel, Reiner; Schutten, Martin

    Despite FDA approval and CE marking of commercial tests, manufacturer-independent testing of the technical aspects of newly developed tests is important. To evaluate the analytical performance and explore the clinical applicability of the new Roche COBAS AmpliPrep COBAS TaqMan HIV-1 test, version

  12. Prevalence of high-risk human papillomavirus by cobas 4800 HPV test in urban Peru

    Directory of Open Access Journals (Sweden)

    Ricardo Iwasaki

    2014-09-01

    Conclusion: Our study showed high prevalence of high-risk human papillomavirus in urban Peru, mainly among young women. In both single and multiple infections other high-risk human papillomavirus were more prevalent than HPV 16 and HPV 18, which might influence vaccine impact in our country. Furthermore, the cobas 4800 HPV test may be considered a useful tool for HPV molecular diagnosis.

  13. NT-proBNP on Cobas h 232 in point-of-care testing

    DEFF Research Database (Denmark)

    Gils, Charlotte; Ramanathan, R.; Breindahl, T.

    2015-01-01

    Background. NT-proBNP may be useful for ruling out heart failure in primary health care. In this study we examined the analytical quality of NT-proBNP in primary health care on the Cobas h 232 point-of-care instrument compared with measurements performed in a hospital laboratory. Materials...

  14. Differences between quantification of genotype 3 hepatitis C virus RNA by Versions 1.0 and 2.0 of the COBAS AmpliPrep/COBAS TaqMan HCV Test.

    Science.gov (United States)

    Pierce, Virginia M; Eversley, Jacqueline S; Tran, Thuy K; Rosenberg, Eric S

    2017-06-27

    Differences between the designs of hepatitis C virus (HCV) viral load assays can result in genotype-related variability in RNA quantification. We tested paired aliquots of plasma specimens from HCV-infected individuals using two versions (v1.0 and v2.0) of the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM HCV) and noted variability between results for a subset of specimens; we then sought to determine whether discrepant results were more prevalent among specific HCV genotypes. Archived and prospectively-collected plasma samples from 114 unique patients were tested using CAP/CTM HCV v1.0 and v2.0. The HCV genotype result for each patient was determined by retrospectively reviewing laboratory records. All (46/46) specimens with quantifiable viral loads from patients with genotype 1 or 2 infection had CAP/CTM HCV v1.0 and v2.0 results that were within 0.5 log10 IU/mL; in contrast, only 3/11 (27.3%) from patients with HCV genotype 3 (mean difference, 0.56 log10 IU/mL higher with v2.0) and 0/3 (0%) from patients with HCV genotype 4 (mean difference, 0.91 log10 IU/mL higher with v2.0) had results within 0.5 log10 IU/mL. Among specimens with detectable HCV RNA below the lower limit of quantification with v1.0, greater proportions of genotype 3 (4/7, 57.1%) and genotype 4 (3/4, 75.0%) specimens than genotype 1 or 2 specimens (6/30, 20.0%) had v2.0 results within the quantifiable range. In patients infected with HCV genotype 3, sequential CAP/CTM HCV viral load results should be compared with caution and interpreted in the context of the specific assay version used.

  15. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four...... of considerable disagreement between human papillomavirus assays. This suggested that the extent of disagreement in primary screening is neither population- nor storage media-specific, leaving assay design differences as the most probable cause. The substantially different selection of women testing positive...... on the various human papillomavirus assays represents an unexpected challenge for the choice of an assay in primary cervical screening, and for follow up of in particular HPV positive/cytology normal women....

  16. Prevalence of high-risk human papillomavirus by cobas 4800 HPV test in urban Peru

    Directory of Open Access Journals (Sweden)

    Ricardo Iwasaki

    Full Text Available Background: Molecular tests allow the detection of high-risk human papillomavirus in cervical samples, playing an important role in the prevention of cervical cancer. Objectives: We performed a study to determine the prevalence of HPV 16, HPV 18 and other high-risk human papillomavirus (pool 12 genotypes in Peruvian females from diverse urban areas using the cobas 4800 HPV test. Methods: Routine cervical samples collected in our laboratory were analyzed by cobas 4800 HPV test. Results: A total of 2247 samples from female patients aged 17–79 years were tested. high-risk human papillomavirus was positive in 775 (34.49% samples. Of these, 641 (82.71% were single infections and 134 (17.29% were multiple infections. The positivity rates for HPV 16, HPV 18, and other high-risk human papillomavirus were 10.77%, 2.0%, and 28.08%, respectively. In multiple high-risk human papillomavirus infections, the concomitance of HPV 16 and other high-risk human papillomavirus was more prevalent (13.42%. Conclusion: Our study showed high prevalence of high-risk human papillomavirus in urban Peru, mainly among young women. In both single and multiple infections other high-risk human papillomavirus were more prevalent than HPV 16 and HPV 18, which might influence vaccine impact in our country. Furthermore, the cobas 4800 HPV test may be considered a useful tool for HPV molecular diagnosis.

  17. Prevalence of high-risk human papillomavirus by cobas 4800 HPV test in urban Peru.

    Science.gov (United States)

    Iwasaki, Ricardo; Galvez-Philpott, Felipe; Arias-Stella, Javier; Arias-Stella, Javier

    2014-01-01

    Molecular tests allow the detection of high-risk human papillomavirus in cervical samples, playing an important role in the prevention of cervical cancer. We performed a study to determine the prevalence of HPV 16, HPV 18 and other high-risk human papillomavirus (pool 12 genotypes) in Peruvian females from diverse urban areas using the cobas 4800 HPV test. Routine cervical samples collected in our laboratory were analyzed by cobas 4800 HPV test. A total of 2247 samples from female patients aged 17-79 years were tested. high-risk human papillomavirus was positive in 775 (34.49%) samples. Of these, 641 (82.71%) were single infections and 134 (17.29%) were multiple infections. The positivity rates for HPV 16, HPV 18, and other high-risk human papillomavirus were 10.77%, 2.0%, and 28.08%, respectively. In multiple high-risk human papillomavirus infections, the concomitance of HPV 16 and other high-risk human papillomavirus was more prevalent (13.42%). Our study showed high prevalence of high-risk human papillomavirus in urban Peru, mainly among young women. In both single and multiple infections other high-risk human papillomavirus were more prevalent than HPV 16 and HPV 18, which might influence vaccine impact in our country. Furthermore, the cobas 4800 HPV test may be considered a useful tool for HPV molecular diagnosis. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

  18. Usefulness of nucleic acid testing to reduce risk of hepatitis B virus transfusion-transmitted infection in Argentina: high rate of recent infections.

    Science.gov (United States)

    Blanco, Sebastián; Balangero, Marcos César; Valle, Mildre Cledy; Montini, Oscar Luis; Carrizo, Luis Horacio; Gallego, Sandra Verónica

    2017-03-01

    Results from 10-year experience using nucleic acid test (NAT) screening in a blood bank of Córdoba are presented, showing the first data on prevalence of recent hepatitis B virus (HBV) infections and occult HBV infections (OBIs) in Argentina. Molecular screening was performed by COBAS AmpliScreen human immunodeficiency virus Type 1 (HIV-1) test Version 1.5 and COBAS AmpliScreen hepatitis C virus (HCV) test Version 2.0 and COBAS TaqScreen MPX and MPX Version 2.0 test (Roche Molecular Systems). To characterize OBI, additional molecular and serologic assays were performed. As results of NAT, 0.075% of the donors (155/205,388) tested positive for HIV, 0.05% (106/205,388) for HCV, and 0.045% (76/168,215) for HBV. Donors who tested positive for HIV or HCV by NAT were also positive by serology. There was one of 33,643 donors recently infected with HBV. At time of donation, six of 76 (7.9%) donors with confirmed HBV infection presented virologic and serologic profiles consistent with OBI. By additional studies three were OBI, two were window period infections, and one remained unclassified. NAT contributed significantly to the reduction of the potential risk of HBV transmission with a frequency of one in 56,072, detecting three in 168,215 donors without serologic evidence of infection. NAT also detected three in 168,215 OBIs. The finding of high frequency of recent infections (1/33,643), unexpected for this country, highlights the need of promoting unified effective regulations that enforce the use of NAT in all blood banks in Argentina and points out the importance of assessing the risk of HBV transmission in blood banks of other countries considered to be low-endemic. © 2016 AABB.

  19. The cobas® 6800/8800 System: a new era of automation in molecular diagnostics.

    Science.gov (United States)

    Cobb, Bryan; Simon, Christian O; Stramer, Susan L; Body, Barbara; Mitchell, P Shawn; Reisch, Natasa; Stevens, Wendy; Carmona, Sergio; Katz, Louis; Will, Stephen; Liesenfeld, Oliver

    2017-02-01

    Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

  20. Clinical Evaluation of COBAS TaqMan PCR for the Detection of Mycobacterium tuberculosis and M. avium Complex

    Science.gov (United States)

    Ikegame, Satoshi; Sakoda, Yoritake; Fujino, Nao; Taguchi, Kazuhito; Kawasaki, Masayuki; Kajiki, Akira

    2012-01-01

    A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation. PMID:23029612

  1. Clinical Evaluation of COBAS TaqMan PCR for the Detection of Mycobacterium tuberculosis and M. avium Complex

    Directory of Open Access Journals (Sweden)

    Satoshi Ikegame

    2012-01-01

    Full Text Available A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87 were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P<0.001 lower than that of smear 2+ (97.3%. From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74; M. avium, 21; M. intracellulare, 43, which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation.

  2. Performance of Aptima and Cobas HPV testing platforms in detecting high-grade cervical dysplasia and cancer.

    Science.gov (United States)

    Ge, Yimin; Christensen, Paul; Luna, Eric; Armylagos, Donna; Schwartz, Mary R; Mody, Dina R

    2017-08-01

    Human papillomavirus (HPV) tests and genotyping have been used in clinical risk assessment. The purpose of this study was to analyze the performance of 2 common HPV testing platforms in detecting high-grade cervical lesions (high-grade squamous intraepithelial lesion [HSIL] or worse [≥HSIL]). Between January 1 and December 31, 2015, 2041 Papanicolaou (Pap) tests with biopsy confirmation were analyzed along with HPV tests performed on Cobas or Aptima platforms. A biopsy diagnosis of grade 2 cervical intraepithelial neoplasia was confirmed with p16/Ki-67 immunohistochemistry. In total, 1866 and 175 Pap cases were tested on Cobas and Aptima platforms, respectively. Both platforms were highly sensitive (97% for both) for biopsy-confirmed ≥HSIL. Cobas HPV testing had higher positive rates for the diagnosis of benign lesions (84% vs 51%) and low-grade squamous intraepithelial lesions (89% vs 63%) on biopsy compared with Aptima. Aptima testing had significantly higher specificity for ≥HSIL than Cobas (41% vs 13%; P cervical lesions, Aptima HPV testing demonstrated significantly higher specificity and positive predictive value than Cobas testing for biopsy-confirmed ≥HSIL. The considerable difference may be related to the significant increase in E6/E7 expression after HPV DNA integration. The significantly higher specificity and overall accuracy of Aptima testing for ≥HSIL, resulting in the identification of high-risk populations that require immediate treatment and close follow-up, may prove useful in clinical risk stratification. Cancer Cytopathol 2017;125:652-7. © 2017 American Cancer Society. © 2017 American Cancer Society.

  3. TELAAH SOAL UJI COBA I BAHASA INDONESIA DI SMP KABUPATEN WONOSOBO TAHUN 2013

    Directory of Open Access Journals (Sweden)

    Mushoffa Mushoffa

    2015-10-01

    Full Text Available Penelitian ini bertujuan untuk mendeskripsikan ketepatan indikator dalam kisi-kisi soal, keterwakilan setiap kompetensi dasar dalam kisi-kisi soal, kesesuaian antara kisi-kisi soal dan soal, serta capaian daya serap setiap kompetensi dasar. Penelitian ini merupakan penelitian deskriptif kualitatif-kuantitatif. Subjek penelitian ini adalah kisi-kisi soal, soal, dan capaian daya serap dari soal uji coba I bahasa Indonesia di SMP kabupaten Wonosobo tahun 2013. Pengumpulan data dilakukan dengan teknik baca dan catat. Hasil penelitian ini adalah sebagai berikut: (1 ketepatan indikator dalam kisi-kisi soal termasuk kategori tepat (86,4% dengan rincian ketepatan indikator pada kategori sangat tepat sebesar 22%, tepat sebesar 68%, cukup tepat sebesar 8%, kurang tepat sebesar 2%, dan tidak tepat sebesar 0%; (2 keterwakilan setiap kompetensi dasar dalam kisi-kisi soal uji coba I bahasa Indonesia Kabupaten Wonosobo dalam kategori sangat terwakili (96,7%; (3 kesesuaian antara kisi-kisi soal dan soal uji coba I bahasa Indonesia Kabupaten Wonosobo tahun 2013 termasuk kategori sesuai (86%; dan capaian daya serap untuk seluruh kompetensi dasar dalam soal uji coba I bahasa Indonesia Kabupaten Wonosobo tahun 2013 termasuk kategori rendah yaitu sebesar 56,89% dengan rincian capaian pada kategori sangat tinggi sebesar 0%, tinggi sebesar 14%,  sedang sebesar 24%, rendah sebesar 30%, dan sangat rendah sebesar 32%. Kata Kunci: telaah, kisi-kisi soal, soal, daya serap   THE ANALYSIS OF THE FIRST TRY OUT OF INDONESIAN LANGUAGE TEST IN JUNIOR HIGH SCHOOLS OF WONOSOBO REGENCY IN 2013 Abstract This study aims to describe the accuracy of the indicators in the table of specifications, the representation of each basic competence in the table of specifications, the compatibility between table of specifications and test items, and the learning outcomes performance of each basic competence of the test items. This research is qualitative-quantitative descriptive. The subjects

  4. Comparison of Architect i2000sr and Cobas e601 Systems for Determining Serum Human Chorionic Gonadotropin-Beta.

    Science.gov (United States)

    Guan, Xiaoyong; Sun, Yifan; Zhang, Hongyu; Liang, Ka; Long, Kang; Li, Jin; Tang, Shifu; Liu, Chunming

    2016-09-01

    Human chorionic gonadotropin-beta (β-hCG) is an important index used to monitor embryonic development following embryo transfer. Architect i2000sr and Cobas e601 are widely used automated immunoassay systems used to measure serum β-hCG concentrations; however, the correlations between serum β-hCG levels measured with these two immunoassays and the accuracy of the immunoassays have not been fully evaluated. Serum β-hCG levels were measured in 133 serum samples using the Architect i2000sr and Cobas e601 automated immunoassay systems. Passing-Bablok regression analysis was used to compare the correlation in serum β-hCG levels obtained using the two immunoassays. A Bland-Altman plot analysis was used to identify mean ratios and 95% CIs of the mean ratios of the β-hCG results between the two immunoassays. In this graphical method the mean ratios between the two techniques were plotted against the averages of the two techniques. The total coefficients of variations (CVs) of serum β-hCG ranged from 3.12 - 4.66% for Cobas e601 and 3.18 - 4.99% for Architect i2000sr. The measured value of serum β-hCG detected by the two immunoassays was statistically significant (p 10000 IU/L, n = 81), the correlation coefficient r was 0.8076. The Bland-Altman plot analysis showed that the measured value of serum β-hCG detected by Architect i2000sr was about 1.25 times higher than that of Cobas e601. The mean ratio was 1.12 at a low concentration of serum β-hCG, and it was 1.33 at a high concentration. Architect i2000sr and Cobas e601 have good concordance for determining serum β-hCG. However, the β-hCG values measured with Architect i2000sr were 25% higher than those obtained using Cobas e601.

  5. A Comparison of the Roche Cobas HPV Test With the Hybrid Capture 2 Test for the Detection of High-Risk Human Papillomavirus Genotypes

    National Research Council Canada - National Science Library

    Levi, Angelique W; Bernstein, Jane I; Hui, Pei; Duch, Kara; Schofield, Kevin; Chhieng, David C

    2016-01-01

    .... To compare the performance of the Roche cobas and Hybrid Capture 2 tests for the detection of high-risk human papillomavirus using both ThinPrep and SurePath preparations as part of a validation study...

  6. Comparison of hybrid capture 2 High-Risk HPV results in the low positive range with cobas® HPV Test results from the ATHENA study.

    Science.gov (United States)

    Rao, Arundhati; Sandri, Maria Teresa; Sideri, Mario; Young, Stephen; Sharma, Abha; Behrens, Catherine

    2013-09-01

    The increasing importance of high-risk human papillomavirus (hrHPV) testing in cervical cancer screening warrants evaluation of HPV DNA tests with an equivocal zone requiring retesting of samples in the low positive range. To compare the results of the digene hc2 High Risk HPV DNA Test (hc2), which has a manufacturer's recommended retesting zone with the cobas HPV Test, a real-time polymerase chain reaction amplification test without an equivocal range. A retrospective subanalysis of the ATHENA study comparing results for hc2 High Risk HPV DNA Test and the cobas HPV Test using the LINEAR ARRAY HPV Genotyping Test (LA) and Sanger sequencing as comparators was performed. The ability of each test to detect high-grade cervical disease in the equivocal range was also evaluated. 5.2% of samples fell within the equivocal zone (RLU/CO 1.0-2.5) and required retesting with the hc2 High Risk HPV DNA Test. In this low-positive range the cobas HPV Test showed better positive percent agreement (PPA) than hc2 High Risk HPV DNA Test for LA and sequencing (84.2% vs.70.9% and 92.1% vs.82.5%, respectively). hc2 High Risk HPV DNA Test and the cobas HPV Test demonstrated comparable sensitivity for detection of high-grade disease in the equivocal range. In the low cobas HPV Test range (cycle threshold [Ct] 40-35), the cobas HPV test again demonstrated a better PPA than hc2 High Risk HPV DNA Test with LA and sequencing as comparators and more high-grade disease was detected by the cobas HPV Test than hc2 High Risk HPV DNA Test. The cobas HPV Test demonstrates reliable performance in the hc2 High Risk HPV DNA Test equivocal zone, thus supporting it as an option for HPV testing that avoids the need for retesting. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Comparison of cobas 4800, m2000, Viper XTR, and Infinity 80 Automated Instruments When Processing Urine Specimens for the Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae.

    Science.gov (United States)

    Chernesky, Max A; Jang, Dan; Gilchrist, Jodi; Smieja, Marek; Arias, Manuel; Hatchette, Todd; Poirier, Andre; Mayne, Donna; Ratnam, Sam

    2017-03-01

    North American and European advisory groups recommend testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) with nucleic acid amplification tests. Testing is often performed on automated instruments. The objectives of this study were to process urines for the diagnosis of CT and NG and to examine workflow procedures and outcomes. While processing 1, 24, 48, 96, and 192 urine specimens on 3 batch-mode systems which use 96-well plates: cobas 4800, m2000, and Viper XTR and the random access cartridge testing GeneXpert Infinity 80, we measured assay performance, hands-on time for processing and maintenance, reagents and plastics consumption, time required to obtain results, and testing accuracy. The Infinity 80 required the least hands-on time for single specimens and smaller batches, whereas the Viper XTR and m2000 required the most hands-on time for all batch sizes. Cumulative daily, weekly, and monthly maintenance was highest for the Viper XTR and lowest for Infinity 80. All batch-mode instruments consumed large amounts of disposables. Time to results was shortest for the Infinity 80, and the Viper XTR provided the shortest time for the batch-mode instruments. All systems showed similar diagnostic accuracy. Because detection performances were similar, issues of hands-on time, maintenance, time to results, and consumables are important operational factors for the diagnosis and treatment of CT/NG infections.

  8. Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

    OpenAIRE

    Sattler, I; Roessner, C A; Stolowich, N J; Hardin, S H; Harris-Haller, L W; Yokubaitis, N T; Murooka, Y; Hashimoto, Y; Scott, A I

    1995-01-01

    We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N...

  9. [Doctor Manuel Cobas Núñez (1885-1956), pioneer of urology and uroradiology in Galicia].

    Science.gov (United States)

    Fariña Pérez, L A

    2004-02-01

    The development of urologic radiology with intracavitary and intravenous contrast agents played an essential role in the rise of Urology as a surgical speciality, in agreement with other circumstances that made it both possible (the invention of endoscopic instruments and laboratory technics to study the urine and the kidney function, advances in anaesthesia, asepsis, control of hemorrhage and infection), and necessary (because of social request for these specialised medical services). Study of the life and work of Manuel Cobas Núñez (1885-1956), whose surgical practice and publications pioneered the modern Urology and Uroradiology in Galicia (Spain). After being trained in Necker Hospital (Paris) and Instituto Rubio (Madrid), Cobas was one of the first to practice urology in Galicia, with a high interest in surgical diseases of the upper urinary tract. His knowledge of renoureteral surgery was outstanding for the time. In august 1931 he published "Pielografia ascendente y pielografia descendente", one of the first and best documented books on upper urinary tract examination using retrograde and intravenous pyelography, written only months after marketing of the first intravenous contrasts agents. Working in private practice, out of the academic fields, Cobas was a protagonists of the beginning of Galician modern urology. His publications and book on pyelography show that he was one of the first experts in renal surgery and must be also recognized as a pioneer of uroradiology in Galicia and Spain.

  10. Comparison of three human papillomavirus DNA assays and one mRNA assay in women with abnormal cytology

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Lynge, Elsebeth; Ejegod, Ditte

    2014-01-01

    OBJECTIVE: To compare the clinical characteristics of four human papillomavirus (HPV) assays: hybrid capture 2 (HC2), cobas, CLART, and APTIMA in Danish women with abnormal cytology. METHODS: SurePath samples from 367 consecutive women from Copenhagen, with atypical squamous cells of undetermined...... between 22% and 35%. Similar results were seen for ≥CIN2. CONCLUSIONS: Small differences in clinical characteristics were found for the four HPV assays in Danish women with abnormal cytology aged ≥30 years. At younger ages, APTIMA was somewhat less sensitive for high-grade CIN than the three HPV DNA...

  11. CLSI-Based Validation of Manufacturer-Derived Reference Intervals on the Cobas 8000 Platform.

    Science.gov (United States)

    Leitner-Ferenc, Veronika; Atamaniuk, Johanna; Jansen-Skoupy, Sonja; Stöckelmeier, Brigitta; Grohs, Katharina; Födinger, Manuela

    2017-05-01

    Reference intervals provided by diagnostic test manufacturers should be transferred to clinical laboratories after validation. Although protocols exist, laboratories rarely perform and report on results of validation studies. We validated reference intervals (RIs) of 87 analytes on a Cobas 8000 platform according to standards published by the Clinical and Laboratory Standards Institute (CLSI). For 8 analytes, decision limits were provided in the package inserts. Among the 79 RIs subjected to transference validation, 8 were found not valid for transference, including lactate dehydrogenase (LDH) among women, and the following among both sexes: potassium, homocysteine, immunoglobulin E (IgE), free lambda light chain (FLC λ), C3 complement (C3c), folate, and 25-hydroxy vitamin D (25[(OH]D). For LDH, potassium, homocysteine, C3c, folate, and 25(OH)D, RIs or thresholds suitable for transference were available in the literature; however, this was not the case for IgE and FLC λ. The present study demonstrates that validation of RIs provided in the manufacturer provided package inserts is indispensable.

  12. Evaluation of clinical sensitivity and specificity of hepatitis B virus (HBV), hepatitis C virus, and human immunodeficiency Virus-1 by cobas MPX: Detection of occult HBV infection in an HBV-endemic area.

    Science.gov (United States)

    Ha, Jihye; Park, Younhee; Kim, Hyon-Suk

    2017-09-21

    Transfusion-transmitted infectious diseases remain a major concern for blood safety, particularly with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Nucleic acid testing (NAT) in donor screening shortens the serologically negative window period and reduces virus transmission. The cobas MPX (Roche Molecular Systems, Inc., Branchburg, New Jersey) is a recently developed multiplex qualitative PCR system that enables the simultaneous detection of HBV, HCV, and HIV with improved sensitivity and throughput using cobas 6800 and 8800 instruments. The aim of this study was to conduct an evaluation of the clinical sensitivity and specificity of cobas MPX detection of HBV, HCV, and HIV in clinical specimens. Among samples referred for HBV, HCV, and HIV-1 quantification at Severance Hospital, Yonsei University College of Medicine, positive samples were selected to evaluate sensitivity. A total of 843 samples was tested using both cobas MPX and COBAS AmpliPrep/COBAS TaqMan Tests for HBV, HCV, and HIV-1 using the cobas 8800 system and a COBAS TaqMan 96 analyzer, respectively. Samples that showed discrepancies were confirmed by nested PCR. The cobas MPX achieved excellent sensitivity and specificity for the detection of HBV, HCV, and HIV-1 in clinical samples. We found that the lower limit of detection (LOD) of blood screening by NAT actually improves clinical sensitivity, and occult HBV infection prevalence among healthy employees of the hospital was rather high. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Multicenter evaluation of a new automated fourth-generation human immunodeficiency virus screening assay with a sensitive antigen detection module and high specificity.

    Science.gov (United States)

    Weber, Bernard; Gürtler, Lutz; Thorstensson, Rigmor; Michl, Ulrike; Mühlbacher, Annelies; Bürgisser, Philippe; Villaescusa, Roberto; Eiras, Adolfo; Gabriel, Christian; Stekel, Herbert; Tanprasert, Srivilai; Oota, Sinenaart; Silvestre, Maria-Jose; Marques, Cristina; Ladeira, Maria; Rabenau, Holger; Berger, Annemarie; Schmitt, Urban; Melchior, Walter

    2002-06-01

    Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody that were available on the international market until now have antigen detection modules with relatively poor sensitivity and produce a higher rate of false-positive results than third-generation enzyme immunoassays (EIAs). The new Cobas Core HIV Combi EIA with an improved sensitivity for HIV p24 antigen was compared to alternative fourth- and third-generation assays, the p24 antigen test, and HIV type 1 (HIV-1) RNA reverse transcriptase PCR (RT-PCR). A total of 94 seroconversion panels (n = 709 sera), samples from the acute phase of infection after seroconversion (n = 32), anti-HIV-1-positive specimens (n = 730) from patients in different stages of the disease, 462 subtyped samples from different geographical locations, anti-HIV-2-positive sera (n = 302), dilutions of cell culture supernatants (n = 62) from cells infected with different HIV-1 subtypes, selected performance panels from Boston Biomedica Inc., 7,579 unselected samples from blood donors, 303 unselected daily routine samples, 997 specimens from hospitalized patients, and potentially interfering samples (n = 1,222) were tested with Cobas Core HIV Combi EIA. The new assay showed a sensitivity comparable to that of the Abbott HIV-1 AG Monoclonal A for early detection of HIV infection in seroconversion panels. The mean time delay of Cobas Core HIV Combi EIA (last negative sample plus 1 day) in comparison to that for HIV-1 RT-PCR for 87 panels tested with both methods was 2.75 days. The diagnostic window was reduced with Cobas Core HIV Combi EIA by between 3.6 and 5.7 days from that for third-generation assays. The specificities of Cobas Core HIV Combi EIA in blood donors were 99.84 and 99.85% (after repeated testing). Overall, 30 repeatedly reactive false-positive results out of 10,031 HIV-negative samples were obtained with Cobas Core HIV Combi EIA. Our results show that a fourth

  14. Performance of the cobas HPV Test for the Triage of Atypical Squamous Cells of Undetermined Significance Cytology in Cervical Specimens Collected in SurePath.

    Science.gov (United States)

    Tewari, Devansu; Novak-Weekley, Susan; Hong, Christina; Aslam, Shagufta; Behrens, Catherine M

    2017-11-02

    Determine performance of the cobas human papillomavirus (HPV) test for triage of atypical squamous cells of undetermined significance (ASC-US) in SurePath. Women presenting for routine screening had cervical specimens collected in SurePath and specimen transport medium (STM); those with ASC-US cytology underwent colposcopy. Performance of cobas HPV in SurePath specimens that had undergone a preanalytic procedure to reverse possible cross-linking of HPV DNA was compared with Hybrid Capture 2 (hc2) specimens in STM. Among 856 women, HPV prevalence was 45.8%; HPV 16 and HPV 18 prevalences were lower than expected in the 21- to 29-year-old group in this highly vaccinated population. cobas HPV performance in SurePath was comparable to hc2 in STM. Sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 3 or worse were 87.5% (95% confidence interval [CI], 71.9%-95.2%) and 55.5% (95% CI, 52.1%-58.9%) for cobas and 85.3% (95% CI, 69.9%-93.6%) and 54.7% (95% CI, 51.4%-57.9%) for hc2. Sensitivity was negatively affected by random biopsies performed at colposcopy; comparable sensitivities were achieved in the nonvaccinated and vaccinated populations with disease determined by directed biopsy only. Performance of cobas HPV for ASC-US triage in pretreated SurePath specimens meets criteria for validation. Preliminary data indicate reliable performance of HPV testing in a highly vaccinated population.

  15. Comparison of Cobas 6500 and Iris IQ200 fully-automated urine analyzers to manual urine microscopy

    Science.gov (United States)

    Bakan, Ebubekir; Ozturk, Nurinnisa; Baygutalp, Nurcan Kilic; Polat, Elif; Akpinar, Kadriye; Dorman, Emrullah; Polat, Harun; Bakan, Nuri

    2016-01-01

    Introduction Urine screening is achieved by either automated or manual microscopic analysis. The aim of the study was to compare Cobas 6500 and Iris IQ200 urine analyzers, and manual urine microscopic analysis. Materials and methods A total of 540 urine samples sent to the laboratory for chemical and sediment analysis were analyzed on Cobas 6500 and Iris IQ200 within 1 hour from sampling. One hundred and fifty three samples were found to have pathological sediment results and were subjected to manual microscopic analysis performed by laboratory staff blinded to the study. Spearman’s and Gamma statistics were used for correlation analyses, and the McNemar test for the comparison of the two automated analyzers. Results The comparison of Cobas u701 to the manual method yielded the following regression equations: y = - 0.12 (95% CI: - 1.09 to 0.67) + 0.78 (95% CI: 0.65 to 0.95) x for WBC and y = 0.06 (95% CI: - 0.09 to 0.25) + 0.66 (95% CI: 0.57 to 0.73) x for RBC. The comparison of IQ200 Elite to manual method the following equations: y = 0.03 (95% CI: - 1.00 to 1.00) + 0.88 (95% CI: 0.66 to 1.00) x for WBC and y = - 0.22 (95% CI: - 0.80 to 0.20) + 0.40 (95% CI: 0.32 to 0.50) x for RBC. IQ200 Elite compared to Cobas u701 yielded the following equations: y = - 0.95 (95% CI: - 2.13 to 0.11) + 1.25 (95% CI: 1.08 to 1.44) x for WBC and y = - 1.20 (95% CI: - 1.80 to -0.30) + 0. 80 (95% CI: 0.55 to 1.00) x for RBC. Conclusions The two analyzers showed similar performances and good compatibility to manual microscopy. However, they are still inadequate in the determination of WBC, RBC, and EC in highly-pathological samples. Thus, confirmation by manual microscopic analysis may be useful. PMID:27812305

  16. Histopathological analysis of UV-B irradiated retina by Cavia Coba'ya and with protection of transmission light lambda 550-600 nm.

    Science.gov (United States)

    Vojniković, Bozidar; Vojniković, Davor; Brcić, Luka

    2007-01-01

    In 14 experimental Cavia Coba'ya eyes were irradiated with UV-B light, lambda 312 nm, 25 J/cm2 in 15 minute exposure. Including the transmission of light through optical media: cornea, lens, humor aqueous and vitreous body, and pupil surface of 7 mm2, we can calculate that in these conditions retina can be really irradiated with 10 J/cm2. The half number of Cavia Coba'ya was simultaneously irradiated with visible light, lambda of 550-600 nm (1000 Lx). Control group was 5 Cavia Coba'ya. Two months after irradiation, eyes were enucleated and fixed in 4% formaldehyde. Histopathological findings showed alterations of all retinal layers: loss of ganglion cells, axons, reduction of photoreceptors, vacuolar degeneration and hyperplasia of retinal pigment epithelium. In the second group of irradiance, the eyes with visible light lambda 550-600 nm, all retinal alterations were in 50% decreased.

  17. EUROarray human papillomavirus (HPV) assay is highly concordant with other commercial assays for detection of high-risk HPV genotypes in women with high grade cervical abnormalities.

    Science.gov (United States)

    Cornall, A M; Poljak, M; Garland, S M; Phillips, S; Machalek, D A; Tan, J H; Quinn, M A; Tabrizi, S N

    2016-06-01

    The purpose of this study was to evaluate the performance of the EUROIMMUN EUROArray HPV genotyping assay against the Roche Cobas 4800, Roche HPV Amplicor, Roche Linear Array and Qiagen Hybrid Capture 2 assays in the detection of high-risk HPV (HR-HPV) from liquid based cervical cytology samples collected from women undergoing follow-up for abnormal cervical cytology results. Cervical specimens from 404 women undergoing management of high-grade cytological abnormality were evaluated by EUROarray HPV for detection of HR-HPV genotypes and prediction of histologically-confirmed cervical intraepithelial neoplasia grade 2 or higher (≥CIN2). The results were compared to Hybrid Capture 2, Cobas 4800 HPV, Amplicor and Linear Array HPV. Positivity for 14 HR-HPV types was 80.0 % for EUROarray (95 % CI; 75.7-83.8 %). Agreement (κ, 95 % CI) between the EUROarray and other HPV tests for detection of HR-HPV was good to very good [Hybrid Capture κ = 0.62 (0.54-0.71); Cobas κ = 0.81 (0.74-0.88); Amplicor κ = 0.68 (0.60-0.77); Linear Array κ = 0.77 (0.70-0.85)]. For detection of HR-HPV, agreement with EUROarray was 87.90 % (Hybrid Capture), 93.58 % (Cobas), 92.84 % (Amplicor) and 92.59 % (Linear Array). Detection of HR-HPV was not significantly different between EUROarray and any other test (p detection of high-risk HPV and showed sensitivity and specificity for detection of ≥ CIN2 of 86 % and 71 %, respectively.

  18. Multicenter Evaluation of a New Automated Fourth-Generation Human Immunodeficiency Virus Screening Assay with a Sensitive Antigen Detection Module and High Specificity

    OpenAIRE

    Weber, Bernard; Gürtler, Lutz; Thorstensson, Rigmor; Michl, Ulrike; Mühlbacher, Annelies; Bürgisser, Philippe; Villaescusa, Roberto; Eiras, Adolfo; Gabriel, Christian; Stekel, Herbert; Tanprasert, Srivilai; Oota, Sinenaart; Silvestre, Maria-Jose; Marques, Cristina; Ladeira, Maria

    2002-01-01

    Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody that were available on the international market until now have antigen detection modules with relatively poor sensitivity and produce a higher rate of false-positive results than third-generation enzyme immunoassays (EIAs). The new Cobas Core HIV Combi EIA with an improved sensitivity for HIV p24 antigen was compared to alternative fourth- and third-generation assays, the p24 ant...

  19. Enzymatic assay for methotrexate in erythrocytes

    DEFF Research Database (Denmark)

    Schrøder, H; Heinsvig, E M

    1985-01-01

    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...... limit was 3 nmol/l. Within run and between-run precision was 7.4% and 13.5% for control 10 nmol/l and 1.2% and 3.2% for control 50 nmol/l. Recovery was 85-115% of MTX added to haemolysed erythrocytes. We found the method useful for pharmacokinetic studies of MTX in erythrocytes in MTX-treated patients...

  20. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  1. Irreproducible positive results on the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test are different qualitatively from confirmed positive results.

    Science.gov (United States)

    Maritz, Jean; van Zyl, Gert U; Preiser, Wolfgang

    2014-01-01

    Criteria that define low positive results on the COBAS® AmpliPrep/COBAS® TaqMan (CAP/CTM) HIV-1 Qual test as inconclusive have been adopted by all academic centres in South Africa that conduct infant HIV PCR, following previous investigations that showed poor specificity of these results. Retesting all inconclusive specimens has considerable cost implications. Therefore, it was attempted to characterise such inconclusive results, by comparing those that prove to be either negative or positive on follow-up testing. This retrospective, laboratory-based study found that 193 of 211 (91.5%) patients with previous inconclusive results (defined as reported positive by CAP/CTM but with cycle threshold [Ct ] values of >32 and/or fluorescence intensity [FI] values of positive using independently obtained follow-up samples after a median of 28 days. The only significant independent predictor of a later positive result was a higher FI value (3.326 vs. 0.495, P positives. As the lower FI values in false-positive compared to true-positive results probably are indicative of a non-specific signal, the incorporation of stringent amplification slope criteria in the assay's test definition file may improve correct classification and thus reduce the need for repeat testing of a large number of inconclusive specimens. © 2013 Wiley Periodicals, Inc.

  2. Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

    Science.gov (United States)

    Sattler, I; Roessner, C A; Stolowich, N J; Hardin, S H; Harris-Haller, L W; Yokubaitis, N T; Murooka, Y; Hashimoto, Y; Scott, A I

    1995-03-01

    We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.

  3. Detection of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues by the New Automated Abbott RealTime MTB Assay.

    Science.gov (United States)

    Fu, Yung-Chieh; Liao, I-Chuang; Chen, Hung-Mo; Yan, Jing-Jou

    2016-07-01

    The Abbott RealTime MTB assay, launched in June 2014, has been shown to have a competitive performance in the detection of the Mycobacterium tuberculosis (MTB) complex in respiratory specimens. The present study was conducted to investigate the usefulness of the Abbott MTB Realtime assay in the detection of MTB in formalin-fixed paraffin-embedded (FFPE) tissues. A total of 96 FFPE specimens obtained from microbiologically proven MTB cases (N=60) and nontuberculous Mycobacterium cases (N=36) were analyzed. The performance of the Abbott MTB Realtime assay was compared with that of the Roche Cobas TaqMan MTB assay. The overall sensitivity and specificity of the Abbott assay were 63.3% and 97.2%, respectively, compared with 11.7% and 100% for the Cobas assay. The detection rate of the Abbott assay was much higher among 37 acid-fast-positive specimens than among 23 acid-fast-negative specimens (89.3% versus 21.7%, respectively). The detection rate of the assay was higher among 29 resection specimens than among 31 small biopsy specimens (86.2% versus 41.9%, respectively). Our results suggest that the Abbott RealTime MTB assay can be used to differentiate MTB from nontuberculous mycobacterial infections in acid-fast-positive FFPE tissues. © 2016 by the Association of Clinical Scientists, Inc.

  4. Quantification of human papilloma virus (HPV) DNA using the Cobas 4800 system in women with and without pathological alterations attributable to the virus.

    Science.gov (United States)

    Álvarez-Argüelles, Marta Elena; de Oña-Navarro, María; Rojo-Alba, Susana; Torrens-Muns, Magdalena; Junquera-Llaneza, Maria Luisa; Antonio-Boga, Jose; Pérez-Castro, Sonia; Melón-García, Santiago

    2015-09-15

    Surrogate markers such as viral load are necessary to follow the evolution of disease resulting from infection with Human Papilloma virus (HPV), especially in this era of vaccination. As such, this paper uses the automated system Cobas-4800-HPV to define viral load as number of HPV copies/cell and apply the results to clinical samples. A curve to determine viral load per cell was constructed from HPV plasmid and cell concentrations using the Cobas-4800-HPV system. According to these curves, HPV viral load was determined in 309 positive endocervical swabs (58 from patients with previous HPV-infection, 118 with current lesions and 133 symptom-free patients presenting for screening) from women attending gynaecology consultations from January to June 2013. In curves with r(2)≥0.95 the Cobas-4800-HPV system has a detection limit of 150 (2.18 log) viral copies, and the limit for β-globin corresponds to that of a single cell. In women reporting for screening, viral load was under 10(4) (4 log) copies/10(3) cells. For women with lesions or previous HPV infection loads were significantly higher particularly in the 30-45 year group (p=0.038). Elevated viral loads were especially noticeable in non-HPV 16/HPV 18. Automated system Cobas-4800-HPV is suitable for define viral load of HPV. Correlation between viral load and number of cells established. Higher viral load in women with disease, and those between 30 and 45 years. Increased viral load of non-16/18 high-risk HPV genotypes detected in patients with lesions compared to screening patients. A difference not observed for HPV 16/18, or in coinfections. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Can Measurement of Progesterone, Estradiol, and Prolactin by Immunoassay be Interchanged? A Comparison of the Roche Cobas e601 vs. Abbott Architect i2000sr.

    Science.gov (United States)

    Yin, Lianli; Chen, Xiang; Tang, Yinghua; Sun, Yifan

    2017-03-01

    Progesterone is a reliable indicator of either natural or induced ovulation, and it plays an important role in preparing for implantation in the uterus and maintaining pregnancy. Estradiol is the most powerful natural estrogen in humans. It adjusts reproductive function in females and, with progesterone, maintains a pregnancy. Prolactin is also an important indicator, and its major physiological action is the initiation and maintenance of lactation in women. Architect i2000sr and Cobas e601 are automated immunoassay systems that are widely used to measure progesterone, estradiol, and prolactin concentrations in the blood. However, there is a dearth of confidence in these methods for comparative research. Therefore, the aim of this study is to investigate the correlation of serum progesterone, estradiol, and prolactin results measured with Architect i2000sr and Cobas e601. Two hundred venous blood samples from routine serum progesterone, estradiol, and prolactin tests were analyzed on the Cobas e601 and the Architect i2000sr in our laboratory within the same day. Passing-Bablok regression analysis and a Bland-Altman plot were used to compare methods. According to the concordance correlation coefficient, the correlation was strong in estradiol, but the correlation of prolactin and progesterone was poor between the two systems. The Bland-Altman plots showed that the measured value of progesterone, estradiol, and prolactin detected by Cobas e601 were about 1.30, 1.24, and 1.10 times higher, respectively, than that measured using Architect i2000sr. The results of progesterone, estradiol, and prolactin of one method should not be directly transferable to the other.

  6. Enzyme assays

    OpenAIRE

    Bisswanger, Hans

    2014-01-01

    The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Neverthele...

  7. Paraprotein interference with turbidimetric gentamicin assay.

    Science.gov (United States)

    Dimeski, Goce; Bassett, Kendra; Brown, Nigel

    2015-01-01

    Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays. A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser. The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery. IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol.

  8. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  9. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik

    2014-01-01

    samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... of mutations in exons 18-21 of the EGFR gene, employing the cobas(®) EGFR Tissue Test and cobas(®) EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). RESULTS: Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  10. Accuracy of self-collected vaginal dry swabs using the Xpert human papillomavirus assay.

    Directory of Open Access Journals (Sweden)

    Rosa Catarino

    Full Text Available Polymerase chain reaction-based Xpert human papillomavirus (HPV assay is a rapid test that detects high-risk HPV (hrHPV infection. This point-of-care test is usually performed by collecting a cervical specimen in a vial of PreservCyt® transport medium. We compared HPV test positivity and accuracy between self-collected sample with a dry swab (s-DRY versus physician-collected cervical sampling using a broom like brush and immediate immersion in PreservCyt (dr-WET.In this cross-sectional study, we recruited 150 women ≥ 18 years old attending the colposcopy clinic in the University Hospital of Geneva. Each participant first self-collected a vaginal sample using a dry swab and then the physician collected a cervical specimen in PreservCyt. HPV analysis was performed with Xpert. Part of the PreservCyt-collected sample was used for hrHPV detection with the cobas® HPV test. HPV test positivity and performance of the two collection methods was compared.HPV positivity was 49.1% for s-DRY, 41.8% for dr-WET and 46.2% for cobas. Good agreement was found between s-DRY and dr-WET samples (kappa±Standard error (SE = 0.64±0.09,, particularly for low-grade squamous intraepithelial lesions (LSIL+ (kappa±SE = 0.80±0.17. Excellent agreement was found between the two samples for HPV16 detection in general (kappa±SE = 0.91±0.09 and among LSIL+ lesions (kappa±SE = 1.00±0.17. Sensitivities and specificities were, respectively, 84.2% and 47.1%(s-DRY, 73.1% and 58.7%. (dr-WET and 77.8% and 45.7% (cobas for CIN2+ detection. The median delay between sampling and HPV analysis was 7 days for the Xpert HPV assay and 19 days for cobas. There were 36 (24.0% invalid results among s-DRY samples and 4 (2.7% among dr-WET (p = 0.001. Invalid results happened due to the long interval between collection and analysis.Self-collected vaginal dry swabs are a valid alternative to collecting cervical samples in PreservCyt solution for HPV testing with the Xpert HPV assay

  11. Laboratory blood analysis in Strigiformes-Part II: plasma biochemistry reference intervals and agreement between the Abaxis Vetscan V2 and the Roche Cobas c501.

    Science.gov (United States)

    Ammersbach, Mélanie; Beaufrère, Hugues; Gionet Rollick, Annick; Tully, Thomas

    2015-03-01

    Limited plasma biochemical information is available in Strigiformes. Only one study investigated the agreement between a point-of-care with a reference laboratory analyzer for biochemistry variables in birds. The objective was to report reference intervals (RI) for plasma biochemistry variables in Strigiformes, and to assess agreement between the Abaxis Vetscan V2 and Roche Cobas c501. A prospective study was designed to assess plasma biochemistry RI for concentration of calcium, phosphorus, total protein, albumin, globulin, glucose, bilirubin, uric acid, bile acids, sodium, potassium, and chloride, and activities of AST, GGT, CK, amylase, lipase, LDH, and GLDH. In addition, the agreement between the Vetscan and the Cobas in owl species was assessed. A total of 190 individuals were sampled belonging to 12 Strigiformes species including Barn Owls, Barred Owls, Great Horned Owls, Eurasian Eagle Owls, Spectacled Owls, Eastern Screech Owls, Long-Eared Owls, Short-Eared Owls, Great Gray Owls, Snowy Owls, Northern Saw-Whet Owls, and Northern Hawk-Owls. Order-, species-, and method-specific RI were determined on both analyzers. Although Vetscan data were not equivalent to the Cobas, 4 analytes (glucose, AST, CK, and total protein, with correction for bias) were within acceptable agreement, 3 analytes (uric acid, calcium, and phosphorus) were within close agreement, and the remaining analytes were in strong disagreement. Species-specific differences were observed notably for the concentration of glucose in Barn Owls and electrolytes in Northern Saw-Whet Owls. Overall, this study suggests that the Vetscan has acceptable clinical performance in Strigiformes for some analytes and highlights discrepancies for several analytes. © 2015 American Society for Veterinary Clinical Pathology.

  12. Evaluation of the Randox colorimetric serum copper and zinc assays against atomic absorption spectroscopy.

    Science.gov (United States)

    Beckett, Jeffrey M; Hartley, Thomas F; Ball, Madeleine J

    2009-07-01

    Analysis of copper and zinc in serum is commonly performed using atomic absorption spectrometry (AAS); however, these methods are often not readily available in smaller laboratories. Randox colorimetric assays for copper and zinc in serum were evaluated on the Thermo Electron Data Pro analyser against flame AAS methods. Copper and zinc were measured in 48 serum samples using the Randox colorimetric copper (CU2340) and zinc (ZN2341) assays on the Data Pro analyser and the results compared with those from a Varian Spectra 880 atomic absorption spectrometer. A smaller set of samples (n = 15) were also analysed colorimetrically for zinc on the Roche Cobas Mira. Linear regression analyses of Bland and Altman plots from the Data Pro - AAS comparison gave the following results for copper: correlation r = 0.6669 (P < 0.01), slope = -0.2499 (P < 0.01), intercept = 3.219 (P < 0.01). For zinc, results were as follows: correlation r = 0.1976, slope = 0.1807, intercept = -1.922. For the smaller set of samples, the Cobas Mira - AAS comparison for zinc gave correlation r = 0.4379, slope = 0.5294, intercept = -4.074. The results indicated significant systematic and fixed bias between the colorimetric copper and the AAS method. Performances in comparison to AAS methods indicated the colorimetric methods, as used, are unsuitable for the accurate determination of copper and zinc in human serum.

  13. Sensibilidad del equipo Cobas AmpliScreenTM HIV-1 Test, v1.5, para la detección de HIV-1

    Directory of Open Access Journals (Sweden)

    Lucía P Gomez

    Full Text Available Las técnicas de amplificación de ácidos nucleicos (NAT se incorporaron en los bancos de sangre para reducir el riesgo residual de transmisión de infecciones por vía transfusional. La cocirculación de distintas variantes del HIV-1 en Argentina indica la necesidad de evaluar la sensibilidad de los ensayos serológicos y moleculares disponibles para su detección. En este trabajo se evaluó la sensibilidad del equipo COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche, para detectar ARN viral en plasmas de individuos infectados con HIV-1 de Argentina. Los resultados demuestran que esta técnica tiene una alta sensibilidad para detectar ARN de HIV-1 en las condiciones ensayadas: para ensayo de mini-pooles (pooles = 50 copias de ARN/ml, la sensibilidad fue = 92 %, y para procedimiento estándar (plasmas = 207 copias de ARN/ml, la sensibilidad fue 100 %. Además, la técnica COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche, es adecuada para la detección de las variantes de HIV-1 prevalentes.

  14. Comparison of two automated assays of BTM (CTX and P1NP) and reference intervals in a Danish population

    DEFF Research Database (Denmark)

    Jørgensen, N R; Møllehave, L T; Hansen, Y B L

    2017-01-01

    the agreement on the two platforms. METHODS: Fasting sera from 2308 individuals (1250 males and 1058 females, age range 24-76 years) participating in the Health2006 study were analyzed for CTX and P1NP using the automated IDS-iSYS analyzer and the automated Cobas e411 analyzer. Participants in anti......-osteoporotic treatment were excluded, while subjects on hormonal contraceptives were included. RESULTS: There was significant disagreement between both the two P1NP assays with a mean difference of -3 μg/L (LoA -19 to 14) (p

  15. Differential Detection of Human Papillomavirus Genotypes and Cervical Intraepithelial Neoplasia by Four Commercial Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah

    2016-01-01

    Laboratories can nowadays choose from >100 Human Papillomavirus (HPV) assays for cervical screening. Our previous analysis based on the data from the Danish Horizon study, however, showed that four widely used assays, Hybrid Capture 2 (HC2), cobas, CLART and APTIMA, frequently do not detect...... intraepithelial neoplasia (CIN) in 2.5 years after the baseline testing were determined from the national pathology register. HPV-positive women undergoing primary screening having concordant samples were more likely to harbor high-risk infections and less likely to harbor only low-risk infections than women...... the younger age and more HPV infections. HPV test result discordance identified a cluster of low-risk HPV infections that were hardly ever associated with high-grade CIN and, almost exclusively, represented false-positive screening findings....

  16. Evaluation of a Novel PCR-Based Assay for Detection and Identification of Chlamydia trachomatis Serovars in Cervical Specimens▿

    Science.gov (United States)

    Quint, Koen; Porras, Carolina; Safaeian, Mahboobeh; González, Paula; Hildesheim, Allan; Quint, Wim; van Doorn, Leen-Jan; Silva, Sandra; Melchers, Willem; Schiffman, Mark; Rodríguez, Ana Cecilia; Wacholder, Sholom; Freer, Enrique; Cortes, Bernal; Herrero, Rolando

    2007-01-01

    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars. PMID:17959760

  17. Negative interference of icteric serum on a bichromatic biuret total protein assay.

    Science.gov (United States)

    Gupta, Aradhana; Stockham, Steven L

    2014-09-01

    Bilirubin is stated to be a negative interferent in some biuret assays and thus could contribute to pseudohypoproteinemia in icteric samples. The purpose of the study was to evaluate the magnitude of and reason for a falsely low total protein concentration in icteric serum when the protein concentration is measured with a bichromatic spectrophotometric biuret assay. Commercially available bilirubin was dissolved in 0.1 M NaOH and mixed with sera from 2 dogs to achieve various bilirubin concentrations of up to 40 mg/dL (first set of samples) and 35 mg/dL (second set of samples, for confirmation of first set of results and to explore the interference). Biuret total protein and bilirubin concentrations were determined with a chemistry analyzer (Cobas 6000 with c501 module). Line graphs were drawn to illustrate the effects of increasing bilirubin concentrations on the total protein concentrations. Specific spectrophotometric absorbance readings were examined to identify the reason for the negative interference. High bilirubin concentrations created a negative interference in the Cobas biuret assay. The detectable interference occurred with a spiked bilirubin concentration of 10.7 mg/dL in one set of samples, 20.8 mg/dL in a second set. The interference was due to a greater secondary-absorbance reading at the second measuring point in the samples spiked with bilirubin, which possibly had converted to biliverdin. Marked hyperbilirubinemia is associated with a falsely low serum total protein concentration when measured with a bichromatic spectrophotometric biuret assay. This can result in pseudohypoproteinemia and pseudohypoglobulinemia in icteric serum. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

  18. Evaluation of fully automated assays for the detection of Rubella IgM and IgG antibodies by the Elecsys(®) immunoassay system.

    Science.gov (United States)

    van Helden, Josef; Grangeot-Keros, Liliane; Vauloup-Fellous, Christelle; Vleminckx, Renaud; Masset, Frédéric; Revello, Maria-Grazia

    2014-04-01

    Screening for acute rubella infection in pregnancy is an important element of antenatal care. This study compared the sensitivity, specificity and reproducibility of two new, fully automated Elecsys(®) Rubella IgM and IgG immunoassays designed for the Elecsys 2010, Modular Analytics E170, COBAS e-411 and COBAS e-601 and e602 analytical platforms, with current assays using serum from patients with primary rubella infections, vaccinated patients, patients with potentially cross-reacting infections and on routine samples in clinical laboratories in France, Germany and Italy. Both assays showed good within-run and within-laboratory precision. A sensitivity of 79.8-96.0% was demonstrated for Elecsys IgM in primary, early acute infection, consistent with existing assays. In samples obtained from routine antenatal screening, the Elecsys Rubella IgM assay revealed high specificity (98.7-99.0%). A significantly (prubella infection was excluded, and the incidence of false positives in patients with potentially cross-reacting infections was lower with Elecsys Rubella IgM compared with other. The Elecsys Rubella IgG assay exhibited a relative sensitivity of 99.9-100.0% and specificity of 97.4-100.0% in samples from routine antenatal screening. The Elecsys Rubella IgM and IgG assays allow convenient, rapid and reliable determination of anti-rubella antibodies. Sensitivity, specificity and reproducibility were comparable with existing assay systems. Assay results were available in approximately half the time required for currently employed methods and the assays are compatible with widely used analytical platforms. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F.

    Science.gov (United States)

    Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

    2017-04-01

    Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.

  20. Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

    Science.gov (United States)

    White, Kirsten; Garner, Will; Wei, Lilian; Eron, Joseph J; Zhong, Lijie; Miller, Michael D; Martin, Hal; Plummer, Andrew; Tran-Muchowski, Cecilia; Lindstrom, Kim; Porter, James; Piontkowsky, David; Light, Angela; Reiske, Heinz; Quirk, Erin

    2018-02-08

    Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. Prospective and retrospective analysis of randomized clinical trial samples and reference standards. To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of at least 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested at least 50 copies/ml. The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low-drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source

  1. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays.

    Science.gov (United States)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik; Wu, Lin; Wei, Wen; Tsai, Julie; Khalil, Azza; Nexo, Ebba; Sorensen, Boe S

    2014-04-28

    Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma samples with allele-specific PCR assays. Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence of mutations in exons 18-21 of the EGFR gene, employing the cobas(®) EGFR Tissue Test and cobas(®) EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue was 179/196 (91%) (kappa value: 0.621). Mutational analysis of the EGFR gene in plasma samples is feasible with allele-specific PCR assays and represents a non-invasive supplement to biopsy analysis. M-20080012 from March 10, 2008 and reported to ClinicalTrials.gov: NCT00815971.

  2. Multicentric performance analysis of HCV quantification assays and its potential relevance for HCV treatment.

    Science.gov (United States)

    Wiesmann, F; Naeth, G; Berger, A; Hirsch, H H; Regenass, S; Ross, R S; Sarrazin, C; Wedemeyer, H; Knechten, H; Braun, P

    2016-06-01

    An accurate quantification of low viremic HCV RNA plasma samples has gained importance since the approval of direct acting antivirals and since only one single measurement predicts the necessity of a prolonged or shortened therapy. As reported previously, HCV quantification assays such as Abbott RealTime HCV and Roche COBAS AmpliPrep/COBAS TaqMan HCV version 2 (CTM v2) may vary in sensitivity and precision particularly in low-level viremia. Importantly, substantial variations were previously demonstrated between some of these assays compared to the Roche High Pure System/COBAS TaqMan assay (HPS) reference assay, which was used to establish the clinical decision points in clinical studies. In this study, the reproducibility of assay performances across several laboratories was assessed by analysing quantification results generated by six independent laboratories (3× RealTime, 3× CTM v2) in comparison with one HPS reference laboratory. The 4th WHO Standard was diluted to 100, 25 and 10 IU/ml, and aliquots were tested in triplicates in 5 independent runs by each assay in the different laboratories to assess assay precision and detection rates. In a second approach, 2 clinical samples (GT 1a & GT 1b) were diluted to 100 and 25 IU/ml and tested as described above. While the result range for WHO 100 IU/ml replicates across all laboratories was similar in this analysis, the CVs of each laboratory ranged from 19.3 to 25.6 % for RealTime laboratories and were lower than CVs of CTM v2 laboratories with a range of 26.1-47.3 %, respectively, and also in comparison with the CV of the HPS reference laboratory (34.9 %). At WHO standard dilution of 25 IU/ml, 24 replicates were quantified by RealTime compared to 8 replicates with CTM v2. Results of clinical samples again revealed a higher variation of CTM v2 results as compared to RealTime values. (CVs at 100 IU/ml: RealTime: 13.1-21.0 % and CTM v2: 15.0-32.3 %; CVs at 25 IU/ml: RealTime 17.6-34.9 % and CTM v2 28

  3. Comparison of four automated serum vitamin B12 assays.

    Science.gov (United States)

    İspir, Emre; Serdar, Muhittin A; Ozgurtas, Taner; Gulbahar, Ozlem; Akın, Kadir Okhan; Yesildal, Fatih; Kurt, İsmail

    2015-07-01

    Diagnosis of vitamin B12 deficiency is generally based on the measurement of serum vitamin B12 levels. However, in selected cases functional indices of vitamin B12, such as methylmalonic acid (MMA) and homocysteine (HCY), are needed. Here we compare the performance of four automated total vitamin B12 assays and also investigate how these assays relate to functional indices of vitamin B12 status. Total vitamin B12, MMA and HCY were measured in 69 serum samples from routine vitamin B12 assay requests. Serum vitamin B12 analysis was performed using four different immunoassay autoanalyzers: DxI 800 Unicel (Beckman Coulter, USA), ADVIA Centaur XP (Siemens Diagnostics, Tarrytown, NY, USA), Roche Cobas E601 (Roche Diagnostics, Germany), Architect i2000sr (Abbott Laboratories, Abbott Park, IL, USA). Serum MMA levels were determined by liquid chromatography-mass spectrometry (LC-MS) and serum homocysteine levels were determined by high pressure liquid chromatography (HPLC) methods. Four immunoassay methods were comparable and correlated with each other. Correlation coefficients (r) ranged from 0.898 to 0.987, pUnicel - ADVIA Centaur comparison. DxI 800 Unicel assay demonstrated high mean bias [-122 pg/mL (-616-125 pg/mL)] and a concordance correlation coefficient (CCC) of 0.9161, lower than the others. MMA and HCY were correlated with the vitamin B12 results. The correlation coefficients with their 95% CI indicated that there was no statistically significant difference between the four methods according to their relationship with MMA and HCY. Total B12 assays correlate very well with each other. However, results of DxI 800 Unicel were lower compared to the other three autoanalyzers. All total vitamin B12 methods show similar relationships with HCY and MMA. Standardization of serum vitamin B12 assays is still not completed and further standardization studies are needed. Laboratory professionals and clinicians should be aware of this disagreement between assay methods and

  4. Comparison of Hemoglobin A1c assay performance on two different commercial systems

    Directory of Open Access Journals (Sweden)

    Jozo Ćorić

    2015-04-01

    Full Text Available Introduction: Glycated hemoglobin (HbA1c is formed by non-enzymatic binding of glucose to the free amino group of the N-terminal end of the ß-chain of hemoglobin A. HbA1c is representative of the mean blood glucose level over three months. The aim of the study was to evaluate the Hemoglobin A1c immunoturbidimetric assay performance on two different commercial systems.Methods: We evaluated the precision and trueness for determination of HbA1c in whole blood. Concentrations of total hemoglobin and HbA1c were evaluated on Dimension Xpand (Siemens and Cobas 501 (Roche analyzers. HbA1c was measured in a latex agglutination inhibition test. Commercial controls Liquichek Diabetes Control Level 1 and Liquichek Diabetes Control Level 2 (Bio Rad at two levels were used for quality control. Analytical validation of HbA1c included: within-run imprecision, between-day imprecision, inaccuracy and comparison determination on the human samples on 2 systems: Dimension Xpand and Cobas 501 analyzers. Results: Within-run imprecision on the commercially controls for Level 1 is 4.5% and Level 2 is 3.2% between-day imprecision on commercially controls is 6.1% Level 1 and 5.1% Level 2 for respectively inac- curacy on commercially controls for Level 1 is 1.8% and Level 2 is 4.8%. Method comparison on human samples shows the correlation coefficient of 0.99.Conclusion: The presented results of the analytical evaluation methods for the determination of HbA1c showed an acceptable accuracy and precision.

  5. A sensitive quantitative test strip based point-of-care albuminuria screening assay.

    Science.gov (United States)

    Decavele, An-Sofie C; Fiers, Tom; Penders, Joris; Delanghe, Joris R

    2012-01-06

    Chronic kidney disease is a major health problem and the global guidelines require screening of albuminuria. Therefore, affordable and sensitive albuminuria screening tests are needed. We explored the potential of urine strips, generally reported in the ordinal scale, measured on an automatic strip reader for reporting quantitative and sensitive albumin results. We compared reflectance data of Combur-Test® strips obtained from the Cobas U411 reader (Roche) with albuminuria data from a nephelometer BNII (Siemens) and with protein concentrations from the pyrogallol red method (Modular P, Roche) for 389/328 non-pathologic and pathologic urine samples, respectively. Imprecision of the reflectance signal of the Cobas U411 was measured with commercial control material (Bio-Rad). Inter-run coefficients of variations (CVs) for reflectance for levels 1 and 2 were 1.7%/4.9%, respectively, and intra-run CVs were 1.8%/4.2%, respectively. Good agreement was obtained between the albumin concentration of the BNII and the protein strip reflectance data (n=389): Y (10,000/protein reflectance, 1/%)=160+0.132·X (albuminuria BNII, mg/L)-0.0000111·X2 (albuminuria BNII, mg/L); r2=0.921. Lower agreement was found between the protein assay (n=328) and the reflectance (r2=0.831). A calibration curve was made between 11.5 mg/L and 121.5 mg/L. The limit of blank (LOB) was 44.7 mg/L. The present study demonstrates that reflectance data generated by a test strip reader allows for quantitative analysis of albumin. Although the lower limit of the microalbumin range (30 mg/L) cannot be achieved with the dye-binding method, the results are satisfactory for screening purposes.

  6. Comparison of the elecsys HBsAg II assay and the architect assay for quantification of hepatitis B surface antigen in chronic hepatitis B patients.

    Science.gov (United States)

    Liao, Chen-Chih; Hsu, Chao-Wei; Gu, Po-Wen; Yeh, Chau-Ting; Lin, Shi-Ming; Chiu, Cheng-Tang

    2015-01-01

    Hepatitis B virus (HBV) infection is one of the infections with a highest prevalence in Taiwan. The most important marker is hepatitis B surface antigen (HBsAg). Using the new generation of HBsAg quantitative assay, HBsAg level may have good correlation with viral activity during different phases of chronic hepatitis B virus infection. This study was conducted to compare two assays of HBsAg level to find if the same results are obtained in HBsAg quantification in treatment-naïve and on-treatment chronic hepatitis B patients. Between March 2012 and June 2012, 90 patients with chronic hepatitis B (68 males and 22 females) were assessed using Abbott Architect HBsAg QT and Roche Elecsys HBsAg II assay. HBV DNA was detected by Roche COBAS TaqMan instrument. HBsAg level measured with Elecsys and Architect assays correlated well in untreated patients (n = 53, γs = 0.997) and on-treatment patients (n = 37, γs = 0.988). Bland-Altman analyses of the discrepancies in HBsAg levels showed a bias of -4.2% in untreated patients and -6.2% in on-treatment patients. Patients with HBeAg-postive chronic hepatitis B had higher HBsAg level than the ones who were HBeAg negative, and both showed statistical differences. Further, HBV DNA concentration analysis also showed higher viral concentration in HBeAg-positive patients, but it revealed no statistical difference. There is a significant correlation between Abbott Architect HBsAg QT assay and Roche Elecsys HBsAg II assay. Moreover, HBsAg quantification may potentially provide complementary information about the deduction of the natural course in chronic hepatitis B infection.

  7. Comparison of DRY and WET vaginal swabs with cervical specimens in Roche Cobas 4800 HPV and Abbott RealTime High Risk HPV tests.

    Science.gov (United States)

    Jun, Jae Kwan; Lim, Myong Cheol; Hwang, Sang-Hyun; Shin, Hye Young; Hwang, Na Rae; Kim, Yeon-Jin; Yoo, Chong Woo; Lee, Dong Ock; Joo, Jungnam; Park, Sang-Yoon; Lee, Do-Hoon

    2016-06-01

    Self-collected vaginal swab samples have been proposed as an alternative specimen collection method for human papillomavirus (HPV) DNA detection. Two vaginal swabs (a cone-shaped flocked swab (DRY) and a L-shape FLOQSwab with 2mL eNAT transport medium (WET)) were compared to standard cervical samples for HPV DNA testing. Additionally, they were also compared by using Roche Cobas 4800 HPV (Roche_HPV) and Abbott Real-time High Risk HPV (Abbott_HPV) tests. Ninety-six women were prospectively enrolled from the National Cancer Center in Korea between June and August 2015. WET and DRY vaginal swabs and cervical specimens were collected. Roche_HPV and Abbott_HPV tests were performed. The Roche_HPV test on cervical specimens was used as reference. The observed agreements (kappa) of Roche_HPV and Abbott_HPV between WET and DRY swabs were 89.6% (0.790, 95% confidence interval (95% CI): 0.667-0.913) and 91.7% (0.833, 95%CI: 0.723-0.943), respectively. No statistical difference was observed between WET and DRY swabs (p>0.05 for all comparisons). For HPV16/18, the sensitivity/specificity of Roche_HPV on the DRY and WET samples presented 93.8%/96.3% and 87.5%/97.5%, respectively. For other High Risk HPV (hrHPV), the sensitivity/specificity of Roche_HPV on the DRY and WET swabs presented 91.9%/91.5% and 97.3%/98.3, respectively. The sensitivity/specificity of the Abbott_HPV on the DRY and WET swabs were 93.8%/98.8%, 87.5%/98.8% for HPV16/18, and 91.9%/93.2%, 100.0%/93.2% for other hrHPV, respectively. HPV tests performed similarly when using vaginal DRY and WET swab samples. Using DRY and WET swabs to collect vaginal specimens could be an alternative to collecting cervical samples for HPV DNA testing. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Comparison of the Abbott RealTime High Risk HPV test and the Roche cobas 4800 HPV test using urine samples.

    Science.gov (United States)

    Lim, Myong Cheol; Lee, Do-Hoon; Hwang, Sang-Hyun; Hwang, Na Rae; Lee, Bomyee; Shin, Hye Young; Jun, Jae Kwan; Yoo, Chong Woo; Lee, Dong Ock; Seo, Sang-Soo; Park, Sang-Yoon; Joo, Jungnam

    2017-05-01

    Human papillomavirus (HPV) testing based on cervical samples is important for use in cervical cancer screening. However, cervical sampling is invasive. Therefore, non-invasive methods for detecting HPV, such as urine samples, are needed. For HPV detection in urine samples, two real-time PCR (RQ-PCR) tests, Roche cobas 4800 test (Roche_HPV; Roche Molecular Diagnostics) and Abbott RealTime High Risk HPV test (Abbott_HPV; Abbott Laboratories) were compared to standard cervical samples. The performance of Roche_HPV and Abbott_HPV for HPV detection was evaluated at the National Cancer Center using 100 paired cervical and urine samples. The tests were also compared using urine samples stored at various temperatures and for a range of durations. The overall agreement between the Roche_HPV and Abbott_HPV tests using urine samples for any hrHPV type was substantial (86.0% with a kappa value of 0.7173), and that for HPV 16/18 was nearly perfect (99.0% with a kappa value of 0.9668). The relative sensitivities (based on cervical samples) for HPV 16/18 detection using Roche_HPV and Abbott_HPV with urine samples were 79.2% (95% CI; 57.9-92.9%) and 81.8% (95% CI; 59.7-94.8%), respectively. When the cut-off CT value for Abbott_HPV was extended to 40 for urine samples, the relative sensitivity of Abbott_HPV increased to 91.7% from 81.8% for HPV16/18 detection and to 87.0% from 68.5% for other hrHPV detection. The specificity was not affected by the change in the CT threshold. Roche_HPV and Abbott_HPV showed high concordance. However, HPV DNA detection using urine samples was inferior to HPV DNA detection using cervical samples. Interestingly, when the cut-off CT value was set to 40, Abbott_HPV using urine samples showed high sensitivity and specificity, comparable to those obtained using cervical samples. Fully automated DNA extraction and detection systems, such as Roche_HPV and Abbott_HPV, could reduce the variability in HPV detection and accelerate the standardization of HPV

  9. Is measurement of TT3 by immunoassay reliable at low concentrations? A comparison of the Roche Cobas 6000 vs. LC-MSMS.

    Science.gov (United States)

    Masika, Likhona Siphe; Zhao, Zhen; Soldin, Steven John

    2016-08-01

    Thyroid dysfunction is a common medical condition affecting an estimated 30 million people in the US alone. Employing gold standard Liquid chromatography-tandem mass spectrometry (LC-MSMS) methods we have examined the extent of inaccuracy of immunoassay (IA) measurement for total T3 (TT3) at low, normal and high concentrations. 268 TT3 Roche Cobas 6000 immunoassay TT3 values (covering the low, normal, and high ranges) were compared with LC-MSMS results. At TT3 concentrations between 50 and 113ng/dL (conversion factor for TT3 to SI Units is ng/dL×0.0154=nmol/L), n=122, LC-MSMS values were lower than immunoassay with 72% found to be below the 2.5th percentile by LC-MSMS compared to 27% for immunoassay. Strikingly 45% of the patients classified as normal TT3 by immunoassay were defined as lower than the 2.5th percentile by LC-MSMS. Only 38 of the 122 patients with low T3's were not receiving T4. In this latter group all of whom had TSH's>3.7mIU/L, 74% of results by LC-MSMS were below the 2.5th percentile while only 21% were below the 2.5th percentile by IA. The clinical consequences of these inaccuracies may affect whether dosing with T4 or combination of T4 with T3 is selected for treatment. Finally the correlation of TT3 with TSH was far superior when TT3 was measured by LC-MSMS. A typical case which demonstrates our message is included. T3 being the active hormone needs to be reliably measured and if the patient has low TT3 and hypothyroid symptoms persist; treatment with T3 should be considered. A typical case report is included to illustrate the problems of inaccurate immunoassay results for TT3. Measurement of TT3 by immunoassay at low concentrations is less than optimal and often provides the clinician with a normal result when the LC-MSMS method and the patient's clinical condition suggests that supplementation with T3 (as in combination therapy) may be required to optimize patient care. Published by Elsevier Inc.

  10. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  11. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  12. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  13. Service impact of a change in HIV-1 viral load quantification assay

    Directory of Open Access Journals (Sweden)

    Craig Tipple

    2014-11-01

    Full Text Available Introduction: Due to discontinuation of the Siemens Versant HIV-1 RNA (bDNA assay in the UK, our laboratory switched to the Roche Cobas Ampliprep/Taqman HIV-1 viral load (VL assay (Roche in April 2013. This assay has a lower cut-off of 20 RNA copies/mL (compared with <50 for the Siemens assay. Our laboratory demonstrated previously that a significant proportion (18% of patients undetectable using bDNA HIV-1 RNA quantification exhibited low level viraemia (LLV using the new assay. Local guidelines recommend that patients stable on therapy receive twice-yearly VLs. We evaluated the impact of the introduction of the new assay on our clinical service. Methods: A retrospective cohort analysis of treated patients with stable undetectable VL by bDNA (<50 copies/mL followed by ≥ one low-level (<400 copies/mL VL with the Roche assay. Demographic data were collected in addition to frequency of VL testing and genotypic resistance assays. Referrals to virtual clinic (VC were recorded. Patients were identified using laboratory data and information collected from electronic patient records. Results were analyzed with SPSS v18. Results: One hundred and ninety patients were included. Demographics: 79.5% male; 60.6% homosexual; mean age of 46 years. Duration on stable treatment was 46.35 (std. dev. 38.15 months. Current treatment regimens were 43.3% PI-based; 43.3% NNRTI-based and 13.7% other. Patients were stratified into VL 20–49 copies/mL (n=109; VL 50–199 copies/mL (n=71 and VL 200–399 copies/mL (n=10. In total, there were 471 VLs measured of which 274 were additional as a result of the assay switch. This resulted in six HIV-1 genotype requests and 16 VC discussions (Table 1. Longer duration on HAART was associated with reduced frequency of VL testing. The relative risk of ongoing detectability according to drug class are: PI 1.62 (95% CI 1.18–2.21; NNRTI 0.507 (95% CI 0.30–0.85 and other 1.09 (95% CI 0.48–2.43. Conclusions: Changes in assay

  14. Multisite analytical evaluation of the Abbott ARCHITECT cyclosporine assay.

    Science.gov (United States)

    Wallemacq, Pierre; Maine, Gregory T; Berg, Keith; Rosiere, Thomas; Marquet, Pierre; Aimo, Giuseppe; Mengozzi, Giulio; Young, Julianna; Wonigeit, Kurt; Kretschmer, Robert; Wermuth, Bendicht; Schmid, Rainer W

    2010-04-01

    The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Cyclosporine (CsA) immunoassay in 7 clinical laboratories in comparison to liquid chromatography/tandem mass spectrometry (LC/MS/MS), Abbott TDx, Cobas Integra 800, and the Dade Dimension Xpand immunoassay. The ARCHITECT assay uses a whole blood specimen, a pretreatment step with organic reagents to precipitate proteins and extract the drug, followed by a 2-step automated immunoassay with magnetic microparticles coated with anti-CsA antibody and an acridinium-CsA tracer. Imprecision testing at the 7 evaluation sites gave a range of total % coefficient of variations of 7.5%-12.2% at 87.5 ng/mL, 6.6%-14.3% at 411 ng/mL, and 5.2%-10.7% at 916 ng/mL. The lower limit of quantification ranged from 12 to 20 ng/mL. Purified CsA metabolites AM1, AM1c, AM4N, AM9, and AM19 were tested in whole blood by the ARCHITECT assay and showed minimal cross-reactivity at all 7 sites. In particular, AM1 and AM9 cross-reactivity in the ARCHITECT assay, ranged from -2.5% to 0.2% and -0.8% to 2.2%, respectively, and was significantly lower than for the TDx assay, in which the values were 3.2% and 16.1%, respectively. Comparable testing of metabolites in the Dade Dimension Xpand assay at 2 evaluation sites showed cross-reactivity to AM4N (6.4% and 6.8%) and AM9 (2.6% and 3.6%) and testing on the Roche Integra 800 showed cross-reactivity to AM1c (2.4%), AM9 (10.7%), and AM19 (2.8%). Cyclosporine International Proficiency Testing Scheme samples, consisting of both pooled specimens from patients receiving CsA therapy as well as whole-blood specimens supplemented with CsA, were tested by the ARCHITECT assay at 6 sites and showed an average bias of -24 to -58 ng/mL versus LC/MSMS CsA and -2 to -37 ng/mL versus AxSYM CsA. Studies were performed with the ARCHITECT CsA assay on patient specimens with the following results: ARCHITECT CsA assay versus LC/MSMS, average bias of 31 ng/mL; ARCHITECT versus the

  15. Differential Detection of Human Papillomavirus Genotypes and Cervical Intraepithelial Neoplasia by Four Commercial Assays.

    Science.gov (United States)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah; Ejegod, Ditte; Rygaard, Carsten; Lynge, Elsebeth

    2016-11-01

    Laboratories now can choose from >100 human papillomavirus (HPV) assays for cervical screening. Our previous analysis based on the data from the Danish Horizon study, however, showed that four widely used assays, Hybrid Capture 2 (HC2), cobas, CLART, and Aptima, frequently do not detect the same HPV infections. Here, we determined the characteristics of the concordant samples (all four assays returning a positive HPV test result) and discordant samples (all other HPV-positive samples) in primary cervical screening at 30 to 65 years of age (n = 2,859) and in a concurrent referral population from the same catchment area (n = 885). HPV testing followed the manufacturers' protocols. Women with abnormal cytology were managed according to the routine recommendations. Cytology-normal/HPV-positive women were invited for repeated testing in 18 months. Screening history and histologically confirmed cervical intraepithelial neoplasia (CIN) in 2.5 years after the baseline testing were determined from the national pathology register. HPV-positive women undergoing primary screening having concordant samples were more likely to harbor high-risk infections and less likely to harbor only low-risk infections than women with discordant samples. Additionally, assay signal strengths were substantially higher in concordant samples. More than 80% of ≥CIN2 results were found for women with concordant samples, and no ≥CIN2 results were found when the infection was detected by only one assay. These patterns were similar in the referral population despite the younger age and higher number of HPV infections. HPV test result discordance identified a cluster of low-risk HPV infections that were hardly ever associated with high-grade CIN and, almost exclusively, represented false-positive screening findings. Copyright © 2016 Rebolj et al.

  16. Paired measurement of urinary creatinine in neonates based on a Jaffe and an enzymatic IDMS-traceable assay.

    Science.gov (United States)

    Allegaert, Karel; Vermeersch, Pieter; Smits, Anne; Mekahli, Djalila; Levtchenko, Elena; Pauwels, Steven

    2014-04-15

    Urinary creatinine can be quantified by Jaffe or enzymatic assays and is commonly used as denominator of urinary excretion of electrolytes or protein. Paired analysis in pediatric and adult samples documented inter-assay differences (up to 80%). We verified the interchangeability of two IDMS-traceable assays (Jaffe and enzymatic) for neonatal urine and report on neonatal urinary creatinine values using these IDMS-traceable methods. Creatinine was measured in 84 neonatal urine samples from 46 neonates by an IDMS traceable Jaffe and enzymatic assay (Roche Diagnostics, Cobas c702 module). Creatinine values, differences in urinary creatinine and clinical characteristics were described and covariates of between assay difference were explored (Wilcoxon, Bland-Altman, correlation, multiple regression). Median Jaffe and enzymatic urinary creatinine concentrations were 9.25 (range 3.7-42.2) and 9.15 (range 3.8-42.9) mg/dL respectively, resulting in a median difference of 0.08 (SD 0.6, range -2.4 to 0.96) mg/dL. In a multiple regression model, urinary enzymatic creatinine concentration (r = 0.45) and postnatal age (r = -0.59) remained independent variables of the difference between both assays (r2 adj = 0.45). The tested IDMS-traceable assays showed interchangeable in heterogeneous neonatal urine samples. Using these assays, neonatal urinary creatinine showed 5-20 fold lower values than those observed in children or adults with a significant negative correlation with postnatal age.

  17. Lateral flow assays.

    Science.gov (United States)

    Koczula, Katarzyna M; Gallotta, Andrea

    2016-06-30

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  18. Lateral flow assays

    Science.gov (United States)

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  19. Tube-Forming Assays.

    Science.gov (United States)

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  20. Modelling the comet assay.

    Science.gov (United States)

    McArt, Darragh G; McKerr, George; Howard, C Vyvyan; Saetzler, Kurt; Wasson, Gillian R

    2009-08-01

    The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

  1. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  2. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R [Danville, CA; Benett, William J [Livermore, CA; Coleman, Matthew A [Oakland, CA; Pearson, Francesca S [Livermore, CA; Nasarabadi, Shanavaz L [Livermore, CA

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  3. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  4. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  5. COBA-Cohort: a prospective cohort of HIV-negative men who have sex with men, attending community-based HIV testing services in five European countries (a study protocol).

    Science.gov (United States)

    Lorente, Nicolas; Fernàndez-López, Laura; Fuertes, Ricardo; Rojas Castro, Daniela; Pichon, François; Cigan, Bojan; Chanos, Sophocles; Meireles, Paula; Lucas, Raquel; Morel, Stéphane; Slaaen Kaye, Per; Agustí, Cristina; Klavs, Irena; Platteau, Tom; Casabona, Jordi

    2016-07-13

    Community-based voluntary counselling and testing (CBVCT) services for men who have sex with men (MSM) can reach those most-at-risk and provide an environment for gay men that is likely to be non-stigmatising. Longitudinal data on the behaviour of HIV-negative MSM are scarce in Europe. The aim of this protocol, developed during the Euro HIV Early Diagnosis And Treatment (EDAT) project, is to implement a multicentre community-based cohort of HIV-negative MSM attending 15 CBVCT services in 5 European countries. (1) To describe the patterns of CBVCT use, (2) to estimate HIV incidence, and to identify determinants of (3) HIV seroconversion and (4) HIV and/or sexually transmitted infection (STI) test-seeking behaviour. All MSM aged 18 years or over and who had a negative HIV test result are invited to participate in the COmmunity-BAsed Cohort (COBA-Cohort). Study enrolment started in February 2015, and is due to continue for at least 12 months at each study site. Follow-up frequency depends on the testing recommendations in each country (at least 1 test per year). Sociodemographic data are collected at baseline; baseline and follow-up questionnaires both gather data on attitudes and perceptions, discrimination, HIV/STI testing history, sexual behaviour, condom use, and pre- and post-exposure prophylaxis. Descriptive, exploratory and multivariate analyses will be performed to address the main research objectives of this study, using appropriate statistical tests and models. These analyses will be performed on the whole cohort data and stratified by study site or country. The study was approved by the Public Health authorities of each country where the study is being implemented. Findings from the COBA-Cohort study will be summarised in a report to the European Commission, and in leaflets to be distributed to study participants. Articles and conference abstracts will be submitted to peer-reviewed journals and conferences. Published by the BMJ Publishing Group Limited

  6. Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?

    Science.gov (United States)

    Arbyn, M; Snijders, P J F; Meijer, C J L M; Berkhof, J; Cuschieri, K; Kocjan, B J; Poljak, M

    2015-09-01

    Several countries are in the process of switching to high-risk human papillomavirus (hrHPV) testing for cervical cancer screening. Given the multitude of available tests, validated assays which assure high-quality screening need to be identified. A systematic review was conducted to answer the question which hrHPV tests fulfil the criteria defined by an international expert team in 2009, based on reproducibility and relative sensitivity and specificity compared to Hybrid Capture-2 or GP5+/6+ PCR-enzyme immunoassay. These latter two hrHPV DNA assays were validated in large randomized trials and cohorts with a follow-up duration of 8 years or more. Eligible studies citing the 2009 guideline were retrieved from Scopus (http://www.scopus.com) and from a meta-analysis assessing the relative accuracy of new hrHPV assays versus the standard comparator tests to detect high-grade cervical intraepithelial neoplasia or cancer in primary screening. The cobas 4800 HPV test and Abbott RealTime High Risk HPV test were consistently validated in two and three studies, respectively, whereas the PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV-Risk assay were validated each in one study. Other tests which partially fulfil the 2009 guidelines are the following: Cervista HPV HR Test, GP5+/6+ PCR-LMNX, an in-house E6/E7 RT quantitative PCR and MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight). The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated. However, the cross-sectional equivalency criteria of the 2009 guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is awaited to inform us about its utility in cervical cancer screening at 5-year or longer intervals. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  7. Global assays of fibrinolysis.

    Science.gov (United States)

    Ilich, A; Bokarev, I; Key, N S

    2017-10-01

    Fibrinolysis is an important and integral part of the hemostatic system. Acting as a balance to blood coagulation, the fibrinolytic system protects the body from unwanted thrombus formation and occlusion of blood vessels. As long as blood coagulation and fibrinolysis remain in equilibrium, response to injury, such as vessel damage, is appropriately regulated. However, alterations in this balance may lead to thrombosis or bleeding. A variety of methods have been proposed to assess fibrinolytic activity in blood or its components, but due to the complexity of the system, the design of a "gold standard" assay that reflects overall fibrinolysis has remained an elusive goal. In this review, we describe the most commonly used methods that have been described, such as thromboelastography (TEG and ROTEM), global fibrinolytic capacity in plasma and whole blood, plasma turbidity methods, simultaneous thrombin and plasmin generation assays, euglobulin clot lysis time and fibrin plate methods. All of these assays have strengths and limitations. We suggest that some methods may be preferable for detecting hypofibrinolytic conditions, whereas others may be better for detecting hyperfibrinolytic states. © 2017 John Wiley & Sons Ltd.

  8. Strong negative interference of ethamsylate (Dicynone®) in serum creatinine quantification via enzymatic assay using Trinder reaction.

    Science.gov (United States)

    Wiewiorka, Ondrej; Dastych, Milan; Čermáková, Zdenka

    2013-08-01

    With discrepancies encountered as early as the verification of enzymatic method for quantification of serum creatinine, our research pointed to a later confirmed interference caused by a compound called ethamsylate present in the commonly used antihemorrhagic drug Dicynone. We measured concentrations of creatinine of 10 patients with blood taken before and 15 minutes after the intravenous administration of a 500 mg dose of Dicynone. The creatinine concentration was determined using Jaffe method and enzymatic method that utilize Trinder reaction (Roche) in analyzer Cobas c 501 (Roche AG, Basel, Switzerland). We also monitored concentration of blood creatinine in three patients before and 15 minutes after application of Dicynone (500 mg i.v.) and in the following 6th, 12th, 18th, and 24th hours. We discovered a significant negative bias in creatinine results using enzymatic assay with Trinder reaction in blood taken 15 min after i.v. application of 500 mg Dicynone to patients compared to their pre-application values (average decrease of 47%). Unlike this, the results of compensated Jaffe method yielded steady results in all samples (average deviation 0.6% from original values). However, 12 h after the drug administration comparable results were seen as before the administration. Considering the strong negative interference of ethamsylate in enzymatic assay using Trinder reaction for creatinine quantification, blood from patients with prescribed Dicynone should be taken at least 12 h after the last application of the drug for obtaining the correct creatinine values.

  9. Use of chimeric influenza viruses as a novel internal control for diagnostic rRT-PCR assays.

    Science.gov (United States)

    Wang, Xueliang; Liu, Fen; Jiang, Lingli; Bao, Yun; Xiao, Yanqun; Wang, Hualiang

    2016-02-01

    Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5′ untranslated region (5′UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays.

  10. Verification of an Assay for Quantification of Hepatitis C Virus RNA by Use of an Analyte-Specific Reagent and Two Different Extraction Methods

    Science.gov (United States)

    Forman, Michael S.; Valsamakis, Alexandra

    2004-01-01

    Protocols were designed for quantification and detection of hepatitis C virus (HCV) RNA by the use of an analyte-specific reagent (ASR) (Roche COBAS TaqMan48 [CTM48] HCV) after manual and automated RNA extraction. The purposes were to determine (i) assay performance characteristics using manual and automated RNA extraction methods, (ii) whether measurable range and limit of detection (LOD) of the ASR assay were influenced by genotype, and (iii) correlation of quantification by CTM48 HCV ASR and COBAS Monitor HCV v. 2.0. For HCV genotype 1 (Gt1), the lower limits of quantification after manual extraction were slightly lower than those for automated extraction (1.0 versus 1.5 log10 IU/ml). Results were linear up to the highest concentration tested after extraction by both methods (manual, 6.1 log10; automated, 6.4 log10). Similar results were obtained for Gt2 (1.8 to 6.8 log10 IU/ml) and Gt3 (1.6 to 6.8 log10 IU/ml) after automated extraction. The LOD of Gt1 virus was 10 IU/ml after manual extraction and between 25 and 37.5 IU/ml after automated extraction. Results with Gt2 and Gt3 viruses were similar after automated extraction (Gt2, between 25 and 50 IU/ml; Gt3, 25 IU/ml). Variability (intrarun and interrun, at concentrations throughout the range of quantification) was ≤13% for both extraction methods. Clinical specimens tested by Monitor were quantified using the CTM48 HCV ASR assay. Characteristics of the regression line included a slope of 0.98 and y intercept of −0.23. Quantification by the two methods was correlated (r = 0.97). CTM48 HCV ASR assay values were on average twofold lower than those for Monitor HCV v. 2.0. These data suggest that our assay combines the characteristics of qualitative and quantitative PCR platforms into a single test. PMID:15297501

  11. Implementación del ensayo de carga viral COBAS Taqman HIV-1 Test, v1.0, para el diagnóstico de la infección congénita por HIV-1

    Directory of Open Access Journals (Sweden)

    Gonzalo M Castro

    2015-03-01

    Full Text Available La transmisión vertical es la principal vía de contagio del HIV en la edad pediátrica. El diagnóstico de la infección congénita antes de los 18 meses se realiza mediante ensayos virológicos: detección de genoma viral como ARN plasmático y ADN proviral. La sensibilidad de estos ensayos varía según la edad del niño, con valores de especificidad mayores al 95 %. El objetivo de este trabajo fue evaluar el desempeño del ensayo de carga viral (CV COBAS Taqman HIV-1 Test, v1.0 (Roche, y su concordancia con una PCR múltiple anidada in-house para la detección del ADN proviral. De 341 muestras procesadas, 15 resultaron positivas y 326 negativas por ambas metodologías. Para la metodología de CV, la sensibilidad general fue del 88,2 % y la especificidad del 100 %. Nuestros resultados indican que la metodología de CV evaluada puede utilizarse como técnica alternativa para el diagnóstico de infección congénita por HIV.

  12. Evaluation of the NucliSens EasyQ v2.0 assay in comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in quantification of C-clade HIV-1 in plasma.

    Directory of Open Access Journals (Sweden)

    Maximilian Muenchhoff

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0, the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0 and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5. Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 - Amplicor v1.5, 0.260 log cp/ml (CAP/CTM v2.0 - Amplicor v1.5 and 0.164 log cp/ml (CAP/CTM v2.0 - NucliSens v2.0, indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml in more than one-third of those in whom viremia had been undetectable (<20 cp/ml in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.

  13. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  14. The Usefulness of Defining Rapid Virological Response by a Very Sensitive Assay (TMA) during Treatment of HCV Genotype 2/3 Infection

    DEFF Research Database (Denmark)

    Dalgard, Olav; Martinot-Peignoux, Michelle; Verbaan, Hans

    2015-01-01

    The aim of this study was to determine in patients with HCV genotype 2 or 3 the performance at week 4 of two assays with different sensitivities for HCV RNA detection, for the prediction of SVR and stratification for treatment duration (14 and 24 weeks). Recruitment was from two trials comparing ...... measured with COBAS Amplicor V2, Roche (CA) (lower detection limit 50 IU/ml) and retrospectively assessed with VERSANT HCV-RNA Qualitative Assay, Siemens (TMA) (lower limit detection 10 IU/ml). Genotype 3 was present in 80% and genotype 2 in 20%. A SVR was achieved in 82%. At week 4 HCV....../TMA positive (p = 0.006). In patients allocated to 14 weeks treatment a relapse was observed in 11% of TMA undetectable patients and 26% of TMA positive (p = 0.031). In genotype 2 patients treated for 14 weeks relapse was observed in 6% of the patients with both CA and TMA undetectable week 4. Assays with high...

  15. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention...... is adapted to receive one or more replaceable solid support(s) (40) onto which chemical entities (41) are attached, said device comprising a base (1, 60, 80, 300, 400, 10, 70, 140, 20, 90, 120, 150, 30, 100), one or more inlet(s) (5), one or more outlet(s) (6). The base and the solid support (40) defines......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  16. Survival assays using Caenorhabditis elegans.

    Science.gov (United States)

    Park, Hae-Eun H; Jung, Yoonji; Lee, Seung-Jae V

    2017-02-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans.

  17. Cell Proliferation and Cytotoxicity Assays.

    Science.gov (United States)

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  18. [Detection of RAS genes mutation using the Cobas®method in a private laboratory of pathology: Medical and economical study in comparison to a public platform of molecular biology of cancer].

    Science.gov (United States)

    Albertini, Anne-Flore; Raoux, Delphine; Neumann, Frédéric; Rossat, Stéphane; Tabet, Farid; Pedeutour, Florence; Duranton-Tanneur, Valérie; Kubiniek, Valérie; Vire, Olivier; Weinbreck, Nicolas

    In France, determination of the mutation status of RAS genes for predictive response to anti-EGFR targeted treatments is carried out by public platforms of molecular biology of cancer created by the French National Cancer Institute. This study aims to demonstrate the feasibility of these analyses by a private pathology laboratory (MEDIPATH) as per the requirements of accreditation. We retrospectively studied the mutation status of KRAS and NRAS genes in 163 cases of colorectal metastatic cancer using the Cobas ® technique. We compared our results to those prospectively obtained through pyrosequencing and allelic discrimination by the genetic laboratory of solid tumors at the Nice University Hospital (PACA-EST regional platform). The results of both series were identical: 98.7% positive correlation; negative correlation of 93.1%; overall correlation of 95.7% (Kappa=0.92). This study demonstrates the feasibility of molecular analysis in a private pathology laboratory. As this practice requires a high level of guarantee, its accreditation, according to the NF-EN-ISO15189 quality compliance French standard, is essential. Conducting molecular analysis in this context avoids the steps of routing the sample and the result between the pathology laboratory and the platform, which reduces the overall time of rendering the result. In conclusion, the transfer of some analysis from these platforms to private pathology laboratories would allow the platforms to be discharged from a part of routine testing and therefore concentrate their efforts to the development of new analyses constantly required to access personalized medicine. Copyright © 2017. Published by Elsevier Masson SAS.

  19. A comparative evaluation of the analytical performances of Capillarys 2 Flex Piercing, Tosoh HLC-723 G8, Premier Hb9210, and Roche Cobas c501 Tina-quant Gen 2 analyzers for HbA1c determination.

    Science.gov (United States)

    Wu, Xiaobin; Chao, Yan; Wan, Zemin; Wang, Yunxiu; Ma, Yan; Ke, Peifeng; Wu, Xinzhong; Xu, Jianhua; Zhuang, Junhua; Huang, Xianzhang

    2016-10-15

    Haemoglobin A1c (HbA1c) is widely used in the management of diabetes. Therefore, the reliability and comparability among different analytical methods for its detection have become very important. A comparative evaluation of the analytical performances (precision, linearity, accuracy, method comparison, and interferences including bilirubin, triglyceride, cholesterol, labile HbA1c (LA1c), vitamin C, aspirin, fetal haemoglobin (HbF), and haemoglobin E (Hb E)) were performed on Capillarys 2 Flex Piercing (Capillarys 2FP) (Sebia, France), Tosoh HLC-723 G8 (Tosoh G8) (Tosoh, Japan), Premier Hb9210 (Trinity Biotech, Ireland) and Roche Cobas c501 (Roche c501) (Roche Diagnostics, Germany). A good precision was shown at both low and high HbA1c levels on all four systems, with all individual CVs below 2% (IFCC units) or 1.5% (NGSP units). Linearity analysis for each analyzer had achieved a good correlation coefficient (R2 > 0.99) over the entire range tested. The analytical bias of the four systems against the IFCC targets was less than ± 6% (NGSP units), indicating a good accuracy. Method comparison showed a great correlation and agreement between methods. Very high levels of triglycerides and cholesterol (≥ 15.28 and ≥ 8.72 mmol/L, respectively) led to falsely low HbA1c concentrations on Roche c501. Elevated HbF induced false HbA1c detection on Capillarys 2FP (> 10%), Tosoh G8 (> 30%), Premier Hb9210 (> 15%), and Roche c501 (> 5%). On Tosoh G8, HbE induced an extra peak on chromatogram, and significantly lower results were reported. The four HbA1c methods commonly used with commercial analyzers showed a good reliability and comparability, although some interference may falsely alter the result.

  20. Analytical evaluation of three enzymatic assays for measuring total bile acids in plasma using a fully-automated clinical chemistry platform.

    Science.gov (United States)

    Danese, Elisa; Salvagno, Gian Luca; Negrini, Davide; Brocco, Giorgio; Montagnana, Martina; Lippi, Giuseppe

    2017-01-01

    Although the clinical significance of measuring bile acids concentration in plasma or serum has been recognized for long in patients with hepatobiliary disease and/or bile acid malabsorption, the reference separation techniques are expensive and mostly unsuitable for early diagnosis and for measuring large volumes of samples. Therefore, this study was aimed to evaluate the analytical performance of three commercial enzymatic techniques for measuring total bile acids in plasma using a fully-automated clinical chemistry platform. Three commercial enzymatic assays (from Diazyme, Randox and Sentinel) were adapted for use on a Cobas Roche c501. We performed imprecision and linearity studies, and we compared results with those obtained using a reference liquid chromatography-mass spectrometry (LC-MS) technique on an identical set of lithium-heparin plasma samples. Total imprecision was optimal, always equal or lower than 3%. All assays had optimal linearity between 3-138 μmol/L. The comparison studies showed good correlation with LC-MS data (Spearman's correlation coefficients always >0.92), but all plasma samples values were significantly underestimated using the commercial enzymatic assays (-44% for Diazyme, -16% for Randox and -12% for Sentinel). The agreement at the 10 and 40 μmol/L diagnostic thresholds of total bile acids in plasma ranged between 86-92%. This discrepancy was found to be mainly attributable to a heterogeneous composition in terms of bile acids content of the three assay calibrators. This study suggests that the analytical performance of the three commercial enzymatic assays is excellent, thus confirming that automation of this important test by means of enzymatic assessment may be feasible, practical, reliable and supposedly cheap. Nevertheless, the underestimation of values compared to the reference LC-MS also suggests that the local definition and validation of reference ranges according to the combination between the specific enzymatic assay

  1. Assay for calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  2. Development of a digital droplet PCR assay to measure HBV DNA in patients receiving long-term TDF treatment.

    Science.gov (United States)

    Liu, Yang; Cathcart, Andrea L; Delaney, William E; Kitrinos, Kathryn M

    2017-11-01

    The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (HBV DNA to 8 copies/mL. HBV DNA levels in plasma from patients with or without HBsAg seroconversion were assessed by ddPCR. For patients who did not achieve HBsAg seroconversion, the majority of TD samples (33/58, 57%) were HBV DNA positive by ddPCR while (10/37, 27%) of TND samples were positive. In contrast, for patients who achieved HBsAg seroconversion, HBV DNA was rarely detected by ddPCR after HBsAg seroconversion (1/28, 3.6%). ddPCR is a sensitive method to evaluate low-level viral replication in plasma samples. Frequent detection of HBV DNA by ddPCR among patients who did not achieve HBsAg seroconversion suggests new agents may be needed to suppress low levels of replicating HBV. Copyright © 2017. Published by Elsevier B.V.

  3. Sulforhodamine B assay and chemosensitivity.

    Science.gov (United States)

    Voigt, Wieland

    2005-01-01

    The sulforhodamine B (SRB) assay was developed by Skehan and colleagues to measure drug-induced cytotoxicity and cell proliferation for large-scale drug-screening applications. Its principle is based on the ability of the protein dye sulforhodamine B to bind electrostatically and pH dependent on protein basic amino acid residues of trichloroacetic acid-fixed cells. Under mild acidic conditions it binds to and under mild basic conditions it can be extracted from cells and solubilized for measurement. Results of the SRB assay were linear with cell number and cellular protein measured at cellular densities ranging from 1 to 200% of confluence. Its sensitivity is comparable with that of several fluorescence assays and superior to that of Lowry or Bradford. The signal-to-noise ratio is favorable and the resolution is 1000-2000 cells/well. It performed similarly compared to other cytotoxicity assays such as MTT or clonogenic assay. The SRB assay possesses a colorimetric end point and is nondestructive and indefinitely stable. These practical advances make the SRB assay an appropriate and sensitive assay to measure drug-induced cytotoxicity even at large-scale application.

  4. Digital Assays Part II: Digital Protein and Cell Assays.

    Science.gov (United States)

    Basu, Amar S

    2017-08-01

    A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

  5. Ethamsylate (Dicynone) interference in determination of serum creatinine, uric acid, triglycerides, and cholesterol in assays involving the Trinder reaction; in vivo and in vitro.

    Science.gov (United States)

    Dastych, Milan; Wiewiorka, Ondrej; Benovská, Miroslava

    2014-01-01

    The aim of our research was the quantification of interfering properties of the haemostatic drug Dicynone (ethamsylate) in serum creatinine, uric acid, cholesterol, and triglyceride assays using the Trinder reaction. Blood from patients was collected before and 15 minutes after administration of 500 mg Dicynone dose i.v. and the above mentioned analytes were quantified using Roche assays (Cobas 8000). In our in vitro experiment, we measured concentrations of the analytes in pooled serum aliquots with final concentrations of Dicynone additions 0, 30, 60, 150, and 300 mg/L. Aliquots with 60 mg/L Dicynone were also measured at 2, 6, and 8 hours after initial measurement when stored in 22 degrees C and 4 degrees C for comparison. Concentrations of the measured analytes in samples from patients administered with a 500 mg dose of Dicynone were lower in all cases (n = 10) when compared to values in samples taken immediately before treatment. The in vitro samples showed that considerable negative interference occurred even with the low concentrations of Dicynone additions (30 and 60 mg/L), showing the strongest negative interference in creatinine values, followed by uric acid, triglycerides, and cholesterol. Using in vitro samples, we showed strong time and temperature dependence on Dicynone interference. We found and proved significant negative interference of the drug Dicynone (ethamsylate) in the clinical analysis of blood using in vivo and in vitro experiments. Furthermore, we observed a change of this effect in serum matrix over time and at different storage temperatures.

  6. Biological assays in microfabricated structures

    Science.gov (United States)

    Fishman, Daniel M.; Fare, Thomas L.; Dong, Qianping; Fan, Z. Hugh; Davis, Timothy J.; Kumar, Rajan

    1999-04-01

    Microfluidic control in microfabricated glass channels enables miniaturized, fast, and multianalyte assays. We are applying this technology in several areas, including real- time environmental monitoring for airborne biological agents. Two complementary approaches are being used in parallel. the first is assaying for the presence of nucleotide sequences that are markers for specific hazardous, engineered bacteria. The second is an assay that monitors the functionality of an in vitro biochemical pathway, in which the pathway that is chosen is sensitive to the presence of the class of toxins to be detected. The first approach is discussed here. The detected signal from the nucleic-acid-based assay is from fluorescently labeled probes that hybridize to bead-bound amplified DNA sequences. Detection approaches and their benefits will be discussed.

  7. Microtiter dish biofilm formation assay.

    Science.gov (United States)

    O'Toole, George A

    2011-01-30

    Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics. The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors. This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes

  8. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  9. Activity Assays for Rhomboid Proteases.

    Science.gov (United States)

    Arutyunova, E; Strisovsky, K; Lemieux, M J

    2017-01-01

    Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein. © 2017 Elsevier Inc. All rights reserved.

  10. CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort.

    Science.gov (United States)

    Estey, Mathew P; Cohen, Ashley H; Colantonio, David A; Chan, Man Khun; Marvasti, Tina Binesh; Randell, Edward; Delvin, Edgard; Cousineau, Jocelyne; Grey, Vijaylaxmi; Greenway, Donald; Meng, Qing H; Jung, Benjamin; Bhuiyan, Jalaluddin; Seccombe, David; Adeli, Khosrow

    2013-09-01

    The CALIPER program recently established a comprehensive database of age- and sex-stratified pediatric reference intervals for 40 biochemical markers. However, this database was only directly applicable for Abbott ARCHITECT assays. We therefore sought to expand the scope of this database to biochemical assays from other major manufacturers, allowing for a much wider application of the CALIPER database. Based on CLSI C28-A3 and EP9-A2 guidelines, CALIPER reference intervals were transferred (using specific statistical criteria) to assays performed on four other commonly used clinical chemistry platforms including Beckman Coulter DxC800, Ortho Vitros 5600, Roche Cobas 6000, and Siemens Vista 1500. The resulting reference intervals were subjected to a thorough validation using 100 reference specimens (healthy community children and adolescents) from the CALIPER bio-bank, and all testing centers participated in an external quality assessment (EQA) evaluation. In general, the transferred pediatric reference intervals were similar to those established in our previous study. However, assay-specific differences in reference limits were observed for many analytes, and in some instances were considerable. The results of the EQA evaluation generally mimicked the similarities and differences in reference limits among the five manufacturers' assays. In addition, the majority of transferred reference intervals were validated through the analysis of CALIPER reference samples. This study greatly extends the utility of the CALIPER reference interval database which is now directly applicable for assays performed on five major analytical platforms in clinical use, and should permit the worldwide application of CALIPER pediatric reference intervals. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  11. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  12. Comet Assay in Cancer Chemoprevention.

    Science.gov (United States)

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  13. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  14. Antioxidants and the Comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

    2009-01-01

    It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

  15. SNO+ Scintillator Purification and Assay

    Science.gov (United States)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  16. Reporter Gene Assays in Ecotoxicology.

    Science.gov (United States)

    Elad, Tal; Belkin, Shimshon

    The need for simple and rapid means for evaluating the potential toxic effects of environmental samples has prompted the development of reporter gene assays, based on tester cells (bioreporters) genetically engineered to report on sample toxicity by producing a readily quantifiable signal. Bacteria are especially suitable to serve as bioreporters owing to their fast responses, low cost, convenient preservation, ease of handling, and amenability to genetic manipulations. Various bacterial bioreporters have been introduced for general toxicity and genotoxicity assessment, and the monitoring of endocrine disrupting and dioxin-like compounds has been mostly covered by similarly engineered eukaryotic cells. Some reporter gene assays have been validated, standardized, and accredited, and many others are under constant development. Efforts are aimed at broadening detection spectra, lowering detection thresholds, and combining toxicity identification capabilities with characterization of the toxic effects. Taking advantage of bacterial robustness, attempts are also being made to incorporate bacterial bioreporters into field instrumentation for online continuous monitoring or on-site spot checks. However, key hurdles concerning test validation, cell preservation, and regulatory issues related to the use of genetically modified organisms still remain to be overcome.

  17. In vivo transgenic mutation assays.

    Science.gov (United States)

    Thybaud, Véronique; Dean, Stephen; Nohmi, Takehiko; de Boer, Johan; Douglas, George R; Glickman, Barry W; Gorelick, Nancy J; Heddle, John A; Heflich, Robert H; Lambert, Iain; Martus, Hans-Jörg; Mirsalis, Jon C; Suzuki, Takayoshi; Yajima, Nobuhiro

    2003-10-07

    Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when

  18. Predictive Assay For Cancer Targets

    Energy Technology Data Exchange (ETDEWEB)

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  19. Predictive assay for cancer targets

    Science.gov (United States)

    Suess, Amanda; Nguyen, Christine; Sorensen, Karen; Montgomery, Jennifer; Souza, Brian; Kulp, Kris; Dugan, Larry; Christian, Allen

    2005-11-01

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  20. Detailed review of transgenic rodent mutation assays.

    Science.gov (United States)

    Lambert, Iain B; Singer, Timothy M; Boucher, Sherri E; Douglas, George R

    2005-09-01

    Induced chromosomal and gene mutations play a role in carcinogenesis and may be involved in the production of birth defects and other disease conditions. While it is widely accepted that in vivo mutation assays are more relevant to the human condition than are in vitro assays, our ability to evaluate mutagenesis in vivo in a broad range of tissues has historically been quite limited. The development of transgenic rodent (TGR) mutation models has given us the ability to detect, quantify, and sequence mutations in a range of somatic and germ cells. This document provides a comprehensive review of the TGR mutation assay literature and assesses the potential use of these assays in a regulatory context. The information is arranged as follows. (1) TGR mutagenicity models and their use for the analysis of gene and chromosomal mutation are fully described. (2) The principles underlying current OECD tests for the assessment of genotoxicity in vitro and in vivo, and also nontransgenic assays available for assessment of gene mutation, are described. (3) All available information pertaining to the conduct of TGR assays and important parameters of assay performance have been tabulated and analyzed. (4) The performance of TGR assays, both in isolation and as part of a battery of in vitro and in vivo short-term genotoxicity tests, in predicting carcinogenicity is described. (5) Recommendations are made regarding the experimental parameters for TGR assays, and the use of TGR assays in a regulatory context.

  1. An ultrafiltration assay for lysyl oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Shackleton, D.R.; Hulmes, D.J. (Univ. of Edinburgh Medical School (England))

    1990-03-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a (4,5-3H)-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.

  2. Development and application of assays for serotonin

    Energy Technology Data Exchange (ETDEWEB)

    Gow, I.F.

    1987-01-01

    In this thesis, two assays for serotonin were developed, validated, and used to investigate the relationship between platelet aggregation, serotonin levels and sodium status and serotonin levels and platelet function in patients with cardiovascular disease. A radioimmunoassay (RIA) using an (/sup 125/I)-labelled tracer was developed and validated for the measurement of serotonin in human platelet-rich plasma (PRP) and rat serum. Antisera were raised against N-succinamylserotonin conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. An assay for serotonin using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) was developed, and used to validate the RIA. The RIA can be used to assay up to 100 samples/day compared with 10-20/day by the HPLC-ECD assay.

  3. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert

    2016-01-01

    OBJECTIVE: Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab......) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...... with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative...

  4. A lateral electrophoretic flow diagnostic assay

    OpenAIRE

    Lin, R; Skandarajah, A; Gerver, RE; Neira, HD; Fletcher, DA; Herr, AE

    2015-01-01

    © 2015 The Royal Society of Chemistry. Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to ...

  5. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

    Science.gov (United States)

    Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David

    2011-08-01

    Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

  6. METHODOLOGICAL ASPECTS OF QUANTITATIVE RECEPTOR ASSAYS

    NARCIS (Netherlands)

    SMISTEROVA, J; ENSING, K; DEZEEUW, RA

    Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand

  7. A lateral electrophoretic flow diagnostic assay.

    Science.gov (United States)

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  8. Assessing sediment contamination using six toxicity assays

    Directory of Open Access Journals (Sweden)

    Allen G. BURTON Jr.

    2001-08-01

    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  9. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    DEFF Research Database (Denmark)

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred

    2016-01-01

    and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2...

  10. Towards a systematic assessment of assay interference: Identification of extensively tested compounds with high assay promiscuity

    National Research Council Canada - National Science Library

    Gilberg Erik; Stumpfe Dagmar; Bajorath Jürgen

    2017-01-01

    A large-scale statistical analysis of hit rates of extensively assayed compounds is presented to provide a basis for a further assessment of assay interference potential and multi-target activities...

  11. Comet assay on mice testicular cells

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar

    2015-01-01

    for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity...... of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided...... with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014). An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster...

  12. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation...... of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...... International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c...

  13. DNA Methyltransferase Activity Assays: Advances and Challenges.

    Science.gov (United States)

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice.

  14. Automation of a chloramphenicol acetyltransferase assay.

    Science.gov (United States)

    Chauchereau, A; Astinotti, D; Bouton, M M

    1990-08-01

    Accurate quantification of chloramphenicol acetyltransferase (CAT) enzyme activity in a large number of samples has been achieved through robotization of a CAT assay on a laboratory workstation (Biomek 1000). The basic principle of this CAT assay relies on the selective diffusion of [3H]acetylchloramphenicol into a water-immiscible liquid scintillation cocktail. This methodology gives unique characteristics to this robotized protocol by allowing complete control over the kinetics of the CAT enzymatic reaction which is a critical parameter in the CAT assay. Thus it has been possible to optimize the CAT assay for every processed sample, through real time monitoring of the enzymatic reaction, and to achieve maximum accuracy in CAT quantification. Moreover the sensitivity of this automated assay is high (detection threshold; 10(-4) CAT unit), and the sample processing is fast (approximately 125 samples per hour). Compared to other CAT assay protocols currently used, our robotized technique offers major advantages in terms of CAT quantification, and sets new standards for CAT assay productivity.

  15. Nano-immunosafety: issues in assay validation

    Energy Technology Data Exchange (ETDEWEB)

    Boraschi, Diana; Italiani, Paola [Institute of Biomedical Technologies, National Research Council, Via G. Moruzzi 1, 56124 Pisa (Italy); Oostingh, Gertie J; Duschl, Albert [Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Casals, Eudald; Puntes, Victor F [Institut Catala de Nanotecnologia, Campus de la UAB - Facultat de Ciencies, Edifici CM7, 08193 Bellaterra (Spain); Nelissen, Inge, E-mail: diana.boraschi@itb.cnr.it [VITO NV, Boeretang 200, BE-2400 Mol (Belgium)

    2011-07-06

    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  16. The comet assay in nanotoxicology research.

    Science.gov (United States)

    Karlsson, Hanna L

    2010-09-01

    Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.

  17. A bioluminescence assay for aldehyde dehydrogenase activity.

    Science.gov (United States)

    Duellman, Sarah J; Valley, Michael P; Kotraiah, Vinayaka; Vidugiriene, Jolanta; Zhou, Wenhui; Bernad, Laurent; Osterman, Jean; Kimball, Joshua J; Meisenheimer, Poncho; Cali, James J

    2013-03-15

    The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay, the 6- and 24-well miniaturized plate incorporation assays and the Ames II™ assay.

    Science.gov (United States)

    Pant, Kamala; Bruce, Shannon; Sly, Jamie; Klug Laforce, Michelle; Springer, Sandra; Cecil, Mark; Andrus, Edgar; Dakoulas, Emily; Wagner, Valentine O; Hewitt, Nicola J; Kulkarni, Rohan

    2016-07-01

    Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non-OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II™ assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre-incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6-well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24-well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II™ assay (TA98 and TAMix). Although miniaturization to a 24-well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II™ assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483-496, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Reporter gene assays for investigating GPCR signaling.

    Science.gov (United States)

    Azimzadeh, Pedram; Olson, John A; Balenga, Nariman

    2017-01-01

    Luciferase-based assays are applied to evaluate various cellular processes due to their sensitivity and feasibility. The field of GPCR research has also benefited from this enzymatic reaction both in deorphanization campaigns and in delineation of the signaling pathways. Here, we describe the details of this assay in GPCR studies in 96-well format and will provide examples where the assay can show constitutive activity of an orphan GPCR and demonstrate the impact of cell type on the efficacy and potency of ligands. © 2017 Elsevier Inc. All rights reserved.

  20. Passive neutron assay of irradiated nuclear fuels

    Energy Technology Data Exchange (ETDEWEB)

    Hsue, S.T.; Stewart, J.E.; Kaieda, K.; Halbig, J.K.; Phillips, J.R.; Lee, D.M.; Hatcher, C.R.

    1979-02-01

    Passive neutron assay of irradiated nuclear fuel has been investigated by calculations and experiments as a simple, complementary technique to the gamma assay. From the calculations it was found that the neutron emission arises mainly from the curium isotopes, the neutrons exhibit very good penetrability of the assemblies, and the neutron multiplication is not affected by the burnup. From the experiments on BWR and PWR assemblies, the neutron emission rate is proportional to burnup raised to 3.4 power. The investigations indicate that the passive neutron assay is a simple and useful technique to determine the consistency of burnups between assemblies.

  1. 21 CFR 866.3210 - Endotoxin assay.

    Science.gov (United States)

    2010-04-01

    ... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a... intended for use in conjunction with other laboratory findings and clinical assessment of the patient to...

  2. NEW INDIRECT IMMUNOFLUORESCENCE ASSAY AS A ...

    African Journals Online (AJOL)

    hi-tech

    2004-05-05

    HIV-1) infection is based on the detection of antibodies to HIV-1 in plasma or serum. Antibodies against various viral structure proteins are measured by a number of simple and sensitive screening tests. These assays include ...

  3. Validation of a new automated renin assay.

    NARCIS (Netherlands)

    Bruin, R.A. de; Bouhuizen, A.; Diederich, S.; Perschel, F.H.; Boomsma, F.; Deinum, J.

    2004-01-01

    BACKGROUND: Measurement of plasma renin is important for the treatment of patients with congenital adrenal hyperplasia (CAH) and in the evaluation of patients with suspected hyperaldosteronism. Immunologic assays for plasma renin offer easier implementation and standardization than enzyme-kinetic

  4. LINE-1 Cultured Cell Retrotransposition Assay

    Science.gov (United States)

    Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

    2016-01-01

    Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

  5. Automated optical sensing system for biochemical assays

    Science.gov (United States)

    Oroszlan, Peter; Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

    1994-03-01

    In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

  6. The comet assay in nanotoxicology research

    OpenAIRE

    Karlsson, Hanna L

    2010-01-01

    Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The ar...

  7. Nuclear Resonance Fluorescence for Materials Assay

    OpenAIRE

    Quiter, Brian

    2010-01-01

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code M...

  8. Nondestructive assay methods for irradiated nuclear fuels

    Energy Technology Data Exchange (ETDEWEB)

    Hsue, S.T.; Crane, T.W.; Talbert, W.L. Jr.; Lee, J.C.

    1978-01-01

    This report is a review of the status of nondestructive assay (NDA) methods used to determine burnup and fissile content of irradiated nuclear fuels. The gamma-spectroscopy method measures gamma activities of certain fission products that are proportional to the burnup. Problems associated with this method are migration of the fission products and gamma-ray attenuation through the relatively dense fuel material. The attenuation correction is complicated by generally unknown activity distributions within the assemblies. The neutron methods, which usually involve active interrogation and prompt or delayed signal counting, are designed to assay the fissile content of the spent-fuel elements. Systems to assay highly enriched spent-fuel assemblies have been tested extensively. Feasibility studies have been reported of systems to assay light-water reactor spent-fuel assemblies. The slowing-down spectrometer and neutron resonance absorption methods can distinguish between the uranium and plutonium fissile contents, but they are limited to the assay of individual rods. We have summarized the status of NDA techniques for spent-fuel assay and present some subjects in need of further investigation. Accuracy of the burnup calculations for power reactors is also reviewed.

  9. Quantum dot-based cell motility assay.

    Science.gov (United States)

    Pellegrino, Teresa; Parak, Wolfgang J; Boudreau, Rosanne; Le Gros, Mark A; Gerion, Daniele; Alivisatos, A Paul; Larabell, Carolyn A

    2003-12-01

    Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.

  10. Thread as a matrix for biomedical assays.

    Science.gov (United States)

    Reches, Meital; Mirica, Katherine A; Dasgupta, Rohit; Dickey, Michael D; Butte, Manish J; Whitesides, George M

    2010-06-01

    This paper describes the use of thread as a matrix for the fabrication of diagnostic assay systems. The kinds of thread used for this study are inexpensive, broadly available, and lightweight; some of them are already familiar materials in healthcare. Fluids wick along these threads by capillary action; no external power source is necessary for pumping. This paper demonstrates three designs for diagnostic assays that use different characteristics of the thread. The first two designs-the "woven array" and the "branching design"-take advantage of the ease with which thread can be woven on a loom to generate fluidic pathways that enable multiple assays to be performed in parallel. The third design-the "sewn array"-takes advantage of the ease with which thread can be sewn through a hydrophobic polymer sheet to incorporate assays into bandages, diapers and similar systems. These designs lead to microfluidic devices that may be useful in performing simple colorimetric assays that require qualitative results. We demonstrate the function of thread-based microfluidic devices in the context of five different colorimetric assays: detection of ketones, nitrite, protein, and glucose in artificial urine, and detection of alkaline phosphatase in artificial plasma.

  11. Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies.

    Science.gov (United States)

    Pauwels, Bea; Korst, Annelies E C; de Pooter, Christel M J; Pattyn, Greet G O; Lambrechts, Hilde A J; Baay, Marc F D; Lardon, Filip; Vermorken, Jan B

    2003-03-01

    Since there is a growing interest in preclinical research on interactions between radiation and cytotoxic agents, this study focused on the development of an alternative to the very laborious clonogenic assay (CA). The colorimetric sulforhodamine B (SRB) assay was compared to the clonogenic assay for radiosensitivity testing in two lung cancer cell lines (A549, H292), one colon cancer cell line (HT-29) and one breast cancer cell line (MCF-7). In addition, the combination of the radiosensitizing agent gemcitabine and radiation was investigated with both assays. The dose-response curves obtained with the SRB assay and the CA were very similar up to 6 Gy. The radiosensitivity parameters (SF(2), alpha, beta, MID and ID(50)) obtained from the SRB assay and the CA were not significantly different between H292, A549 and MCF-7 cells. The radiation dose-response curves for A549 and H292 cells pretreated with 4 n M gemcitabine for 24 h clearly showed a radiosensitizing effect with both assays. The dose-enhancement factors obtained with the SRB assay and the CA were 1.80 and 1.76, respectively, for A549 cells, and 1.52 and 1.41 for H292 cells. The SRB assay was shown to be as useful as the more traditional CA for research on chemotherapy/radiotherapy interactions in cell lines with moderate radiosensitivity. This assay will be used for more extensive in vitro research on radiosensitizing compounds in these cell lines.

  12. A multi-centre evaluation of the intra-assay and inter-assay variation of commercial and in-house anti-cardiolipin antibody assays.

    Science.gov (United States)

    Wong, Richard; Favaloro, Emmanuel; Pollock, Wendy; Wilson, Robert; Hendle, Michelle; Adelstein, Stephen; Baumgart, Karl; Homes, Paul; Smith, Stuart; Steele, Richard; Sturgess, Allan; Gillis, David

    2004-04-01

    To assess the intra-assay (intra-run) and inter-assay (inter-run) variation of commercial and in-house IgG and IgM anti-cardiolipin antibody (aCL) assays/kits, and to determine an appropriate maximum value for inclusion in consensus guidelines. Frozen aliquots of two patient specimens and one commercial control were sent to nine laboratories for the evaluation of eight commercial kits and one in-house assay. Intra-assay and inter-assay evaluations were performed with all three samples for IgG aCL, and one patient specimen for IgM aCL. The IgG and IgM aCL values varied considerably between the nine assays/kits. The majority of assays/kits demonstrated less than 20% intra-assay and inter-assay variation, with lower intra-assay and inter-assay variation observed with the commercial control. Single calibrator assays were not consistently associated with higher inter-assay variation than multi-point calibrator assays. An inter-assay coefficient of variation of 20% was determined to be an appropriate maximum value for inclusion in the Australasian aCL Working Party consensus guidelines. Improved standardisation between different assay/kits is still required.

  13. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  14. The comet assay in male reproductive toxicology.

    Science.gov (United States)

    Baumgartner, A; Cemeli, E; Anderson, D

    2009-02-01

    Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.

  15. Controlling variation in the comet assay

    Directory of Open Access Journals (Sweden)

    Andrew Richard Collins

    2014-10-01

    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  16. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Directory of Open Access Journals (Sweden)

    Bas-jan Van Der Leede

    2015-08-01

    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

  17. Comparison of Elecsys Anti-HCV II Assay With Other HCV Screening Assays.

    Science.gov (United States)

    Li, Dongdong; Zhu, Siyuan; Wang, Tingting; An, Jingna; Wang, Lanlan; Tao, Chuanmin

    2016-09-01

    Early detection of hepatitis C virus (HCV) is an important step in preventing progression to cirrhosis and hepatocellular carcinoma. Serologic assays for anti-HCV antibody are valuable first-line tests in the screening and diagnosis of HCV infection. This study's aim was to evaluate the sensitivity and specificity of Elecsys Anti-HCV II assay for HCV screening. A total of 1,044 routine sera, 20 known HCV-positive samples, plus 54 preselected weakly positive samples were tested for anti-HCV with Elecsys Anti-HCV II assay, Elecsys Anti-HCV assays, InTec HCV enzymoimmunoassay (EIA), and Livzon Anti-HCV EIA. Interference test was assessed with additional 423 specimens without clinical evidence of HCV infection: preselected HCV weak reactive samples; dialysis samples; anti-HBc (antibody to HBV core antigen) (+), anti-Treponema pallidum (+), and anti-HIV (+) sera; and samples form autoimmune/alcoholic hepatitis or systemic Lupus erythematosus (SLE). Discrepant results were evaluated with recombinant immunoblot assay. The seroconversion panels were evaluated to assess how early each assay could detect HCV infection. The specificity (99.81%) of the Elecsys Anti-HCV II assay was less than that with the two EIA comparison methods. However, false-negative results were easily seen in the EIA assays. When serial bleeds of HCV panels were compared with the above-mentioned methods, the assay detected acute HCV infection only 3.5 days after a positive HCV-RNA nucleic acid test and earlier than the comparator assays. Sensitivities and specificities of the anti-HCV assays were sufficiently high for use in this study. The Elecsys Anti-HCV II assay is suitable for screening and reliable early detection of HCV infection. © 2015 Wiley Periodicals, Inc.

  18. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Energy Technology Data Exchange (ETDEWEB)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.

    2016-02-12

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  19. Random assay in radioimmunoassay: Feasibility and application compared with batch assay

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

    2016-12-15

    The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

  20. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  1. Comet assay in genetic ecotoxicology: a review.

    Science.gov (United States)

    Cotelle, S; Férard, J F

    1999-01-01

    The Comet assay, also called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay, primarily measures DNA strand breakage in single cells. Since the protocol was published by Singh et al. [1988], its use has increased in different topic areas: clinical applications, human monitoring, radiation biology, genetic toxicology, and genetic ecotoxicology. This study is a review of the investigations that have involved the alkaline version of the Comet assay in genetic ecotoxicology. It focuses mainly on the type of organisms (plants, worms, molluscs, fish, amphibians, and mammalians) but also on the type of cells that have been used for ecotoxicological studies. In the 23 papers published since 1993 and presented here, the original test procedure may have been slightly modified according to the cell type. In vitro and in vivo experiments as well as in situ studies have been carried out in various environments (water, soil, and air). Although the Comet assay is able to detect genotoxic effects of chemical and physical agents, only chemical substances and environmental complex mixtures will be considered in this review. Copyright 1999 Wiley-Liss, Inc.

  2. Assay Portal | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

  3. Assay Characterization Guidance Documents | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    CPTAC characterized assays are defined as those that meet the criteria described in the Assay Characterization Guidance Document. This guidance document aligns with recommendations by the research community as “fit-for-purpose” validation requirements of targeted proteomics assays.

  4. Competitive protein binding assay for piritrexim

    Energy Technology Data Exchange (ETDEWEB)

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. (Wellcome Research Laboratories, Research Triangle Park, NC (USA))

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  5. Nuclear Resonance Fluorescence for Materials Assay

    Science.gov (United States)

    Quiter, Brian J.; Ludewigt, Bernhard A.; Mozin, Vladimir V.; Prussin, Stanley G.

    2011-04-01

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  6. Mathematical Model of Serodiagnostic Immunochromatographic Assay.

    Science.gov (United States)

    Sotnikov, Dmitriy V; Zherdev, Anatoly V; Dzantiev, Boris B

    2017-04-18

    This article describes the mathematical model for an immunochromatographic assay for the detection of specific immunoglobulins against a target antigen (antibodies) in blood/serum (serodiagnosis). The model utilizes an analytical (non-numerical) approach and allows the calculation of the kinetics of immune complexes' formation in a continuous-flow system using commonly available software, such as Microsoft Excel. The developed model could identify the nature of the influence of immunochemical interaction constants and reagent concentrations on the kinetics of the formation of the detected target complex. On the basis of the model, recommendations are developed to decrease the detection limit for an immunochromatographic assay of specific immunoglobulins.

  7. A new fluorescent assay for enalapril maleate.

    Science.gov (United States)

    de los A Oliva, María; Sombra, Lorena L; Olsina, Roberto A; Masi, Adriana N

    2005-09-01

    A new spectrofluorimetric method for the enalapril maleate monitoring was studied. Enalapril maleate was found to be highly photolabile. This drug was evaluated according to photodegradation assay at pH 2.5 and 6. Enalapril maleate was exposed to UVA-UVB radiations. Under these specific conditions was found as degradation product, the diketopiperazine. The modification of the fluorescent properties of enalapril maleate in solution after exposure UV-radiation and the degradation mechanisms were studied. The photodegradation was followed by the developed spectrofluorimetric assay.

  8. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

    Directory of Open Access Journals (Sweden)

    Angela Filomena

    2017-10-01

    Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  9. Increased sensitivity of SPR assays in plasma through efficient parallel assay optimization.

    Science.gov (United States)

    Moberg, Anna; Lager, Anna; Hämäläinen, Markku D; Jarhede, Tanja

    2013-05-05

    The sensitivity of biosensor assays in complex media such as plasma or serum is often limited by non-specific binding. The degree of binding often varies between individuals and therefore a large number of different plasma samples have to be used during assay development. Some surface plasmon resonance (SPR) biosensors allow for parallel screening of several running buffer compositions, with a number of different immobilization levels for each buffer. These technical possibilities combined with statistical design of experiments (DoE) enable efficient parallel optimization of multiple assay conditions. In this paper we outline how to increase the sensitivity in SPR-based assays by minimizing background binding and variability from negative control plasma while retaining high signals from positive samples. To mimic immunogenicity studies of biotherapeutics we have used a model assay with anti-rituximab as an anti-drug antibody to be detected in plasma by binding to immobilized rituximab. Immobilization level and sodium chloride concentration were found to be the most important factors to optimize. There were also a number of significant interaction effects and strong non-linearites between the buffer composition/immobilization level and the assay performance, which necessitated DoE based optimization strategies. The applicability of the optimized conditions was verified with several assays (anti-erythropoietin, omalizumab, anti-IgE and anti-myoglobin) in spiked plasma samples resulting in detection levels in the range of 80-170 ng ml(-1). The buffer conditions presented in this paper can be used for other immunogenicity assays on biosensor platforms or as a good starting point for further assay development for new immunogenicity assays. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Performance and logistical challenges of alternative HIV-1 virological monitoring options in a clinical setting of Harare, Zimbabwe

    NARCIS (Netherlands)

    Ondoa, Pascale; Shamu, Tinei; Bronze, Michelle; Wellington, Maureen; Boender, Tamara Sonia; Manting, Corry; Steegen, Kim; Luethy, Rudi; Rinke de Wit, Tobias

    2014-01-01

    We evaluated a low-cost virological failure assay (VFA) on plasma and dried blood spot (DBS) specimens from HIV-1 infected patients attending an HIV clinic in Harare. The results were compared to the performance of the ultrasensitive heat-denatured p24 assay (p24). The COBAS AmpliPrep/COBAS TaqMan

  11. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Science.gov (United States)

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  12. Development and validation of ultraviolet spectrophotometric assay ...

    African Journals Online (AJOL)

    In order to validate the method, the timeabsorbance relationship, optical characteristics (limit of detection, limit of quantitation, wavelength of maximum absorption, slope, linearity, intercept and Beer's law limits), accuracy, inter-day precision and intra-day precision of the assay method were studied. The absorbance of ...

  13. A novel fluorescent assay for sucrose transporters

    Directory of Open Access Journals (Sweden)

    Gora Peter J

    2012-04-01

    Full Text Available Abstract Background We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs to transport the fluorescent coumarin β-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae is routinely used for the heterologous expression of SUTs and does not take up esculin. Results When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS. Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant and BY4742. Conclusions The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.

  14. Assay for Angiotensin-Converting Enzyme.

    Science.gov (United States)

    Russo, Salvatore F.

    1983-01-01

    Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

  15. Optimization of the sulforhodamine B colorimetric assay.

    Science.gov (United States)

    Papazisis, K T; Geromichalos, G D; Dimitriadis, K A; Kortsaris, A H

    1997-10-27

    Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays. However, the cell fixation step in the original assay is subject to error. We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells. A panel of adherent cell lines was used. Signal-to-noise ratios were significantly increased in the new assay. Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities. The linearity of the method improved, with absolute linearity over the whole range of cell densities. The aspiration procedure dislodged only negligible numbers of cells. Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol. We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.

  16. Relationships between ytterbium precipitation assay, colorimetric ...

    African Journals Online (AJOL)

    vitro biological activities of tannins and the commonly used, rapid colorimetric and gravimetric assays of phenolic concentration was of interest. An in vitro gas production bioassay was used to assess the potential biological effect of tannins by incubating tree fruits with and without tannin-binding PEG. Material and Methods.

  17. Wafer-scale Mitochondrial Membrane Potential Assays

    Science.gov (United States)

    Lim, Tae-Sun; Davila, Antonio; Zand, Katayoun; Douglas, Wallace C.; Burke, Peter J.

    2012-01-01

    It has been reported that mitochondrial metabolic and biophysical parameters are associated with degenerative diseases and the aging process. To evaluate these biochemical parameters, current technology requires several hundred milligrams of isolated mitochondria for functional assays. Here, we demonstrate manufacturable wafer-scale mitochondrial functional assay lab-on-a-chip devices, which require mitochondrial protein quantities three orders of magnitude less than current assays, integrated onto 4” standard silicon wafer with new fabrication processes and materials. Membrane potential changes of isolated mitochondria from various well-established cell lines such as human HeLa cell line (Heb7A), human osteosarcoma cell line (143b) and mouse skeletal muscle tissue were investigated and compared. This second generation integrated lab-on-a-chip system developed here shows enhanced structural durability and reproducibility while increasing the sensitivity to changes in mitochondrial membrane potential by an order of magnitude as compared to first generation technologies. We envision this system to be a great candidate to substitute current mitochondrial assay systems. PMID:22627274

  18. Endoproteolytic activity assay in malting barley

    Directory of Open Access Journals (Sweden)

    Blanca Gómez Guerrero

    2013-12-01

    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  19. Protein biomarker validation via proximity ligation assays.

    Science.gov (United States)

    Blokzijl, A; Nong, R; Darmanis, S; Hertz, E; Landegren, U; Kamali-Moghaddam, M

    2014-05-01

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

  20. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

  1. 21 CFR 866.3305 - Herpes simplex virus serological assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices... herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus antisera...

  2. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

  3. Identification of irradiated pepper with comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear. (CEADEN), Ciudad de La Habana (Cuba)]. E-mail: efprieto@ceaden.edu.cu; damaris@ceaden.edu.cu; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)

    2007-07-01

    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  4. A Quantitative Fluorescence-Based Lipase Assay

    Directory of Open Access Journals (Sweden)

    Giovanna Lomolino

    2012-01-01

    Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

  5. Delayed gamma technique for fissile material assay

    Energy Technology Data Exchange (ETDEWEB)

    Mozin, Vladimir [Los Alamos National Laboratory; Tobin, Stephen [Los Alamos National Laboratory; Vujie, Jasmina [UC BERKELEY; Hunt, Alan [IDAHO ACCELERATOR CENTER

    2010-01-01

    Research sponsored by the Next Generation Safeguards Initiative are investigating several non-destructive assay techniques for the quantification of fissile plutonium mass in spent nuclear fuel assemblies. AppHcation of the delayed gamma signatures is investigated in this context. The objective of the research is to assess whether the delayed gamma assay instrument can provide sufficient sensitivity, isotope specificity and accuracy as required in nuclear material safeguards. This effort includes theoretical and experimental components for the optimal combination of interrogation parameters. A new modeling algorithm offering a high level of detail was developed specifically for this purpose and was validated in series of benchmark experiments. Preliminary modeling of the delayed gamma response from spent fuel assemblies was accomplished offering a future direction for the design process.

  6. DNA Origami Seesaws as Comparative Binding Assay.

    Science.gov (United States)

    Nickels, Philipp C; Høiberg, Hans C; Simmel, Stephanie S; Holzmeister, Phil; Tinnefeld, Philip; Liedl, Tim

    2016-06-16

    The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami-based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw-like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  7. Quantifiable Lateral Flow Assay Test Strips

    Science.gov (United States)

    2003-01-01

    As easy to read as a home pregnancy test, three Quantifiable Lateral Flow Assay (QLFA) strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the middle left and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations. The color intensity correlates with concentration of contamination.

  8. Carbohydrate microarrays for assaying galactosyltransferase activity.

    Science.gov (United States)

    Park, Sungjin; Shin, Injae

    2007-04-26

    [reaction: see text] Carbohydrate microarrays have been used recently for the rapid analysis of glycan-protein or glycan-cell interactions and for the detection of pathogens. As a demonstration of its significance and versatility, the microarray technology has been applied in this effort to assay glycosyltransferase activities. In addition, carbohydrate microarray based methods have been employed to quantitatively determine binding affinities between lectins and carbohydrates.

  9. Clinical mutation assay of tumors: new developments.

    Science.gov (United States)

    Starostik, Petr

    2017-01-01

    Mutation detection in tumors started with classical cytogenetics as the method of choice more than 50 years ago. Karyotyping proved to be sensitive enough to detect deletions or duplications of large chromosome segments, and translocations. Over time, new techniques were developed to detect mutations that are much smaller in scope. The availability of Sanger sequencing and the invention of the PCR improved the discriminatory power of mutation detection to just one base change in the genomic DNA sequence. Techniques derived from PCR (allele-specific PCR, qPCR) and improved or modified sequencing methods (capillary electrophoresis, pyrosequencing) considerably increased the efficiency and sample throughput of mutation detection assays. With the advent of massive parallel sequencing [also called next-generation sequencing (NGS)] in the past decade, a major shift to even higher sample throughput and a significant decrease in cost per sequenced base occurred. The application of the new technology provided a whole slew of novel biomarkers and potential therapy targets to improve diagnosis and treatment. It even led to changes in cancer classification as new information on the mutation profile of tumors became available that characterizes some disease entities better than morphology. NGS, which usually interrogates multiple genes at once and is a prime example of a multianalyte assay, started to replace older single analyte assays focused on analysis of one target, one gene. However, the transition to these extremely complex NGS-based assays is associated with multiple challenges. There are issues with adequate tissue source of nucleic acids, sequencing library preparation, bioinformatics, government regulations and oversight, reimbursement, and electronic medical records that need to be resolved to successfully implement the new technology in a clinical laboratory.

  10. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  11. Radon assay and radioactivity database in Kamioka

    Science.gov (United States)

    Takeuchi, Yasuo; Tasaka, Shigeki; Nakano, Yuuki

    2017-09-01

    A new cooperative program among underground experiments, theorists, and low-background researchers was started in 2014 in Japan. The purpose of this program is to achieve technical and scientific synergies among underground researchers. In this report, the R&D of radon assay technique as an activity in this program is shown. The current status of the radioactivity database system in Kamioka is also shown.

  12. Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice.

    NARCIS (Netherlands)

    J. Groen (Jan); A.D.M.E. Osterhaus (Albert); H.W.J. Broeders; H.E.M. Spijkers (Ine)

    1989-01-01

    textabstractThe sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks

  13. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier

    2014-01-01

    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  14. The Engineered SVA Trans-mobilization Assay.

    Science.gov (United States)

    Bock, Anja; Schumann, Gerald G

    2016-01-01

    Mammalian genomes harbor autonomous retrotransposons coding for the proteins required for their own mobilization, and nonautonomous retrotransposons, such as the human SVA element, which are transcribed but do not have any coding capacity. Mobilization of nonautonomous retrotransposons depends on the recruitment of the protein machinery encoded by autonomous retrotransposons. Here, we summarize the experimental details of SVA trans-mobilization assays which address multiple questions regarding the biology of both nonautonomous SVA elements and autonomous LINE-1 (L1) retrotransposons. The assay evaluates if and to what extent a noncoding SVA element is mobilized in trans by the L1-encoded protein machinery, the structural organization of the resulting marked de novo insertions, if they mimic endogenous SVA insertions and what the roles of individual domains of the nonautonomous retrotransposon for SVA mobilization are. Furthermore, the highly sensitive trans-mobilization assay can be used to verify the presence of otherwise barely detectable endogenously expressed functional L1 proteins via their marked SVA trans-mobilizing activity.

  15. New Application of the Comet Assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

  16. Screening of fullerene toxicity by hemolysis assay.

    Science.gov (United States)

    Tramer, Federica; Da Ros, Tatiana; Passamonti, Sabina

    2012-01-01

    Fullerene is a compound formed during carbon burst that has been produced synthetically starting from the 1990s. The spherical shape and the characteristic carbon bonds of this allotrope (C(60)) have made it a suitable molecule for many applications. During the last decade, the low aqueous solubility of this molecule has been improved by chemical functionalization allowing the use of fullerene derivatives in biological fluids. The characterization of the toxicity potential of fullerenes is therefore of growing interest for any biomedical application. Intravenous injection is one of the possible routes of their administrations and therefore red blood cells are among the first targets of fullerene cytotoxicity. Human red blood cells are easily available and separated from plasma. Membrane disruption by toxic compounds is easily detected in red blood cells as release of hemoglobin in the cell medium, which can be assayed spectrophotometrically at λ = 415 nm. Due to the high molar extinction coefficient of hemoglobin, the assay can be performed on a small amount of both red blood cells and the test compounds, which might be available only in small quantities. So, the hemolysis assay is a simple screening test, whose results can guide further investigations on cytotoxicity in more complex experimental models.

  17. Variant Interpretation: Functional Assays to the Rescue.

    Science.gov (United States)

    Starita, Lea M; Ahituv, Nadav; Dunham, Maitreya J; Kitzman, Jacob O; Roth, Frederick P; Seelig, Georg; Shendure, Jay; Fowler, Douglas M

    2017-09-07

    Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of "lookup tables" of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  18. Comparative Evaluation of Various Total Antioxidant Capacity Assays Applied to Phenolic Compounds with the CUPRAC Assay

    Directory of Open Access Journals (Sweden)

    Dilek Özyurt

    2007-07-01

    Full Text Available It would be desirable to establish and standardize methods that can measure the total antioxidant capacity level directly from vegetable extracts containing phenolics. Antioxidant capacity assays may be broadly classified as electron transfer (ET− and hydrogen atom transfer (HAT−based assays. The majority of HAT assays are kinetics-based, and involve a competitive reaction scheme in which antioxidant and substrate compete for peroxyl radicals thermally generated through the decomposition of azo compounds. ET−based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced. ET assays include the ABTS/TEAC, CUPRAC, DPPH, Folin-Ciocalteu and FRAP methods, each using different chromogenic redox reagents with different standard potentials. This review intends to offer a critical evaluation of existing antioxidant assays applied to phenolics, and reports the development by our research group of a simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E, and human serum antioxidants, utilizing the copper(II-neocuproine reagent as the chromogenic oxidizing agent, which we haved named the CUPRAC (cupric ion reducing antioxidant capacity method. This method offers distinct advantages over other ET−based assays, namely the selection of working pH at physiological pH (as opposed to the Folin and FRAP methods, which work at alkaline and acidic pHs, respectively, applicability to both hydrophilic and lipophilic antioxidants (unlike Folin and DPPH, completion of the redox reactions for most common flavonoids (unlike FRAP, selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly contained in foodstuffs and the capability to assay –SH bearing antioxidants (unlike FRAP. Other similar ET–based antioxidant assays that we have developed or modified for phenolics are the Fe

  19. The in vitro and in vivo comet assays.

    Science.gov (United States)

    Burlinson, Brian

    2012-01-01

    The strategy for testing for genotoxicity covers three main areas, namely gene mutation, chromosome aberration or breakage (clastogenicity), and chromosome loss or gain (aneuploidy). The current generalized strategy consists of assays capable of detecting all of these endpoints using in vitro assays such as the Ames test for detecting gene mutations in bacteria, the human peripheral lymphocyte chromosome aberration (CA) test for detecting clastogenicity, and the in vitro micronucleus test for clastogenicity and aneuploidy. The primary in vivo assay, and generally the only in vivo assay required, is the in vivo rodent bone marrow micronucleus assay. However, there are instances when these assays alone are inadequate and further testing is required, especially in vivo. Historically, the preferred second assay has been the rodent liver unscheduled DNA synthesis assay but recently this has been superseded by the rodent single cell gel electrophoresis or Comet assay. This assay has numerous advantages especially in vivo, where virtually any tissue can be examined. The status of the in vitro comet assay in regulatory testing is much less clear although a preliminary review of data from the assay has shown it to be more specific than other in vitro genotoxicity tests and less prone to false positives.Detailed here are general protocols for both the in vitro and in vivo comet assays which will form the basis of the pending OECD guideline for the assay.

  20. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  1. Assaying the Potency of Influenza Vaccines

    Directory of Open Access Journals (Sweden)

    Philip D. Minor

    2015-02-01

    Full Text Available The potency of vaccines must be determined to ensure that the appropriate dose is given. The manufacture and assessment of influenza vaccines are complicated by the continuously changing nature of the pathogen, which makes efficacy estimates difficult but also confounds attempts to produce a well-validated, consistent potency assay. Single radial diffusion has been used for decades and provides a relatively simple way to measure the amount of biologically active materials present in the vaccine. It requires reagents, which are updated on a regular, frequently yearly, basis and alternative methods continue to be sought.

  2. Bicarbonate alters cellular responses in respiration assays.

    Science.gov (United States)

    Krycer, James R; Fisher-Wellman, Kelsey H; Fazakerley, Daniel J; Muoio, Deborah M; James, David E

    2017-08-05

    Metabolic assay buffers often omit bicarbonate, which is susceptible to alkalinisation in an open environment. Here, we assessed the effect of including bicarbonate in respirometry experiments. By supplementing HEPES-buffered media with low concentrations of bicarbonate, we found increased respiration in adipocytes and hepatocytes, but not myotubes. This was observed across multiple respirometry platforms and was independent of effects on enhanced insulin sensitivity, pH drift, or mitochondrial function. Permeabilised cell experiments suggest that bicarbonate increases substrate availability, likely by acting as a cofactor for carboxylase enzymes. This emphasises the importance of buffer choice in experimental biology. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Sperm DNA damage measured by comet assay.

    Science.gov (United States)

    Simon, Luke; Carrell, Douglas T

    2013-01-01

    Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidation-mediated DNA damage. The Comet assay or single-cell gel electrophoresis is a relatively simple and sensitive method for measuring strand breaks in DNA in individual sperm. During this procedure, sperm cells are embedded in a thin layer of agarose on a microscope slide and lysed with detergent under high salt conditions. This process removes protamines and histones allowing the nucleus to form a nucleoid-like structure containing supercoiled loops of DNA. Alkaline pH conditions result in unwinding of double-stranded DNA, and subsequent electrophoresis results in the migration of broken strands towards the anode, forming a comet tail, when observed under fluorescence microscope. The amount of DNA in the head and tail is reflected by its fluorescent intensity. The relative fluorescence in the tail compared with its head serves as a measure of the level of DNA damage. In this chapter, we describe the alkaline version of the Comet assay, which is highly sensitive for measuring single- and double-strand DNA breaks.

  4. Sulforhodamine B colorimetric assay for cytotoxicity screening.

    Science.gov (United States)

    Vichai, Vanicha; Kirtikara, Kanyawim

    2006-01-01

    The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.

  5. Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay.

    Science.gov (United States)

    Lah, B; Vidic, T; Glasencnik, E; Cepeljnik, T; Gorjanc, G; Marinsek-Logar, Romana

    2008-04-01

    Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO(2)) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.

  6. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    Science.gov (United States)

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  7. Evaluation of an immunoradiometric assay protocol for determining ...

    African Journals Online (AJOL)

    BDL) for prostate-specific antigen (PSA) by an assay system are essential for successful prediction of recurrent disease after radical prostatectomy. A study was conducted to evaluate an Immunoradiometric Assay (IRMA) system to establish the ...

  8. The Statistics of wood assays for preservative retention

    Science.gov (United States)

    Patricia K. Lebow; Scott W. Conklin

    2011-01-01

    This paper covers general statistical concepts that apply to interpreting wood assay retention values. In particular, since wood assays are typically obtained from a single composited sample, the statistical aspects, including advantages and disadvantages, of simple compositing are covered.

  9. a positive control plasmid for reporter gene assay

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-04

    903 for ... qualification as a positive control for luciferase reporter gene assays. Key words: Reporter gene plasmid, luciferase assay, .... tissue-specific promoters by gene gun. Br. J. Dermatol. 144: 34-39. Naylor LH (1999).

  10. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    Science.gov (United States)

    Kawaguchi, Satomi; Nakamura, Takanori; Yamamoto, Ayumi; Honda, Gisho; Sasaki, Yu F.

    2010-01-01

    Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were

  11. Analytical evaluation of nine serological assays for diagnosis of syphilis.

    Science.gov (United States)

    Malm, K; Andersson, S; Fredlund, H; Norrgren, H; Biague, A; Månsson, F; Ballard, R; Unemo, M

    2015-12-01

    The diagnosis of syphilis is most frequently dependent on antibody detection with serological assays. Assays for both treponemal and non-treponemal antibodies are needed to provide a sensitive and specific diagnosis. For decades, a first screening has been done with non-treponemal assays, followed by treponemal. However, in recent years, following laboratory automation, the reverse sequence screening algorithms have been developed, using a treponemal assay as the initial screening test. To evaluate serological assays for treponemal and non-treponemal antibodies, to use in reverse algorithm screening of syphilis. Six treponemal assays (one IgM-specific assay), two non-treponemal assays and one novel dual point-of-care (POC) assay for serological diagnosis of syphilis were evaluated. Serum samples from Guinea-Bissau and Sweden were examined, as well as two performance panels and samples from blood donors. Sensitivity and specificity were calculated for each assay, using different assays as gold standard test. The Macro-Vue RPR Card test was the most sensitive non-treponemal test and the TrepSure Anti-Treponema EIA Screen and the SeroDia TP-PA were the most sensitive and specific treponemal assays. Among the automated assays, both the Liaison Treponema Screen and Architect Syphilis TP showed high sensitivity, however, the former had clearly higher specificity. In resourced settings, where the reverse sequence algorithm is preferred for screening, an automated treponemal immunoassay for initial screening subsequently followed by the TrepSure test or TP-PA assay as a second treponemal assay appear highly effective. Finally, a quantitative highly sensitive non-treponemal assay, e.g. the Macro-Vue RPR Card test, could then be used as a supplementary test to evaluate activity of the syphilis infection. © 2015 European Academy of Dermatology and Venereology.

  12. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  13. 21 CFR 864.7415 - Abnormal hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864... hemoglobin assay. (a) Identification. An abnormal hemoglobin assay is a device consisting of the reagents... hemoglobin types. (b) Classification. Class II (performance standards). [45 FR 60618, Sept. 12, 1980] ...

  14. 21 CFR 864.7470 - Glycosylated hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section... Glycosylated hemoglobin assay. (a) Identification. A glycosylated hemoglobin assay is a device used to measure the glycosylated hemoglobins (A1a, A1b, and A1c) in a patient's blood by a column chromatographic...

  15. Systems, devices, and methods for agglutination assays using sedimentation

    Energy Technology Data Exchange (ETDEWEB)

    Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.

    2016-01-26

    Embodiments of the present invention include methods for conducting agglutination assays using sedimentation. Aggregates may be exposed to sedimentation forces and travel through a density medium to a detection area. Microfluidic devices, such as microfluidic disks, are described for conducting the agglutination assays, as are systems for conducting the assays.

  16. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation...

  17. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, Thomas R. (Somerville, MA); Liber, Howard L. (Brookline, MA); Penman, Bruce W. (Cambridge, MA); Thilly, William G. (Cambridge, MA); Hoppe, IV, Henry (Arlington, MA)

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  18. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Skopek, T.R.; Liber, H.L.; Penman, B.W.; Thilly, W.G.; Hoppe, H. IV.

    1981-11-24

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay. 7 figs.

  19. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Glial fibrillary acidic protein assay... Glial fibrillary acidic protein assay. (a) Purpose. Chemical-induced injury of the nervous system, i.e... paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate...

  20. 21 CFR 866.3402 - Plasmodium species antigen detection assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866... Plasmodium species antigen detection assays. (a) Identification. A Plasmodium species antigen detection assay... malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum, Plasmodium...

  1. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione reductase assay. (a) Identification. A glutathione reductase assay is a device used to determine the...

  2. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT PCR assays and two new assays that we have developed. To determine the analytical sensitivity of

  3. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  4. Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays.

    Science.gov (United States)

    Singer, Timothy M; Lambert, Iain B; Williams, Andrew; Douglas, George R; Yauk, Carole L

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently--the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  5. Abbott ARCHITECT iPhenytoin assay versus similar assays for measuring free phenytoin concentrations.

    Science.gov (United States)

    Tacker, Danyel Hermes; Robinson, Randy; Perrotta, Peter L

    2014-01-01

    To measure free phenytoin (FP) concentrations in filtered specimens using the Abbott ARCHITECT iPhenytoin assay and to compare results from this method with results from the Abbott TDx/FLx assays. We verified accuracy, analytic measurement range, and precision for FP measurements. For correlation and therapeutic interval studies, we used filtered calibrators, controls, proficiency-testing materials, and surplus clinical samples. After implementation, we determined proficiency testing results. The analytic measurement range was 2.0 to 25.0 micromol/L. Quality control materials (6.1, 12.6, and 20.1 micromol/L) provided mean (SD) recoveries of 96.1 (5.0%), 99.2 (5.0%), and 99.3 (5.7%), respectively, and coefficients of variation of 5.2%, 5.0%, and 5.8%, respectively. Clinical specimens produced mean (SD) FP recovery levels of 103.7 (10.6%) (bias, 0.1 [0.3] micromol/L). Altering the FP therapeutic range (4.0-8.0 micromol/L) was unnecessary. Proficiency testing yielded consistently acceptable results. Our accuracy, precision, and correlation results were similar for the TDx/FLx and ARCHITECT assays, which demonstrates that the ARCHITECT iPhenytoin assay is acceptable for clinical FP measurements.

  6. Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.

    Science.gov (United States)

    2017-07-27

    The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

  7. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed.

  8. Lab-on-a-Chip Multiplex Assays.

    Science.gov (United States)

    Peter, Harald; Wienke, Julia; Bier, Frank F

    2017-01-01

    Lab-on-a-chip multiplex assays allow a rapid identification of multiple parameters in an automated manner. Here we describe a lab-based preparation followed by a rapid and fully automated DNA microarray hybridization and readout in less than 10 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable rapid identification of bacterial species and detection of antibiotic resistance. The use of DNA microarrays allows a fast adaptation of new biomarkers enabling the identification of different genes as well as single-nucleotide-polymorphisms (SNPs) within these genes. In this protocol we describe a DNA microarray developed for identification of Staphylococcus aureus and the mecA resistance gene.

  9. Stable isotope dilution assays in mycotoxin analysis.

    Science.gov (United States)

    Rychlik, Michael; Asam, Stefan

    2008-01-01

    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis.

  10. COMPARATIVE STUDY ON MILK CASEIN ASSAY METHODS

    Directory of Open Access Journals (Sweden)

    RODICA CĂPRIłĂ

    2008-05-01

    Full Text Available Casein, the main milk protein was determined by different assay methods: the gravimetric method, the method based on the neutralization of the NaOH excess used for the casein precipitate solving and the method based on the titration of the acetic acid used for the casein precipitation. The last method is the simplest one, with the fewer steps, and also with the lowest error degree. The results of the experiment revealed that the percentage of casein from the whole milk protein represents between 72.6–81.3% in experiment 1, between 73.6–81.3% in experiment 2 and between 74.3–81% in experiment 3.

  11. Rapid Automated Sample Preparation for Biological Assays

    Energy Technology Data Exchange (ETDEWEB)

    Shusteff, M

    2011-03-04

    Our technology utilizes acoustic, thermal, and electric fields to separate out contaminants such as debris or pollen from environmental samples, lyse open cells, and extract the DNA from the lysate. The objective of the project is to optimize the system described for a forensic sample, and demonstrate its performance for integration with downstream assay platforms (e.g. MIT-LL's ANDE). We intend to increase the quantity of DNA recovered from the sample beyond the current {approx}80% achieved using solid phase extraction methods. Task 1: Develop and test an acoustic filter for cell extraction. Task 2: Develop and test lysis chip. Task 3: Develop and test DNA extraction chip. All chips have been fabricated based on the designs laid out in last month's report.

  12. Nondestructive Assay Options for Spent Fuel Encapsulation

    Energy Technology Data Exchange (ETDEWEB)

    Tobin, Stephen J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Jansson, Peter [Uppsala Univ. (Sweden)

    2014-10-02

    This report describes the role that nondestructive assay (NDA) techniques and systems of NDA techniques may have in the context of an encapsulation and deep geological repository. The potential NDA needs of an encapsulation and repository facility include safeguards, heat content, and criticality. Some discussion of the facility needs is given, with the majority of the report concentrating on the capability and characteristics of individual NDA instruments and techniques currently available or under development. Particular emphasis is given to how the NDA techniques can be used to determine the heat production of an assembly, as well as meet the dual safeguards needs of 1) determining the declared parameters of initial enrichment, burn-up, and cooling time and 2) detecting defects (total, partial, and bias). The report concludes with the recommendation of three integrated systems that might meet the combined NDA needs of the encapsulation/repository facility.

  13. Stable isotope dilution assays in mycotoxin analysis

    Energy Technology Data Exchange (ETDEWEB)

    Rychlik, Michael; Asam, Stefan [Universitaet Muenchen, Lehrstuhl fuer Lebensmittelchemie der Technischen, Garching (Germany)

    2008-01-15

    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis. (orig.)

  14. Development of a Radioactive Waste Assay System

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Duck Won; Song, Myung Jae; Shin, Sang Woon; Sung, Kee Bang; Ko, Dae Hach [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Kil Jeong; Park, Jong Mook; Jee, Kwang Yoong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-12-31

    Nuclear Act of Korea requires the manifest of low and intermediate level radioactive waste generated at nuclear power plants prior to disposal sites.Individual history records of the radioactive waste should be contained the information about the activity of nuclides in the drum, total activity, weight, the type of waste. A fully automated nuclide analysis assay system, non-destructive analysis and evaluation system of the radioactive waste, was developed through this research project. For the nuclides that could not be analysis directly by MCA, the activities of the representative {gamma}-emitters(Cs-137, Co-60) contained in the drum were measured by using that system. Then scaling factors were used to calculate the activities of {alpha}, {beta}-emitters. Furthermore, this system can automatically mark the analysis results onto the drum surface. An automated drum handling system developed through this research project can reduce the radiation exposure to workers. (author). 41 refs., figs.

  15. Universal fieldable assay with unassisted visual detection

    Science.gov (United States)

    Chelyapov, Nicolas (Inventor)

    2012-01-01

    A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

  16. Die Sulforhodamien B Seltellingsmetode in vergelyking met drie algemene seltellingsmetodes vir sitotoksisiteitstoetse/The Sulforhodamine B Assay in comparison with three commonly used cytotoxicity assays

    National Research Council Canada - National Science Library

    A van Tonder; A Joubert; A D Cromarty

    2014-01-01

      The sulforhodamine B (SRB) assay was compared to the MTT assay, the neutral red uptake assay and the resazurin reduction assay with regards to sensitivity, interference with glycolysis inhibitors, reproducibility and cost-effectiveness...

  17. A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

    Science.gov (United States)

    Faulkner, Ashton; Purcell, Robert; Hibbert, Andrew; Latham, Sally; Thomson, Scott; Hall, Wendy L; Wheeler-Jones, Caroline; Bishop-Bailey, David

    2014-12-05

    Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

  18. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    Pas, S D; Fries, E; De Man, R A; Osterhaus, A D; Niesters, H G

    A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV

  19. Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

    NARCIS (Netherlands)

    S.D. Pas (Suzan); E. Fries; H.G.M. Niesters (Bert); R.A. de Man (Robert); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractA highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel,

  20. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  1. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    Science.gov (United States)

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay.

    Science.gov (United States)

    McKenzie, Jennifer Helen; Alwis, K Udeni; Sordillo, Joanne E; Kalluri, Kesava Srinivas; Milton, Donald Kirby

    2011-06-01

    Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.

  3. Reliability of mycotoxin assays--an update.

    Science.gov (United States)

    Horwitz, W; Albert, R; Nesheim, S

    1993-01-01

    The precision parameters of the method-performance (collaborative) studies for mycotoxins published in the literature through 1991 have been recalculated on a uniform basis by following the International Union of Pure and Applied Chemistry protocol. About 80% of the 793 accepted assays for mycotoxins, almost all of which have been conducted by thin-layer chromatography (TLC), liquid chromatography (LC), and enzyme-linked immunosorbent assays (ELISA), exhibit relative standard deviations among laboratories (RSDR) that are less than 2 times the values predicted from the Horwitz equation: RSDR, % = 2(1-0.5log10C) where C is the concentration expressed as a decimal fraction. The precision of TLC and LC methods is about the same, but that of ELISA is somewhat poorer. For those commodities for which sufficient data exist to provide a meaningful comparison, the methods applied to cottonseed products have the best precision and corn the worst, with peanuts intermediate. Overall, however, the primary factor affecting RSDR is concentration, more or less independent of analyte, method, matrix, and age of the study. If it is assumed that the test results are normally distributed and that an RSDR of 50% is the point where effective control of the results begins to be lost (a value equivalent to the production of 2% false-negative values), then relying on the Horwitz curve, the limit of quantitative measurement is the single digit, i.e., 5, micrograms/kg (10(-9); ppb) concentration for solid food commodities. Such a value must be considered as a limit applicable to a single analyte, aflatoxin B1, and not as a mean, and not applicable to the sum of the individual components, each of whose associated standard deviation would lie in the unacceptable region. Enforcement of a 5 micrograms aflatoxin B1/kg limit, under the assumptions made, requires that a responsible manufacturer and a prudent regulator operate at opposite extremes of tolerance limits: e.g., the producer at 2

  4. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay.

    Science.gov (United States)

    Boe-Hansen, Gry B; Morris, Ian D; Ersbøll, Annette K; Greve, Torben; Christensen, Preben

    2005-04-01

    During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.

  5. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Science.gov (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  6. A quantitative comet infection assay for influenza virus

    Science.gov (United States)

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

  7. Mouse lung adhesion assay for Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Burns, K.A.; Freer, J.H. (Department of Microbiology, Alexander Stone Building, Bearsden, Glasgow, Scotland)

    1982-03-01

    The ability of Bordetella pertussis to adhere to cell surfaces has been demonstrated by adhesion to tissue culture cells and adhesion to chicken, hamster or rabbit trachea in organ culture. In this report a mouse lung assay for adhesion is described and the results obtained using two virulent strains of B. pertussis and their avirulent counterparts. These were a C modulation of one of the original virulent strains and a phase IV variant of the other virulent strain. Organisms were radiolabelled by adding 1 ..mu..Ci (37 K Bq) of (/sup 14/C)glutamic acid per 10 ml of culture medium before inoculation and incubation for 5 days. The lungs were washed by perfusion in situ with at least two volumes (1 ml) of sterile 1% (w/v) casamino acids. The percentage of the inoculated organisms retained in the lungs was determined, after removal of the lungs, by one of the following two methods: viable count or radioactive count. Results for both methods were expressed as the percentage of the inoculum retained in the lungs plus or minus one standard deviation.

  8. Prandiology of Drosophila and the CAFE assay

    Science.gov (United States)

    Ja, William W.; Carvalho, Gil B.; Mak, Elizabeth M.; de la Rosa, Noelle N.; Fang, Annie Y.; Liong, Jonathan C.; Brummel, Ted; Benzer, Seymour

    2007-01-01

    Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7× their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant. PMID:17494737

  9. Fluorometric enzymatic assay of L-arginine

    Science.gov (United States)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

  10. Virus Characterization by FFF-MALS Assay

    Science.gov (United States)

    Razinkov, Vladimer

    2009-03-01

    Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

  11. Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

    Science.gov (United States)

    Huang, Kai-Wen; Chieh, Jen-Jie; Shi, Jin-Cheng; Chiang, Ming-Hsien

    2015-07-01

    Biofunctionalized magnetic nanoparticles (BMNs) that provide unique advantages have been extensively used to develop immunoassay methods. However, these developed magnetic methods have been used only for specific immunoassays and not in studies of magnetic characteristics of materials. In this study, a common vibration sample magnetometer (VSM) was used for the measurement of the hysteresis loop for different carcinoembryonic antigens (CEA) concentrations ( Φ CEA) based on the synthesized BMNs with anti-CEA coating. Additionally, magnetic parameters such as magnetization ( M), remanent magnetization ( M R), saturation magnetization ( M S), and normalized parameters (Δ M R/ M R and Δ M S/ M S) were studied. Here, Δ M R and Δ M s were defined as the difference between any ΦCEA and zero Φ CEA. The parameters M, Δ M R, and Δ M S increased with Φ CEA, and Δ M S showed the largest increase. Magnetic clusters produced by the conjugation of the BMNs to CEAs showed a Δ M S greater than that of BMNs. Furthermore, the relationship between Δ M S/ M S and Φ CEA could be described by a characteristic logistic function, which was appropriate for assaying the amount of CEAs. This analytic Δ M S/ M S and the BMNs used in general magnetic immunoassays can be used for upgrading the functions of the VSM and for studying the magnetic characteristics of materials.

  12. Assaying Visual Memory in the Desert Locust

    Directory of Open Access Journals (Sweden)

    Senne Dillen

    2015-04-01

    Full Text Available The involvement of associative learning cues has been demonstrated in several stages of feeding and food selection. Short neuropeptide F (sNPF, an insect neuropeptide whose effects on feeding behavior have previously been well established, may be one of the factors bridging feeding and learning behavior. Recently, it was shown in Drosophila melanogaster that the targeted reduction of Drome-sNPF transcript levels significantly reduced sugar-rewarded olfactory memory. While Drosophila mainly relies on olfactory perception in its food searching behavior, locust foraging behavior is likely to be more visually orientated. Furthermore, a feeding-dependent regulation of Schgr-sNPF transcript levels has previously been observed in the optic lobes of the locust brain, suggesting a possible involvement in visual perception of food and visual associative memory in this insect species. In this study, we describe the development of a robust and reproducible assay allowing visual associative memory to be studied in the desert locust, Schistocerca gregaria. Furthermore, we performed an exploratory series of experiments, studying the role of Schgr-sNPF in this complex process.

  13. Evaluation of a MTT assay in measurement of radiosensitizing effect

    Energy Technology Data Exchange (ETDEWEB)

    Higuchi, Keiko; Mitsuhashi, Norio; Saitoh, Jun-ichi; Maebayashi, Katsuya; Sakurai, Hideyuki; Akimoto, Tetsuo; Niibe, Hideo [Gunma Univ., Maebashi (Japan). School of Medicine

    1999-11-01

    The usefulness of a MTT assay by measuring the radiosensitizing effect of caffeine on rat yolk sac tumor cell line with a mutant-type p53 in vitro was evaluated. A rat yolk sac tumor cell line with a mutant-type p53, NMT-1R, was used in this study. The radiosensitivity of NMT-1R with or without caffeine was measured with a MTT assay. The results were compared with those by a clonogenic assay. Caffeine at a concentration of 2.0 mM which released radiation-induced G{sub 2} block demonstrated a radiosensitizing effect, but caffeine at a concentration of 0.5 mM did not. The radiosensitizing effect of caffeine measured by a MTT assay correlated with that measured by a clonogenic assay. A MTT assay was useful to measure radiosensitivity and/or a radiosensitizing effect in vitro. (author)

  14. An assay to screen bacterial adhesion to mucus biomolecules.

    Science.gov (United States)

    Tkachenko, A; Da Silva, L; Hearne, J; Parveen, S; Waguespack, Y

    2013-01-01

    To develop an assay for rapid screening of bacterial adhesion to various groups of biomolecules present in fish mucus. A novel assay was developed for investigation of bacterial adhesion to various groups of mucus biomolecules from fish. Lipid-, protein-, carbohydrate- and nucleic acid-rich constituents of mucus were separated using isopycnic density gradient centrifugation techniques. Separated mucus fractions were assayed for bacterial adhesion using a blotting apparatus. The assay was validated using Vibrio vulnificus and skin mucus from hybrid tilapia. A novel assay was developed for the screening of bacterial adhesion to major groups of mucus biomolecules. Adhesion of V. vulnificus MLT403 positively correlated with lipid- and protein-rich mucus constituents and negatively correlated with carbohydrate-rich mucus constituents. The assay can be used as an initial approach in a systematic identification of mucus constituent(s) exhibiting the most favourable adhesion properties for bacteria. © 2012 The Society for Applied Microbiology.

  15. Multiplex cytological profiling assay to measure diverse cellular states.

    Directory of Open Access Journals (Sweden)

    Sigrun M Gustafsdottir

    Full Text Available Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

  16. Comparison of assay formats for drug-tolerant immunogenicity testing.

    Science.gov (United States)

    Butterfield, Anthony M; Chain, Jana S; Ackermann, Bradley L; Konrad, Robert J

    2010-12-01

    Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development. Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(®) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive. Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.

  17. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

    Directory of Open Access Journals (Sweden)

    KHUSNUL YAQIN

    2006-09-01

    Full Text Available Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The applications of this assay suggest that it is a useful assay to assess aquatic genotoxicants. However, there are some factors, which should be taken into account when using this assay as aquatic ecotoxicological assessment device such as inter-animal and cell variability.

  18. Proteomics: from hypothesis to quantitative assay on a single platform. Guidelines for developing MRM assays using ion trap mass spectrometers.

    Science.gov (United States)

    Han, Bomie; Higgs, Richard E

    2008-09-01

    High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.

  19. Epithelial cells as alternative human biomatrices for comet assay

    OpenAIRE

    Emilio eRojas; Yolanda eLorenzo; Kristiane eHuag-Berg; Bjørn eNicolaissen; Mahara eValverde

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many org...

  20. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

    OpenAIRE

    YAQIN, KHUSNUL

    2006-01-01

    Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The appli...

  1. Enzyme Assay Methods to Validate DIGE Proteomics Data.

    Science.gov (United States)

    Dowd, Andrew

    2018-01-01

    Enzyme activity assay methods can be used to corroborate the results generated by Difference Gel Electrophoresis (DIGE) proteomic experiments. Two assay methods were chosen to demonstrate how this can be achieved. Assays for determining the activity of superoxide dismutase and NADH dehydrogenase are outlined in detail in this paper. These methods were chosen as examples because they are frequently used in conjunction with DIGE proteomics.

  2. Absolute nuclear material assay using count distribution (LAMBDA) space

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Mano K.; Snyderman, Neal J.; Rowland, Mark S.

    2015-12-01

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  3. Comparative investigation of various cellulase assay procedures

    Energy Technology Data Exchange (ETDEWEB)

    Canevascini, G.; Gattlen, C.

    1981-07-01

    The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. 1) Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis, analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present. (Refs. 39).

  4. BROMATOMATRIC ASSAY OF GATIFLOXACIN IN PHARMACEUTICALS

    Directory of Open Access Journals (Sweden)

    KALSANG THARPA

    2008-09-01

    Full Text Available Three new, simple, and cost-effective visible spectrophotometric methods are proposed for determination of gatifloxacin (GTF using bromate-bromide mixture, and three dyes, methyl orange, indigocarmine and thymol blue, as reagents.The methods engross the addition of a known excess of bromate-bromide mixture to GTF in hydrochloric acid medium followed by determination of residual bromine by reacting with a fixed amount of either methyl orange andmeasuring the absorbance at 520 nm (method A or indigo carmine and measuring the absorbance at 610 nm (method B or thymol blue and measuring the absorbance at 550 nm (method C. In all the methods, the amount of brominereacted corresponds to the amount of GTF, and the absorbance is found to increase linearly with the concentration of GTF. Under the optimum conditions, GTF could be assayed in the concentration range 0.25-1.5, 0.5-6.0, and 0.5-10μg/mL by method A, method B and method C, respectively. The apparent molar absorptivities are calculated to be 1.6x105, 4.0x104 and 3.2x104 L mol-1 cm-1 for the method A, method B and method C, respectively, and the corresponding Sandell sensitivity values are 0.0025, 0.010 and 0.012 μg/cm2. The intra-day and inter-day precision, and the accuracy of the methods were evaluated as per the current ICH guidelines. The methods were successfully applied to the determination of GTF in pharmaceutical preparations without the interference from any of the pharmaceutical adjuvants.

  5. Functional assays for hematopoietic stem cell self-renewal.

    Science.gov (United States)

    Perry, John M; Li, Linheng

    2010-01-01

    Stem cells are defined by the ability to self-renew. Specific functional assays have been developed for the rigorous identification and quantification of hematopoietic stem cells (HSCs), making these cells the benchmark in studies of self-renewal. Here, we review the theory behind these functional stem cell tests and discuss important considerations in choosing and designing these assays. Finally, we provide a basic protocol for the serial-dilution assay, a quantitative assay for HSCs, from which individual researchers can construct their own customized protocols utilizing the guidelines discussed.

  6. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

    DEFF Research Database (Denmark)

    Møller, Peter; Möller, Lennart; Godschalk, Roger W L

    2010-01-01

    The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data...... are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level...... reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation....

  7. Cell Enumeration Assays: Application of the MTT and Sulforhodamine B Assays to Lipopolysaccharide-Stimulated Neonatal Rodent Microglia.

    Science.gov (United States)

    Facci, Laura; Skaper, Stephen D

    2018-01-01

    Glial cell activation, in particular microglia, plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter compares two established cell enumeration assays, namely, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the protein-binding sulforhodamine B assay for microglia as a function of culture condition and activation state. The pros and cons of each are then described.

  8. [Evaluation of Abbott Fourth Generation HIV Antigen and Antibody Assays.].

    Science.gov (United States)

    Kang, Hee Jung; Yoo, Kyeong Ha; Kim, Han Sung; Cho, Hyoun Chan

    2006-02-01

    In order to reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and serological diagnosis, new fourth generation screening assays which detect HIV p24 antigen and specific antibody simultaneously have been developed. In this study, we evaluated the performance of a new fourth generation assay. We compared a new fourth generation assay, Architect HIV Ag/Ab combo, with another fourth generation assay AxSYM HIV Ag/Ab combo and a third generation assay, AxSYM HIV 1/2 gO for their performance. The assays were evaluated using 3 HIV seroconversion panels, 305 sera of healthy subjects and 100 sera of patients with HBsAg or anti-HCV antibodies. Within-run and total coefficient variations of the three screening assays were analyzed for the evaluation of precision. Architect HIV Ag/Ab combo shortened the window period by 8.7+/-2.1 days relative to AxSYM HIV 1/2 gO and 2.0+/-2.0 days relative to AxSYM HIV Ag/Ab combo in seroconversion panels. Architect HIV Ag/Ab combo presented the best performance in precision among the three reagents; total CV for positive control was 3.6%, 9.6% and 4.6% for Architect HIV Ag/Ab combo, AxSYM HIV Ag/Ab combo and AxSYM HIV 1/2 gO, respectively. Specificities of three assays were not different in this study. HIV Ag/Ab combined assays reduced the diagnostic window as compared to the third generation screening assays, enabling an earlier diagnosis of HIV infection. A new fourth generation assay, Architect HIV Ag/Ab combo presents a better performance than AxSYM HIV Ag/Ab combo, showing improved seroconversion sensitivity and precision.

  9. Assay optimization for molecular detection of Zika virus

    Science.gov (United States)

    Corman, Victor M; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal BEM; Pas, Suzan D; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M; Koopmans, Marion P; Schmidt-Chanasit, Jonas; Grobusch, Martin P; de Lamballerie, Xavier; Drosten, Christian

    2016-01-01

    Abstract Objective To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. Methods We compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays. Findings Oligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine. Conclusion We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results. PMID:27994281

  10. Genetic damage in oligozoospermic patients detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the Comet assay.

    Science.gov (United States)

    Schmid, T E; Kamischke, A; Bollwein, H; Nieschlag, E; Brinkworth, M H

    2003-07-01

    The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring. Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay. FISH analysis showed a significant increase in gonosomal X,Y,18 (P sperm with X,Y,18,18 (P sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P assay. The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

  11. Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis

    NARCIS (Netherlands)

    Smits, H. L.; van der Hoorn, M. A.; Goris, M. G.; Gussenhoven, G. C.; Yersin, C.; Sasaki, D. M.; Terpstra, W. J.; Hartskeerl, R. A.

    2000-01-01

    A newly developed latex agglutination assay for the detection of genus-specific Leptospira antibodies in human sera was evaluated. The assay is performed by mixing, on an agglutination card, serum with equal volumes of stabilized antigen-coated, dyed test and control latex beads and is read within 2

  12. Reliability of mutagen sensitivity assay: an inter-laboratory comparison.

    Science.gov (United States)

    Erdei, Esther; Lee, Sang-Joon; Wei, Qingyi; Wang, Li-E; Song, Yan-S; Bovbjerg, Dana; Berwick, Marianne

    2006-07-01

    Mutagen sensitivity is regarded as a genetic susceptibility phenotype for various cancers; it is cytogenetically based and probably involves a number of genes from different DNA repair pathways. This assay has been used in a number of laboratories in the field of epidemiology, where it has been investigated and appears to be a useful susceptibility biomarker for epidemiological studies assessing cancer risks at the population level. One concern about phenotypic assays, such as the mutagen sensitivity assay, has been that there could be wide variation in results depending on the timing of the assay (within individual variation), the individual performing the assay (within observer variation) and the laboratory where the assay has been performed (inter-laboratory variation). We conducted an inter-laboratory comparison study between the Memorial Sloan-Kettering Cancer Center and M. D. Anderson, in which we assessed all these concerns. We did not find any significant variation in any of the assays. The correlation was high for all tests. The good concordance rate between laboratories supports the continued use of the mutagen sensitivity assay by different laboratories, and demonstrates its potential to identify at-risk subgroups among normal individuals and cancer patients alike.

  13. Mutation assay in diploid human lymphoblasts: methodological aspects

    Energy Technology Data Exchange (ETDEWEB)

    Thilly, W.G.; DeLuca, J.G.; Hoppe, H. IV; Liber, H.L.; Penman, B.W.

    1977-01-01

    The protocol for a recently developed quantitative assay for mutation at the hgprt locus of human lymphoblasts is presented. Practical problems affecting ease of performance and reliability are discussed with the aim of making the assay available for assessment and possible use in other laboratories.

  14. The Efficiency of the Assay for Haemopietic Colony Forming Cells

    NARCIS (Netherlands)

    Lahiri, S.K.; Keizer, H.J.; Putten, L.M.v. (L. M. van)

    1970-01-01

    textabstractThe quantitative efficiency of the spleen colony assay in mice is discussed in the light of recent findings on the kinetics of colony forming cells. Arguments are presented showing that the f factor, the 2 hr CFU recovery fraction in the spleen, markedly over‐estimates the assay

  15. A new spectrofluorimetric assay method for vandetanib in tablets ...

    African Journals Online (AJOL)

    ... two methods in terms of accuracy and precision. Conclusion: The proposed method is simple and highly sensitive and, consequently, can be applied to assay VDB in biological samples as well as in dosage form. Keywords: Vandetanib, Spectrofluorimetry, Assay, Validation, Human plasma, Human urine, Dosage forms ...

  16. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei, E-mail: kwng@ntu.edu.sg; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

    2015-01-15

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

  17. Evaluation and improvement of LAMP assays for detection of

    African Journals Online (AJOL)

    Abstract: Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseases, and LAMP assays have been developed for detection of the seven leading STEC serogroups. Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, ...

  18. Specific gravity and antibacterial assays of some synthetic industrial ...

    African Journals Online (AJOL)

    Samples of synthetic industrial essential oil (SIEO) were assayed to provide basis for establishing correlation and validation of claims of its density, disinfectant and germicidal properties with its natural counterparts. Specific gravity (SG) and antibacterial assays were carried out on twenty typical SIEO samples that are ...

  19. ToxCast HTS Assay Development and Retrofitting: Strategies ...

    Science.gov (United States)

    A presentation to EC JRC partners on new ToxCast HTS assay methods and strategies to address current limitations to HTS methods Slide presentation to EC JRC partners on new ToxCast HTS assay methods and strategies to address current limitations to HTS methods.

  20. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...

  1. Colorimetric micro-assay for accelerated screening of mould inhibitors

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2013-01-01

    Since current standard laboratory methods are time-consuming macro-assays that rely on subjective visual ratings of mould growth, rapid and quantitative laboratory methods are needed to screen potential mould inhibitors for use in and on cellulose-based products. A colorimetric micro-assay has been developed that uses XTT tetrazolium salt to enzymatically assess...

  2. Radioimmunoprecipitation polyethylene glycol assay for circulating Entamoeba histolytica antigens

    Energy Technology Data Exchange (ETDEWEB)

    Pillai, S.; Mohimen, A.; Mehra, S. (Calcutta Medical Research Inst., Calcutta (India). Kothari Centre of Gastroenterology)

    1982-12-17

    An assay capable of detecting circulating Entamoeba histolytica antigens in amoebiasis is described. This assay utilised a radiolabelled affinity purified rabbit anti-E. histolytica antibody that had been depleted of antibodies that cross-react with human serum proteins, and a polyethylene glycol precipitation step.

  3. Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

    Science.gov (United States)

    High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

  4. Evaluation and improvement of LAMP assays for detection of ...

    African Journals Online (AJOL)

    Abstract: Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseas- es, and LAMP assays have been developed for detection of the seven leading STEC serogroups. Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, ...

  5. Development and evaluation of an in vivo assay in Caenorhabditis ...

    Indian Academy of Sciences (India)

    We developed a fluorescence-based in vivo assay using transgenic Caenorhabditis elegans worms that express the green fluorescent protein (GFP) under the control of various CYP promoters. Using this assay, we found striking similarities between the worm CYPs and their human orthologs in their response to treatment ...

  6. ABCs of DNA aptamer and related assay development.

    Science.gov (United States)

    Sharma, Tarun Kumar; Bruno, John G; Dhiman, Abhijeet

    This review is intended to guide the novice in aptamer research and development to understand virtually all of the aptamer development options and currently available assay modalities. Aptamer development topics range from discussions of basic and advanced versions of Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and SELEX variations involving incorporation of exotic unnatural nucleotides to expand library diversity for even greater aptamer affinity and specificity to improved next generation methods of DNA sequencing, screening and tracking aptamer development throughout the SELEX process and characterization of lead aptamer candidates. Aptamer assay development topics include descriptions of various colorimetric and fluorescent assays in microplates or on membranes including homogeneous beacon and multiplexed Fluorescence Resonance Energy Transfer (FRET) assays. Finally, a discussion of the potential for marketing successful aptamer-based assays or test kits is included. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The development of SRM assays is transforming proteomics research.

    Science.gov (United States)

    Manes, Nathan P; Nita-Lazar, Aleksandra

    2017-04-01

    Bottom-up targeted proteomics using SRM is a powerful analytical technology, but it requires the development of SRM assays, which is a complex procedure. Whereas proteome-wide SRM assays have recently been developed for a small number of species, this is not so for the mouse. In this issue, Percy et al. report the development of hundreds of mouse SRM assays. Their development required shotgun MS to identify proteotypic peptides, synthesis, and LC-MS characterization of peptide standards, and interlaboratory SRM to robustly assess the quality of the assays. The resulting SRM assays are intended to be used to analyze mouse plasma and cardiac tissue, primarily for cardiovascular disease and cancer research. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  8. A more sensitive and specific radioenzymatic assay for catecholamines

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, B.; Ziegler, M.G. (Univ. of California, San Diego (USA))

    1990-01-01

    This modification of the catechol-O-methyltransferase (COMT) based radioenzymatic assay for norepinephrine (NE) and epinephrine (E) improves sensitivity, selectivity and eliminates many inhibitors of COMT. Prior to assay, samples are extracted into heptane with diphenylborate, then into dilute acetic acid. This extraction procedure has an efficiency of 78% for NE but less than 2% for S-adenosylmethionine (SAM). The extraction procedure also excludes calcium and other COMT inhibitors present in urine, plasma and every tissue tested. This eliminates the requirement for individual standardization of tissue and urine samples. Sensitivity of the assay for NE and E is 10 and 6 pg/ml respectively in 1 ml of plasma. The intraassay coefficients of variation for NE and E are 4 and 13% and the interassay coefficients of variation for NE and E are 10 and 16% in a human plasma sample containing low catecholamine levels. The assay permits quantitation of plasma E levels that were undetectable in prior assays.

  9. Environmental and occupational biomonitoring using the Comet assay.

    Science.gov (United States)

    Valverde, Mahara; Rojas, Emilio

    2009-01-01

    Biomonitoring of human populations exposed to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation in the development of cancer. In recent years, the Comet assay, also known as a "single cell gel" (SCG) electrophoresis assay, has become an important tool for assessing DNA damage in exposed populations. This is the method of choice for population-based studies of environmental and occupational exposure to air pollutants, metals, pesticides, radiation, and other xenobiotics as we show in this review. To appreciate the role of the Comet assay in the field of biomonitoring, we review data from 122 studies that employed the assay. These studies evaluated environmental versus occupational exposures and the levels of DNA damage in cells of individuals exposed in each case. Our review of the literature reveals the importance of the need to establish standard methodological conditions that affect unwinding and electrophoresis times and tail values (tail length, tail DNA, tail moment), with the goal of being able to compare data collected in different laboratories throughout the world. The Comet assay is susceptible to subtle artifacts of manipulation depending on the type and timing of sampling performed. Therefore, in the reporting of DNA damage detected by the Comet assay, the context of how the DNA damage was created also needs to be reported and considered in the interpretation of Comet assay results. The success of the Comet assay is reflected by its use over the past 20 years in the field of biomonitoring, and by the increasing number of studies that continue to report its use. As the shortcomings of the assay are identified and considered in the interpretation of DNA damage detection, the Comet assay will continue to provide improved reliability as a biomarker in human biomonitoring studies.

  10. The comet assay: ready for 30 more years.

    Science.gov (United States)

    Møller, Peter

    2018-01-06

    During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years. © The Author(s) 2018. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Development and Validation of a HPV-32 Specific PCR Assay

    Directory of Open Access Journals (Sweden)

    Leigh Janet

    2009-06-01

    Full Text Available Abstract Background Human Papillomavirus-32 (HPV-32 has traditionally been associated with focal-epithelial-hyperplasia (FEH. It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects. Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

  12. Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans

    NARCIS (Netherlands)

    van Doorn, H. Rogier; Koelewijn, Rob; Hofwegen, Henk; Gilis, Henk; Wetsteyn, Jose C. F. M.; Wismans, Pieter J.; Sarfati, Claudine; Vervoort, Tony; van Gool, Tom

    2007-01-01

    A homemade enzyme-linked immunosorbent assay (ELISA) (Academic Medical Center ELISA [AMC-ELISA]) and a dipstick assay for the detection of anti-Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA [IVD Research, Inc.

  13. APPLICATION OF RADIORECEPTOR ASSAYS FOR SYSTEMATIC TOXICOLOGICAL ANALYSIS .1. PROCEDURES FOR RADIORECEPTOR ASSAYS FOR ANTIHISTAMINICS, ANTICHOLINERGICS AND BENZODIAZEPINES

    NARCIS (Netherlands)

    ENSING, K; BOSMAN, IJ; EGBERTS, ACG; FRANKE, JP; DEZEEUW, RA

    Radioreceptor assays can be a useful tool for systematic toxicological analysis in that they can be applied for the detection of an entire pharmacological class of drugs. In the present paper procedures for radioreceptor assays for benzodiazepines, anticholinergics and antihistaminics have been

  14. Evaluation of the IMMULITE® 2000 CMV IgM assay.

    Science.gov (United States)

    Bal, Tricia A; Armstrong, Glenn; Han, Xiang Y

    2012-02-29

    Diagnosis of cytomegalovirus (CMV) infection is challenging because of the high rate of asymptomatic infection and the low specificity of associated symptoms and signs. As a result, laboratory testing is an essential aid in making an accurate diagnosis. The presence of CMV IgM is indicative of primary CMV infection. In pregnancy, diagnosis of primary infection is important because primary maternal infection increases fetal infection risk substantially. Fetal infection can result in serious sequelae ranging from neurological deficits to death. Diagnosis among the immunocompromised is also critical for the timely initiation of therapy that can reduce morbidity and mortality risk. The IMMULITE® 2000 CMV IgM assay qualitatively detects CMV IgM antibodies in human serum or plasma to aid in the diagnosis of current or recent CMV infection. To determine expected values in apparently healthy subjects, 136 samples were tested. Reproducibility, normal range, and method comparison studies were also performed to evaluate the assay's performance. The assay's reproducibility was evaluated across three sites. Seven hundred and eighteen (n = 718) individual patient serum samples, which included samples from CMV IgM-positive (n = 109, determined by the Abbott IMx CMV or the Diamedix CMV IgM assays), pregnant (n = 210), HIV-positive (n = 30), immunosuppressed (n = 102), and transplant patients (n = 17) and from patients with potentially cross-reacting conditions (n = 136) were evaluated in the method comparison study. The positive, negative, and overall agreement between the IMMULITE 2000 CMV IgM assay and the VIDAS CMV IgM assay (predicate assay) were determined. The assay demonstrated excellent reproducibility with a total CV of less than 10%. The positive, negative, and overall agreement between the IMMULITE 2000 assay and the VIDAS assay were > 95% for the method comparison samples. Among potentially cross-reactive samples, the overall agreement between the two assays was 96

  15. Evaluation of boron neutron capture effects in cell culture using sulforhodamine-B assay and a colony assay.

    Science.gov (United States)

    Wittig, A; Sauerwein, W; Pöller, F; Fuhrmann, C; Hideghéty, K; Streffer, C

    1998-06-01

    The purpose of this study was to find an in vitro method for determining the cytotoxicity of boronated drugs as well as their potential suitability for neutron capture therapy. The survival of human melanoma cells has been determined by a colony assay and the sulforhodamine-B assay after X-irradiation and irradiation with fast d(14) + Be-neutrons using the boronated compound borocaptate sodium (BSH). The cytotoxic effects of BSH have been studied using both methods. Under well-defined experimental conditions, and after a sufficient amount of time for the expression of radiation damage, the results of the sulforhodamine-B assay are qualitatively comparable with the results of the colony assay. The sulforhodamine-B assay is suitable for the screening of compounds for potential use in neutron capture therapy because it is a fast and efficient method that is reproducible and technically advantageous.

  16. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

    Science.gov (United States)

    Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

    2013-09-01

    Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men. © 2013 American Society of Andrology and European Academy of Andrology.

  17. The application of protein microarray assays in psychoneuroimmunology.

    Science.gov (United States)

    Ayling, K; Bowden, T; Tighe, P; Todd, I; Dilnot, E M; Negm, O H; Fairclough, L; Vedhara, K

    2017-01-01

    Protein microarrays are miniaturized multiplex assays that exhibit many advantages over the commonly used enzyme-linked immunosorbent assay (ELISA). This article aims to introduce protein microarrays to readers of Brain, Behavior, and Immunity and demonstrate its utility and validity for use in psychoneuroimmunological research. As part of an ongoing investigation of psychological and behavioral influences on influenza vaccination responses, we optimized a novel protein microarray to quantify influenza-specific antibody levels in human sera. Reproducibility was assessed by calculating intra- and inter-assay coefficients of variance on serially diluted human IgG concentrations. A random selection of samples was analyzed by microarray and ELISA to establish validity of the assay. For IgG concentrations, intra-assay and inter-assay precision profiles demonstrated a mean coefficient of variance of 6.7% and 11.5% respectively. Significant correlations were observed between microarray and ELISA for all antigens, demonstrating the microarray is a valid alternative to ELISA. Protein microarrays are a highly robust, novel assay method that could be of significant benefit for researchers working in psychoneuroimmunology. They offer high throughput, fewer resources per analyte and can examine concurrent neuro-immune-endocrine mechanisms. Copyright © 2016. Published by Elsevier Inc.

  18. Quantitative Fissile Assay In Used Fuel Using LSDS System

    Directory of Open Access Journals (Sweden)

    Lee YongDeok

    2017-01-01

    Full Text Available A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241 was done at Korea Atomic Energy Research Institute (KAERI, using lead slowing down spectrometer (LSDS. The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with ~2% uncertainty for Pu239. By applying the covering (neutron absorber, the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

  19. A High-Throughput Screening Assay to Detect ...

    Science.gov (United States)

    In support of the Endocrine Disruption Screening Program (EDSP21), the US EPA ToxCast program is developing assays to enable screening for chemicals that may disrupt thyroid hormone synthesis. Thyroperoxidase (TPO) is critical for TH synthesis and is a known target of thyroid-disrupting chemicals that adversely impact neurodevelopment. The AUR-TPO assay was recently developed to screen >1,900 ToxCast chemicals for potential TPO inhibition activity. Parallel assays were used to determine which AUR-TPO actives were more selective for TPO inhibition. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO assay and an orthogonal peroxidase oxidation assay using guaiacol as substrate to confirm putative TPO inhibition profiles. Bioactivity results from the AUR-TPO assay were used to identify chemical substructures associated with in vitro TPO inhibition. Substructure profiles were generated for each chemical in the ToxCast test set using the publicly-available ToxPrint 2.0 chemotypes. Chemotypes enriched among the putative TPO inhibitors were identified using a cumulative hypergeometric probability (p assay:

  20. Quantitative Fissile Assay In Used Fuel Using LSDS System

    Science.gov (United States)

    Lee, YongDeok; Jeon, Ju Young; Park, Chang-Je

    2017-09-01

    A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241) was done at Korea Atomic Energy Research Institute (KAERI), using lead slowing down spectrometer (LSDS). The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with 2% uncertainty for Pu239. By applying the covering (neutron absorber), the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

  1. Usefulness of human coagulation and fibrinolysis assays in domestic pigs

    DEFF Research Database (Denmark)

    Münster, Anna-Marie Bloch; Olsen, Aage Kristian; Bladbjerg, Else-Marie

    2002-01-01

    Pigs are often used as animal models in research on blood coagulation and fibrinolysis. The usefulness of the assays applied within this field, and the knowledge of reference intervals are therefore essential and of utmost importance. In the study reported here, we investigated the applicability...... of commercial human coagulation and fibrinolysis assays for use with porcine plasma. In total, 22 functional and immunologic assays were applied to plasma obtained from domestic pigs, and the following blood coagulation and fibrinolysis variables were measured: prothrombin time, activated partial thromboplastin...

  2. Ethanal assay, using an enzymo-conductimetric method

    Energy Technology Data Exchange (ETDEWEB)

    Saad, I.; Wallach, J.M. (I.C.B.M.C., Villeurbanne (France))

    1992-01-01

    An enzymo-conductimetric method for the assay of ethanol is described. It is based on ethanol oxidation is presence of yeast aldehyde dehydrogenase. Experimental conditions compatible with conductimetry were optimized and kinetic parameters of the enzymatic reaction determined. Due to the low K{sub M} value of ethanol for the enzyme, an end-point assay was proposed. A linear relationship was demonstrated between experimental conductance changes and ethanol concentration up to 25 {mu}M. Validation of the assay was made on wines by comparison with a spectrophotometric method. Both methods gave similar results, but conductimetry avoids sample treatment if solutions are colored or turbid.

  3. In vitro JAK kinase activity and inhibition assays.

    Science.gov (United States)

    Babon, Jeffrey J; Murphy, James M

    2013-01-01

    The discovery that a range of myeloproliferative diseases and leukemias are associated with Janus Kinase (JAK) mutations has highlighted the importance of JAK/STAT signalling in disease and sparked a renewed interest in developing JAK inhibitors. In vitro kinase assays are the most direct and quantitative method to assess mutant forms of JAK for altered enzymatic properties as well as verifying and quantifying the affinity and efficacy of potential inhibitors. Here, we describe protocols for heterologous expression and purification of JAK kinases from insect cells, assays to determine the activity of these purified kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.

  4. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

    Directory of Open Access Journals (Sweden)

    Chen Zhang

    2013-01-01

    Full Text Available Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis. In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

  5. Application of radioreceptor assay of benzodiazepines for toxicology

    Energy Technology Data Exchange (ETDEWEB)

    Aaltonen, L.; Scheinin, M. (Department of Pharmacology and the Department of Internal Medicine, University of Turku, Finland)

    1982-01-01

    A radioreceptor assay (RRA) for determining benzodiazepines (BZ) has been developed and applied to toxicological analysis of serum samples from 21 patients with acute BZ overdosage. The method was sensitive (e.g., lorazepam 17 nM, diazepam 41 nM), and specific for pharmacologically active BZ derivatives. The reproducibility of the results was good (intra-assay variation < 8%, inter-assay variation < 10%). Concentrations measured by the RRA showed a good correlation with those obtained by gas-liquid chromatographic analysis of the same samples. The quantitative results represent the sum of one or several parent substances and all biologically active metabolites, in proportion to their receptor binding affinities.

  6. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H2O2) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H2O2, less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier B

  7. The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop.

    Science.gov (United States)

    Koppen, Gudrun; Azqueta, Amaya; Pourrut, Bertrand; Brunborg, Gunnar; Collins, Andrew R; Langie, Sabine A S

    2017-05-01

    The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity. After 30 years of experimentation with the comet assay, we are in a position to control the important experimental parameters and make the comet assay a truly reliable method with a wealth of possible applications. © The Author 2017. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test

    National Research Council Canada - National Science Library

    Resende, Flavia Aparecida; Vilegas, Wagner; Dos Santos, Lourdes Campaner; Varanda, Eliana Aparecida

    2012-01-01

    The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles...

  9. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Science.gov (United States)

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  10. Simple sensitive radioreceptor assay for calcium antagonist drugs

    Energy Technology Data Exchange (ETDEWEB)

    Gould, R.J.; Murphy, K.M.M.; Snyder, S.H.

    1983-12-26

    A radioreceptor assay for calcium channel antagonist drugs described here is based on the ability of these drugs to affect /sup 3/H-nitrendipine binding to calcium channels. All the known calcium channel antagonists may be assayed in this manner. The assay can detect 10-100 nM nimodipine, 10-100 nM nifedipine, 3-30 ..mu..M prenylamine, 0.1 - 1.0 ..mu..M verapamil and 3-30 ..mu..M diltiazem. These values cover the range of concentrations of calcium channel antagonists that are clinically important. As the radioreceptor assay detects active metabolites as well as the parent drugs, it should prove a useful adjunct in cardiovascular therapy. The method is more reproducible, simpler and less expensive than other methods such as high pressure liquid chromatography.

  11. Study of Salmonella typhimurium mutagenicity assay of (E ...

    African Journals Online (AJOL)

    Study of Salmonella typhimurium mutagenicity assay of (E)-piplartine by the Ames test. AA Morandim-Giannetti, F Cotinguiba, LO Regasini, MC Frigieri, EA Varanda, A Coqueiro, MJ Kato, VS Bolzani, M Furlan ...

  12. A novel behavioral assay for measuring cold sensation in mice.

    Directory of Open Access Journals (Sweden)

    Daniel S Brenner

    Full Text Available Behavioral models of cold responses are important tools for exploring the molecular mechanisms of cold sensation. To complement the currently cold behavioral assays and allow further studies of these mechanisms, we have developed a new technique to measure the cold response threshold, the cold plantar assay. In this assay, animals are acclimated on a glass plate and a cold stimulus is applied to the hindpaw through the glass using a pellet of compressed dry ice. The latency to withdrawal from the cooled glass is used as a measure of the cold response threshold of the rodents, and the dry ice pellet provides a ramping cold stimulus on the glass that allows the correlation of withdrawal latency values to rough estimates of the cold response threshold temperature. The assay is highly sensitive to manipulations including morphine-induced analgesia, Complete Freund's Adjuvant-induced inflammatory allodynia, and Spinal Nerve Ligation-induced neuropathic allodynia.

  13. DNA Y structure: a versatile, multidimensional single molecule assay.

    Science.gov (United States)

    Inman, James T; Smith, Benjamin Y; Hall, Michael A; Forties, Robert A; Jin, Jing; Sethna, James P; Wang, Michelle D

    2014-11-12

    Optical trapping is a powerful single molecule technique used to study dynamic biomolecular events, especially those involving DNA and DNA-binding proteins. Current implementations usually involve only one of stretching, unzipping, or twisting DNA along one dimension. To expand the capabilities of optical trapping for more complex measurements would require a multidimensional technique that combines all of these manipulations in a single experiment. Here, we report the development and utilization of such a novel optical trapping assay based on a three-branch DNA construct, termed a "Y structure". This multidimensional assay allows precise, real-time tracking of multiple configurational changes. When the Y structure template is unzipped under both force and torque, the force and extension of all three branches can be determined simultaneously. Moreover, the assay is readily compatible with fluorescence, as demonstrated by unzipping through a fluorescently labeled, paused transcription complex. This novel assay thus allows for the visualization and precision mapping of complex interactions of biomechanical events.

  14. A comprehensive review on clinical applications of comet assay

    National Research Council Canada - National Science Library

    Gunasekarana, Vidya; Raj, Gladwin Victor; Chand, Parkash

    2015-01-01

    .... Among the various methods employed in the estimation of DNA damage, alkaline comet assay is proven to be a relatively simple and versatile tool in the assessment of DNA damage and also in determining...

  15. Development of fluorescent methods for DNA methyltransferase assay

    Science.gov (United States)

    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  16. Battery operated preconcentration-assisted lateral flow assay.

    Science.gov (United States)

    Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

    2017-07-11

    Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

  17. Radiometric microbiologic assay for the biologically active forms of niacin

    Energy Technology Data Exchange (ETDEWEB)

    Kertcher, J.A.; Guilarte, T.R.; Chen, M.F.; Rider, A.A.; McIntyre, P.A.

    1979-05-01

    A radiometric microbiologic assay has been developed for the determination of niacin in biologic fluids. Lactobacillus plantarum produced /sup 14/CO/sub 2/ from L-(U-/sup 14/C) malic acid in quantities proportional to the amount of niacin present. The assay is specific for the biologically active forms of niacin in humans. Thirty normal hemolysates were analyzed and the values ranged from 13.0 to 17.8 ..mu..g niacin/ml RBC (mean = 15.27 +- 1.33 s.d.). Good recovery and reproducibility studies were obtained with this assay. On thirty blood samples, correlation was excellent between the radiometric and the conventional turbidimetric assays.

  18. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

  19. Development of novel IVD assays: a manufacturer's perspective.

    Science.gov (United States)

    Metcalfe, Thomas A

    2010-01-01

    IVD manufacturers are heavily reliant on novel IVD assays to fuel their growth and drive innovation within the industry. They represent a key part of the IVD industry's value proposition to customers and the healthcare industry in general, driving product differentiation, helping to create demand for new systems and generating incremental revenue. However, the discovery of novel biomarkers and their qualification for a specific clinical purpose is a high risk undertaking and the large, risky investments associated with doing this on a large scale are incompatible with IVD manufacturer's business models. This article describes the sources of novel IVD assays, the processes for discovering and qualifying novel assays and the reliance of IVD manufacturers on collaborations and in-licensing to source new IVD assays for their platforms.

  20. Liquid chromatographic-tandem mass spectrometric assay for ...

    African Journals Online (AJOL)

    Liquid chromatographic-tandem mass spectrometric assay for simultaneous quantitation of tofacitinib, cabozantinib and afatinib in human plasma and urine. Adnan A. Kadi, Ali S. Abdelhameed, Hany W. Darwish, Mohamed W. Attwa, Ahmed H. Bakheit ...

  1. Click Chemistry-Mediated Nanosensors for Biochemical Assays.

    Science.gov (United States)

    Chen, Yiping; Xianyu, Yunlei; Wu, Jing; Yin, Binfeng; Jiang, Xingyu

    2016-01-01

    Click chemistry combined with functional nanoparticles have drawn increasing attention in biochemical assays because they are promising in developing biosensors with effective signal transformation/amplification and straightforward signal readout for clinical diagnostic assays. In this review, we focus on the latest advances of biochemical assays based on Cu (I)-catalyzed 1, 3-dipolar cycloaddition of azides and alkynes (CuAAC)-mediated nanosensors, as well as the functionalization of nanoprobes based on click chemistry. Nanoprobes including gold nanoparticles, quantum dots, magnetic nanoparticles and carbon nanomaterials are covered. We discuss the advantages of click chemistry-mediated nanosensors for biochemical assays, and give perspectives on the development of click chemistry-mediated approaches for clinical diagnosis and other biomedical applications.

  2. A sensitive and selective assay for chloramine production by myeloperoxidase.

    Science.gov (United States)

    Dypbukt, Jeannette M; Bishop, Cynthia; Brooks, Wendy M; Thong, Bob; Eriksson, Håkan; Kettle, Anthony J

    2005-12-01

    We describe a new assay for the chlorination activity of myeloperoxidase and detection of chloramines. Chloramines were detected by using iodide to catalyze the oxidation of either 3,3',5,5'-tetramethylbenzidine (TMB) or dihydrorhodamine to form strongly absorbing or fluorescent products, respectively. With TMB as little as 1 muM taurine chloramine could be detected. The sensitivity of the dihydrorhodamine assay was about 10-fold greater. The chlorination activity of myeloperoxidase was measured by trapping hypochlorous acid with taurine and subsequently using iodide to promote the oxidation reactions of the accumulated taurine chloramine. A similar approach was used to detect hypochlorous acid production by stimulated human neutrophils. Iodide-dependent catalysis distinguished N-chloramines from N-bromamines. This allows for discrimination between heme peroxidases that generate either hypochlorous acid or hypobromous acid. The assay has distinct advantages over existing assays for myeloperoxidase with regard to sensitivity, specificity, and its ease and versatility of use.

  3. Application of neutron multiplicity counting to waste assay

    Energy Technology Data Exchange (ETDEWEB)

    Pickrell, M.M.; Ensslin, N. [Los Alamos National Lab., NM (United States); Sharpe, T.J. [North Carolina State Univ., Raleigh, NC (United States)

    1997-11-01

    This paper describes the use of a new figure of merit code that calculates both bias and precision for coincidence and multiplicity counting, and determines the optimum regions for each in waste assay applications. A {open_quotes}tunable multiplicity{close_quotes} approach is developed that uses a combination of coincidence and multiplicity counting to minimize the total assay error. An example is shown where multiplicity analysis is used to solve for mass, alpha, and multiplication and tunable multiplicity is shown to work well. The approach provides a method for selecting coincidence, multiplicity, or tunable multiplicity counting to give the best assay with the lowest total error over a broad spectrum of assay conditions. 9 refs., 6 figs.

  4. Multiplex polymerase chain reaction (PCR) assay for simultaneous ...

    African Journals Online (AJOL)

    Multiplex polymerase chain reaction (PCR) assay for simultaneous detection of shiga-like toxin ( stx 1 and stx 2), intimin (eae) and invasive plasmid antigen H ( ipaH ) genes in diarrheagenic Escherichia coli.

  5. Cholinesterase assay by an efficient fixed time endpoint method

    Directory of Open Access Journals (Sweden)

    Mónica Benabent

    2014-01-01

    The method may be adapted to the user needs by modifying the enzyme concentration and applied for simultaneously testing many samples in parallel; i.e. for complex experiments of kinetics assays with organophosphate inhibitors in different tissues.

  6. PubChem BioAssay: 2017 update

    Science.gov (United States)

    Wang, Yanli; Bryant, Stephen H.; Cheng, Tiejun; Wang, Jiyao; Gindulyte, Asta; Shoemaker, Benjamin A.; Thiessen, Paul A.; He, Siqian; Zhang, Jian

    2017-01-01

    PubChem's BioAssay database (https://pubchem.ncbi.nlm.nih.gov) has served as a public repository for small-molecule and RNAi screening data since 2004 providing open access of its data content to the community. PubChem accepts data submission from worldwide researchers at academia, industry and government agencies. PubChem also collaborates with other chemical biology database stakeholders with data exchange. With over a decade's development effort, it becomes an important information resource supporting drug discovery and chemical biology research. To facilitate data discovery, PubChem is integrated with all other databases at NCBI. In this work, we provide an update for the PubChem BioAssay database describing several recent development including added sources of research data, redesigned BioAssay record page, new BioAssay classification browser and new features in the Upload system facilitating data sharing. PMID:27899599

  7. Bacterial enzyme biosyntheses inhibition: a tool for ecotoxicity assay ...

    African Journals Online (AJOL)

    hydrocarbon fuels, oil spill dispersants, household detergents, drilling chemicals and crude oils) were examined as a tool for ecotoxicity assay. The enzyme systems were two extracellular inducible enzymes tryptophanase and a - glucosidase ...

  8. Comparison of the direct enzyme assay method with the membrane ...

    African Journals Online (AJOL)

    Comparison of the direct enzyme assay method with the membrane filtration technique in the quantification and monitoring of microbial indicator organisms – seasonal variations in the activities of coliforms and E. coli, temperature and pH.

  9. Evaluation and potential of Double Immunodifusion Gel Assay for ...

    African Journals Online (AJOL)

    Evaluation and potential of Double Immunodifusion Gel Assay for serological characterization of rice yellow mottle virus isolates in West Africa. Yacouba Séré, Amos Onasanya, Abolade S Afolabi, Emmanuel M Abo ...

  10. Spectrophotometric assay of catalase with perborate as substrate

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, J.F.; Nance, S.L.; Tollaksen, S.L.

    1978-01-01

    A spectrophotometric assay for catalase has been developed in which sodium perborate, rather than hydrogen peroxide, is used as the substrate. The method is convenient, rapid, and readily adapted to the measurement of catalase in subcellular fractions.

  11. Colorimetric Assay Of Naproxen Tablets by Derivatization Using 4 ...

    African Journals Online (AJOL)

    A colorimetric assay of naproxen tablets based on chemical derivatization with the .... reported to couple with some phenol ethers. (anisole .... Properties, Applications 2nd ... Binding in: Physical Pharmacy: Physical. Chemical. Principles in the.

  12. Nondestructive assay needs at the Los Alamos Plutonium Facility

    Energy Technology Data Exchange (ETDEWEB)

    Cremesa, T.L.; Longmire, V.L. [Los Alamos National Laboratory, NM (United States)

    1993-12-31

    The Los Alamos National Laboratory Plutonium Facility is the leading facility for research and development in the area of plutonium processing. The diverse aspects of the activities at this facility require a large and varied effort in the area of non-destructive assay (NDA). The NDA activities at the plutonium facility fall into three different categories: accountability, safeguards, and process control. This talk will describe assay methods, instruments, and future needs and directions of NDA at the plutonium facility.

  13. Uranium internal exposure evaluation based on urine assay data

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, J.N.P.

    1984-09-01

    The difficulties in assessing internal exposures to uranium from urine assay data are described. A simplified application of the ICRP-30 and ICRP Lung Model concepts to the estimation of uranium intake is presented. A discussion follows on the development of a computer code utilizing the ICRP-30-based uranium elimination model with the existing urine assay information. The calculated uranium exposures from 1949 through 1983 are discussed. 13 references, 1 table.

  14. THE ISOLATION AND ASSAY OF HYPERTENSIN FROM BLOOD

    Science.gov (United States)

    Skeggs, Leonard T.; Kahn, Joseph R.; Shumway, Norman P.

    1952-01-01

    A method has been described for isolation and assay of hypertensin from the blood of dogs. An ethanol filtrate of blood drawn from the femoral artery is prepared and concentrated by evaporation. The hypertensin is extracted into n-butanol from which it is adsorbed into alumina, and subsequently eluted with dilute ethanol. The eluate is then evaporated to dryness, dissolved in water, and assayed by intravenous injection into rats. PMID:14927788

  15. Pitfalls in the assay of carboxymethylcellulase activity. [Sclerotium rolfsii

    Energy Technology Data Exchange (ETDEWEB)

    Lindner, W.A.; Dennison, C.; Quicke, G.V.

    1983-02-01

    A purified endocellulase from Sclerotium rolfsii and a crude cellulase preparation from Trichoderma reesei are used to illustrate several pitfalls associated with the assay of carboxymethylcellulase activity and the subsequent attainment of linear enzyme dilution curves. It is shown that the nature of both the enzymes and the substrate make the assay unsuitable for use in the calculation of enzyme recovery and purity. (Refs. 16).

  16. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Chinen, Isabel; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  17. A rapid assay for the biological evaluation of helicase activity.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Dimitrios Vlachakis, Andrea Brancale, Colin Berry & Sophia Kossida ### Abstract A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5’ termini. This ...

  18. A Quantitative Electrochemiluminescence Assay for Clostridium perfringens alpha toxin

    Science.gov (United States)

    2006-08-10

    Clostridium perfringens alpha toxin , Gerald A. Merrill a,b,¤, Victor R. Rivera b, Dwayne D. Neal b, Charles Young c, Mark A. Poli b a Department of...format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha...matrix eVects. © 2006 Elsevier Inc. All rights reserved. Keywords: Electrochemiluminescence (ECL); Immunoassay; Clostridium perfringens ; Alpha toxin

  19. Smartphone instrument for portable enzyme-linked immunosorbent assays

    OpenAIRE

    Long, Kenneth D.; Yu, Hojeong; Cunningham, Brian T.

    2014-01-01

    We demonstrate the utilization of a smartphone camera as a spectrometer that is capable of measuring Enzyme Linked Immunosorbent Assays (ELISA) at biologically-relevant concentrations with the aid of a custom cradle that aligns a diffraction grating and a collimating lens between a light source and the imaging sensor. Two example biomarkers are assayed using conventional ELISA protocols: IL-6, a protein used diagnostically for several types of cancer, and Ara h 1, one of the principle peanut ...

  20. Evaluation of The Alamarblue Assay for Adherent Cell Irradiation Experiments

    OpenAIRE

    Zachari, Maria A.; Chondrou, Panagiota S.; Pouliliou, Stamatia E.; Mitrakas, Achilleas G; Abatzoglou, Ioannis; Zois, Christos E.; Koukourakis, Michael I

    2013-01-01

    The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the...

  1. An o-phthalaldehyde spectrophotometric assay for proteinases.

    Science.gov (United States)

    Church, F C; Porter, D H; Catignani, G L; Swaisgood, H E

    1985-05-01

    A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.

  2. Murine High Specificity/Sensitivity Competitive Europium Insulin Autoantibody Assay

    Science.gov (United States)

    Babaya, Naru; Liu, Edwin; Miao, DongMei; Li, Marcella; Yu, Liping

    2009-01-01

    Abstract Background Most insulin autoantibody assays for both human and animal models are in a radioassay format utilizing 125I-insulin, but despite the radioassay format international workshops have documented difficulty in standardization between laboratories. There is thus a need for simpler assay formats that do not utilize radioactivity, yet retain the high specificity and sensitivity of radioassays. Methods To establish an easier enzyme-linked immunosorbent assay (ELISA) for insulin autoantibodies of non-obese diabetic (NOD) mice, we used an ELISA format, competition with unlabeled insulin, europium-avidin, and time-resolved fluorescence detection (competitive europium insulin autoantibody assay). Results The competitive europium assay of insulin autoantibodies when applied to sera from NOD mice had high sensitivity and specificity (92% sensitivity, 100% specificity) compared to our standard insulin autoantibody radioassay (72% sensitivity, 100% specificity) in analyzing blind workshop sera. It is noteworthy that though the assay has extremely high sensitivity for murine insulin autoantibodies and utilizes human insulin as target autoantigen, human sera with high levels of insulin autoantibodies are not detected. Conclusions Our results clearly indicate that low levels of insulin autoantibodies can be detected in an ELISA-like format. Combining a europium-based ELISA with competition with fluid-phase autoantigen can be applicable to many autoantigens to achieve high specificity and sensitivity in an ELISA format. PMID:19344197

  3. A Simple Assay for Measuring Catalase Activity: A Visual Approach

    Science.gov (United States)

    Iwase, Tadayuki; Tajima, Akiko; Sugimoto, Shinya; Okuda, Ken-ichi; Hironaka, Ippei; Kamata, Yuko; Takada, Koji; Mizunoe, Yoshimitsu

    2013-01-01

    In this study, an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity was developed. The assay reagents comprised only hydrogen peroxide and Triton X-100. The enzyme-generated oxygen bubbles trapped by Triton X-100 were visualized as foam, whose height was estimated. A calibration plot using the defined unit of catalase activity yielded the best linear fit over a range of 20–300 units (U) (y = 0.3794x − 2.0909, r2 = 0.993). The assay precision and reproducibility at 100 U were 4.6% and 4.8%, respectively. The applicability of the assay for measuring the catalase activity of various samples was assessed using laboratory strains of Escherichia coli, catalase-deficient isogenic mutants, clinically isolated Shiga toxin-producing E. coli, and human cells. The assay generated reproducible results. In conclusion, this new assay can be used to measure the catalase activity of bacterial isolates and human cells. PMID:24170119

  4. Determining the Degree of Promiscuity of Extensively Assayed Compounds.

    Science.gov (United States)

    Jasial, Swarit; Hu, Ye; Bajorath, Jürgen

    2016-01-01

    In the context of polypharmacology, an emerging concept in drug discovery, promiscuity is rationalized as the ability of compounds to specifically interact with multiple targets. Promiscuity of drugs and bioactive compounds has thus far been analyzed computationally on the basis of activity annotations, without taking assay frequencies or inactivity records into account. Most recent estimates have indicated that bioactive compounds interact on average with only one to two targets, whereas drugs interact with six or more. In this study, we have further extended promiscuity analysis by identifying the most extensively assayed public domain compounds and systematically determining their promiscuity. These compounds were tested in hundreds of assays against hundreds of targets. In our analysis, assay promiscuity was distinguished from target promiscuity and separately analyzed for primary and confirmatory assays. Differences between the degree of assay and target promiscuity were surprisingly small and average and median degrees of target promiscuity of 2.6 to 3.4 and 2.0 were determined, respectively. Thus, target promiscuity remained at a low level even for most extensively tested active compounds. These findings provide further evidence that bioactive compounds are less promiscuous than drugs and have implications for pharmaceutical research. In addition to a possible explanation that drugs are more extensively tested for additional targets, the results would also support a "promiscuity enrichment model" according to which promiscuous compounds might be preferentially selected for therapeutic efficacy during clinical evaluation to ultimately become drugs.

  5. Detection of Streptococcus pyogenes using rapid visual molecular assay.

    Science.gov (United States)

    Zhao, Xiangna; He, Xiaoming; Li, Huan; Zhao, Jiangtao; Huang, Simo; Liu, Wei; Wei, Xiao; Ding, Yiwei; Wang, Zhaoyan; Zou, Dayang; Wang, Xuesong; Dong, Derong; Yang, Zhan; Yan, Xiabei; Huang, Liuyu; Du, Shuangkui; Yuan, Jing

    2015-09-01

    Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64°C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Hemizona Assay and Sperm Penetration Assay in the Prediction of IVF Outcome: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Paraskevi Vogiatzi

    2013-01-01

    Full Text Available The limited predictive value of semen analysis in achieving natural conception or in IVF outcome confirms the need for sperm function tests to determine optimal management. We reviewed HZA and SPA predictive power in IVF outcome, with statistical significance of diagnostic power of the assays. HZA was readily efficient in predicting IVF outcome, while evident inconsistency among the studies analysed framed the SPA’s role in male fertility evaluation. Considerable variation was noted in the diagnostic accuracy values of SPA with wide sensitivity (52–100%, specificity (0–100%, and PPV (18–100% and NPV (0–100% together with fluctuation and notable differentiation in methodology and cutoff values employed by each group. HZA methodology was overall consistent with minor variation in cutoff values and oocyte source, while data analysis reported strong correlation between HZA results with IVF outcome, high sensitivity (75–100%, good specificity (57–100%, and high PPV (79–100% and NPV (68–100%. HZA correlated well with IVF outcome and demonstrated better sensitivity/specificity and positive/negative predictive power. Males with normal or slightly abnormal semen profiles could benefit by this intervention and could be evaluated prior to referral to assisted reproduction. HZA should be used in a sequential fashion with semen analysis and potentially other bioassays in an IVF setting.

  7. Clonogenic assays measure leukemia stem cell killing not detectable by chromium release and flow cytometric cytotoxicity assays.

    Science.gov (United States)

    Williams, Brent A; Wang, Xing-Hua; Keating, Armand

    2010-11-01

    NK-92, a permanent natural killer (NK) cell line, shows cytotoxicity against a variety of tumors and has been tested in a phase I trial. We tested the toxicity of NK-92 and chemotherapy drugs against the stem cell capacity of the acute leukemia cell line, KG1. While the chromium-release assay is the most common method for assessing cytotoxicity of immune effectors, and flow cytometry is increasingly used, the relationship of either assay to clonogenic readouts remains unknown. KG1 was assessed for stem cell frequency by serial dilution, single-cell sorting and colony growth in methylcellulose. KG1 was sorted into CD34(+) CD38(+) and CD34(+) CD38⁻ populations and recultured in liquid medium or methylcellulose to determine the proliferative capacity of each fraction. Cytotoxicity of NK-92, daunorubicin and cytarabine against KG1 was measured using the chromium-release assay, flow cytometry and clonogenic assays. The culture-initiating cell frequency of whole KG1 was between 1 in 100 to 1000 by serial dilution and single-cell sorting. Although a rare (1-3%) CD34(+) CD38⁻ population could be demonstrated in KG1, both fractions had equivalent proliferative capacity. The cumulative flow cytotoxicity assay was more sensitive than the chromium-release assay in detecting target cell killing. At a 10:1 ratio NK-92 eliminated the clonogenic capacity of KG1, which was not predicted by the chromium-release assay. Clonogenic assays provide a more sensitive means of assessing the effect of a cytotoxic agent against putative cancer stem cells within cell lines, provided that they grow well in liquid culture medium or methylcellulose.

  8. Assessment of the integrity of compounds stored in assay-ready plates using a kinase sentinel assay.

    Science.gov (United States)

    Chen, Ada; Zhao, Xiaoning; Mercer, Laina; Su, Cheng; Zalameda, Leeanne; Liu, Yichin; Lembke, Amanda; Eastwood, Heather; Dang, Son; Oung, Thim; Xia, Xiaoyang; Young, Stephen W; Xiao, Shouhua; McCarter, John D

    2013-09-01

    Sentinel assays are a convenient adjunct to LC-MS purity assessment to monitor the integrity of compounds in pharmaceutical screening collections over time. To assess the stability of compounds stored both at room temperature and at -20°C in assay-ready plates that were either vacuum pack-sealed using a convenient industrial vacuum sealing method or individually sealed using conventional foil seals, a diverse collection of ~ 5,000 compounds was assayed using a robust biochemical kinase assay at intervals over a one year period. Assay results at each time point were compared to those of initial assay using a series of correlations of compound Percent of Control (POC) values as well as IC50 values of a subset of compounds in 200 nL or 500 nL plates. The fraction of hits in common between initial assays and assays at later time points ranged from 82% to 95% over one year and remained relatively constant over time with all storage temperatures or sealing methods tested. A majority of the hits that exhibited a consistent gradual trend to lower potency over one year storage were shifted to lower potency upon the rapid removal of DMSO solvent. Compound precipitation rather than compound decomposition is the likely reason for trends to lower potency for most such compounds over the storage period. Plates stored at room temperature featured a significantly higher fraction of hits that exhibited a trend to lower apparent potency than those stored at -20°C suggesting that this lower temperature is preferable for longer-term storage.

  9. Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs.

    Science.gov (United States)

    Hanko, Valoran P; Rohrer, Jeffrey S

    2010-01-05

    The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.

  10. The Architect Syphilis assay for antibodies to Treponema pallidum: an automated screening assay with high sensitivity in primary syphilis.

    Science.gov (United States)

    Young, H; Pryde, J; Duncan, L; Dave, J

    2009-02-01

    To determine the sensitivity and specificity of the Architect Syphilis Chemiluminescence Assay (CLIA): a new highly automated screening test for syphilis. To establish the sensitivity of the Architect Syphilis assay we tested 129 stored sera from serologically characterised cases of untreated syphilis. The sera were selected to contain a disproportionately high number of primary infections. There were 79 primary infections, 29 secondary infections, 9 early latent infections and 12 latent syphilis of unknown duration. To establish the specificity of the assay we tested 1107 sera that had been submitted for routine syphilis serology. The Architect CLIA and the Treponema pallidum particle agglutination test (TPPA) were in total agreement for all untreated infection with sensitivity of 98.4%. This was significantly higher than the sensitivity of the Murex immune capture enzyme (ICE) immunoassay (86%, pArchitect and ICE assays was entirely due to primary stage syphilis (97.5% vs 77.2%, pArchitect CLIA was very high (99.1%, 1049/1059) it was significantly lower (p = 0.016) than that of the Murex ICE assay (99.9%). The Architect CLIA is significantly more sensitive than the Murex ICE screening assay in detecting primary syphilis but it is significantly less specific. Given the relatively high levels of early syphilis, we consider a small increase in the number of confirmatory tests required to exclude false-positive results is worthwhile to increase the detection of primary syphilis by 20%.

  11. Neutron Resonance Transmission Analysis (NRTA): A Nondestructive Assay Technique for the Next Generation Safeguards Initiative’s Plutonium Assay Challenge

    Energy Technology Data Exchange (ETDEWEB)

    J. W. Sterbentz; D. L. Chichester

    2010-12-01

    This is an end-of-year report for a project funded by the National Nuclear Security Administration's Office of Nuclear Safeguards (NA-241). The goal of this project is to investigate the feasibility of using Neutron Resonance Transmission Analysis (NRTA) to assay plutonium in commercial light-water-reactor spent fuel. This project is part of a larger research effort within the Next-Generation Safeguards Initiative (NGSI) to evaluate methods for assaying plutonium in spent fuel, the Plutonium Assay Challenge. The first-year goals for this project were modest and included: 1) developing a zero-order MCNP model for the NRTA technique, simulating data results presented in the literature, 2) completing a preliminary set of studies investigating important design and performance characteristics for the NRTA measurement technique, and 3) documentation of this work in an end of the year report (this report). Research teams at Los Alamos National Laboratory (LANL), Lawrence Berkeley National Laboratory (LBNL), Pacific Northwest National Laboratory (PNNL), and at several universities are also working to investigate plutonium assay methods for spent-fuel safeguards. While the NRTA technique is well proven in the scientific literature for assaying individual spent fuel pins, it is a newcomer to the current NGSI efforts studying Pu assay method techniques having just started in March 2010; several analytical techniques have been under investigation within this program for two to three years or more. This report summarizes a nine month period of work.

  12. Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

    Science.gov (United States)

    Serafini, Rosanna; Romano, Juan E; Varner, Dickson D; Di Palo, Rossella; Love, Charles C

    2015-08-01

    The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (PComet head measures (diameter, area, and intensity) and positively with percentage Ghosts (Psperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Epithelial cells as alternative human biomatrices for comet assay

    Directory of Open Access Journals (Sweden)

    Emilio eRojas

    2014-11-01

    Full Text Available The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes.Over a thirty year period, the comet assay in epithelial cells has been litlle employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  14. Worldwide interest in the comet assay: a bibliometric study.

    Science.gov (United States)

    Neri, Monica; Milazzo, Daniele; Ugolini, Donatella; Milic, Mirta; Campolongo, Alessandra; Pasqualetti, Patrizio; Bonassi, Stefano

    2015-01-01

    The comet assay is a rapid, sensitive and relatively simple method for measuring DNA damage. A bibliometric study was performed to evaluate temporal and geographical trends, research quality and main areas of interest in scientific production in this field. A PubMed search strategy was developed and 7674 citations were retrieved in the period 1990-2013. Notably, the MeSH (Medical Subject Headings) term 'comet assay', officially introduced in 2000, is used by indexers only in two thirds of papers retrieved. Articles on the comet assay were published in 78 countries, spread over the 5 continents. The EU contributed the greatest output, producing >2900 articles with IF (42.0%) and totalling almost 10000 IF points, and was followed by USA. In the new millennium, research with this assay reached a plateau or slow decline in the most industrialised areas (USA, Germany, UK, Italy), while its use has boomed in emerging countries, with increases of 5- to 7-fold in the last 10 years in China, India and Brazil, for instance. This transition resulted in a slow decrease of scientific production quality, as the countries that increased their relative weight typically had lower mIFs. The most common MeSH terms used in papers using the comet assay referred to wide areas of interest, such as DNA damage and repair, cell survival and apoptosis, cancer and oxidative stress, occupational and environmental health. Keywords related to humans, rodents and cell culture were also frequently used. The top journal for the comet assay articles was found to be Mutation Research, followed by Mutagenesis. Most papers using the comet assay as a biomarker were published in genetic and toxicology journals, with a stress on environmental and occupational disciplines. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Recommendations for safety testing with the in vivo comet assay.

    Science.gov (United States)

    Vasquez, Marie Z

    2012-08-30

    While the in vivo comet assay increases its role in regulatory safety testing, deliberations about the interpretation of comet data continue. Concerns can arise regarding comet assay publications with limited data from non-blind testing of positive control compounds and using protocols (e.g. dose concentrations, sample times, and tissues) known to give an expected effect. There may be a tendency towards bias when the validation or interpretation of comet assay data is based on results generated by widely accepted but non-validated assays. The greatest advantages of the comet assay are its sensitivity and its ability to detect genotoxicity in tissues and at sample times that could not previously be evaluated. Guidelines for its use and interpretation in safety testing should take these factors into account. Guidelines should be derived from objective review of data generated by blind testing of unknown compounds dosed at non-toxic concentrations and evaluated in a true safety-testing environment, where the experimental design and conclusions must be defensible. However, positive in vivo comet findings with such compounds are rarely submitted to regulatory agencies and this data is typically unavailable for publication due to its proprietary nature. To enhance the development of guidelines for safety testing with the comet assay, and with the permission of several sponsors, this paper presents and discusses relevant data from multiple GLP comet studies conducted blind, with unknown pharmaceuticals and consumer products. Based on these data and the lessons we have learned through the course of conducting these studies, I suggest significant adjustments to the current conventions, and I provide recommendations for interpreting in vivo comet assay results in situations where risk must be evaluated in the absence of carcinogenicity or clinical data. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Epithelial cells as alternative human biomatrices for comet assay.

    Science.gov (United States)

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  17. Phosphatidylglycerol in amniotic fluid. Comparison of an "ultrasensitive" immunologic assay with TLC and enzymatic assay.

    Science.gov (United States)

    Eisenbrey, A B; Epstein, E; Zak, B; McEnroe, R J; Artiss, J D; Kiechle, F L

    1989-03-01

    Phosphatidylglycerol (PG) in amniotic fluid is recognized as a good indicator of fetal lung maturity and is unaffected by moderate amounts of blood or meconium contamination. A rapid immunologic agglutination assay, Ultrasensitive AmnioStat-FLM (FLM), was compared with two-dimensional thin-layer chromatography (TLC) and an enzymic, colorimetric procedure (E-PG). Eighty amniotic fluid specimens were analyzed. FLM results were reported as high (H), intermediate (I), or low positive (L). TLC was compared with FLM:H (n = 27), mean 0.14 (fraction of total phospholipids); I (n = 7), mean 0.11; L (n = 9), mean 0.03; negative results had no detectable PG by TLC. In 33 cases E-PG was compared with FLM:H (n = 9), mean 7.0 mumol/L; I (n = 5), mean 8.1 mumol/L; L (n = 3), mean 3.0 mumol/L; negative (n = 16), mean 3.2 mumol/L. Records were reviewed in 70 cases. Thirty cases were excluded: sample to delivery time was greater than 72 hours; steroids were given or sepsis was documented. Fetal lung immaturity was clinically present in six cases: respiratory distress syndrome in three cases and transient tachypnea of the newborn (TTN) in three cases. One false positive result was identified (TTN, FLM:H). FLM sensitivity for fetal lung maturity was 85.3%, specificity was 83.3%, and the positive predictive value for fetal lung maturity was 96.7%. FLM is a fast, reliable indicator of fetal lung maturity.

  18. The comet assay in testing the potential genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Amaya Azqueta

    2015-06-01

    Full Text Available In the last two decades the production and use of nanomaterials (NMs has impressively increased. Their small size, given a mass equal to that of the corresponding bulk material, implies an increase in the surface area and consequently in the number of atoms that can be reactive. They possess different physical, chemical and biological properties compared to bulk materials of the same composition, which makes them very interesting and valuable for many different applications in technology, energy, construction, electronics, agriculture, optics, paints, textiles, food, cosmetics, medicine... Toxicological assessment of NMs is crucial; the same properties that make them interesting also make them potentially harmful for health and the environment. However, the term NM covers many different kinds of particle , and so there is no simple, standard approach to assessing their toxicity. NMs can enter the cell, interact with cell components and even penetrate the nucleus and interfere with the genetic material. Among the different branches of toxicology, genotoxicity is a main area of concern since it is closely related with the carcinogenic potential of compounds. The Organisation for Economic Co-operation and Development (OECD has published internationally agreed in vitro and in vivo validated test methods to evaluate different genotoxic endpoints of chemicals, including chromosome and gene mutations, and DNA breaks. However not all the assays are suitable to study the genotoxic potential of NMs as has been shown by the OECD Working Party on Manufactured Nanomaterials (WPMN. Moreover, alterations to DNA bases, which are precursors to mutations and of great importance in elucidating the mechanism of action of NMs, are not covered by the OECD guidelines. The in vivo standard comet assay (which measures DNA breaks and alkali-labile sites was included in the OECD assays battery in September 2014 while the in vitro standard comet assay is currently under

  19. A homogeneous biochemiluminescent assay for detection of influenza

    Science.gov (United States)

    Hui, Kwok Min; Li, Xiao Jing; Pan, Lu; Li, X. J.

    2015-05-01

    Current methods of rapid detection of influenza are based on detection of the nucleic acids or antigens of influenza viruses. Since influenza viruses constantly mutate leading to appearance of new strains or variants of viruses, these detection methods are susceptible to genetic changes in influenza viruses. Type A and B influenza viruses contain neuraminidase, an essential enzyme for virus replication which enables progeny influenza viruses leave the host cells to infect new cells. Here we describe an assay method, the homogeneous biochemiluminescent assay (HBA), for rapid detection of influenza by detecting viral neuraminidase activity. The assay mimics the light production process of a firefly: a viral neuraminidase specific substrate containing a luciferin moiety is cleaved in the presence of influenza virus to release luciferin, which becomes a substrate to firefly luciferase in a light production system. All reagents can be formulated in a single reaction mix so that the assay involves only one manual step, i.e., sample addition. Presence of Type A or B influenza virus in the sample leads to production of strong, stable and easily detectable light signal, which lasts for hours. Thus, this influenza virus assay is suitable for use in point-of-care settings.

  20. Reliability of the comet assay in cryopreserved human sperm.

    Science.gov (United States)

    Duty, S M; Singh, N P; Ryan, L; Chen, Z; Lewis, C; Huang, T; Hauser, R

    2002-05-01

    Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.

  1. Quantitative CrAssphage PCR Assays for Human Fecal ...

    Science.gov (United States)

    Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools. To inform the public.

  2. Evaluation of the Determine™ fourth generation HIV rapid assay.

    Science.gov (United States)

    Brauer, Marieke; De Villiers, Johanna C; Mayaphi, Simnikiwe H

    2013-04-01

    Assays that detect p24 antigen reduce the diagnostic window period of HIV testing. Most point-of-care HIV assays have poor sensitivity to diagnose acute HIV infection as they only detect antibodies against HIV-1 and HIV-2 (HIV-1/2). This was a cross-sectional laboratory-based study that evaluated the performance of the Determine™ HIV-1/2 Ag/Ab Combo fourth generation rapid strip - currently the only rapid assay that detects both HIV-1/2 antibodies and p24 antigen. A total of 79 serum specimens (29 positive for HIV antibodies only, 14 positive for HIV antibodies and p24 antigen, 20 HIV-negative, and 16 positive for p24 antigen only) were used for the evaluation. Results were compared with those from validated fourth generation HIV ELISAs. The Determine™ Combo rapid strips had a sensitivity of 90.7% and a specificity of 100% for the detection of HIV-1/2 antibodies. Its sensitivity for the detection of p24 antigen was only 10% (3 out of 30 p24 antigen positive specimens). This implies that most acute HIV infections will be missed with this assay. The need for a point-of-care assay which can detect acute HIV infection reliably still remains, particularly for use in a high prevalence setting such as South Africa. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Evaluation of a radioreceptor assay for TSH receptor autoantibodies

    Energy Technology Data Exchange (ETDEWEB)

    Rootwelt, K.

    1988-02-01

    A commercial radioreceptor assay for TSH receptor autoantibodies (TRAb), based on solubilized porcine receptor and purified radio-iodinated bovine TSH, was tested in 264 subjects with a variety of thyroid disorders. The sensitivity of the assay for the detection of hyperthyroid Graves' disease was 91%. The assay specificity for Graves' disease was 95%. With the exception of one patient with Hashimoto's disease and one patient with de Quervain's subacute thyroiditis no subjects other than Graves' patients had detectable TRAb. Thus purely blocking TSII receptor autoantibodies were not detected with the assay. One female with thyroxine-treated idiopathic primary hypothyroidism who had given birth to two children with transiently elevated TSH, was found to have a circulating TSH-binding substance that resulted in an abnormally negative TRAb value, and highly discrepant results when TSH was measured with a double antibody TSH radioimmunoassay and an immunoradiometric assay. The TSH-binding substance was precipitated like a protein, but was not IgG. Similar findings have not previously been reported.

  4. Comment on The Ecstasy and Agony of Assay Interference Compounds.

    Science.gov (United States)

    Kenny, Peter W

    2017-11-27

    A recent editorial (Aldrich et al. The Ecstasy and Agony of Assay Interference Compounds . J. Chem. Inf. 2017 , 57 , 387 - 390 ) is examined critically. When assessing assay hits from screening, it is important to draw a distinction between false positives, that have no effect on target function, and compounds that affect target function through an undesirable mechanism of action. Observation of frequent-hitter behavior for a compound should be regarded as circumstantial evidence, rather than definitive proof, that the compound has interfered with assay readouts or acted through an undesirable mechanism of action. The applicability domain of published (Baell and Holloway J. Med. Chem. 2010 , 53 , 2719 - 2740 ) Pan Assay INterference compoundS (PAINS) filters is limited by the narrow scope of the proprietary data used to derive them. It is suggested that journal guidelines for authors should not prescribe, as those for the Journal of Medicinal Chemistry appear to do, that activity in assays reported for compounds that match PAINS filters be treated any differently from that for compounds that do not match PAINS filters. It is argued that use of models based on proprietary data in the evaluation of manuscripts would contradict the editorial policy of any journal that deemed the use of proprietary data to be unacceptable in modeling studies.

  5. Improvements in Mass Spectrometry Assay Library Generation for Targeted Proteomics.

    Science.gov (United States)

    Teleman, Johan; Hauri, Simon; Malmström, Johan

    2017-07-07

    In data-independent acquisition mass spectrometry (DIA-MS), targeted extraction of peptide signals in silico using mass spectrometry assay libraries is a successful method for the identification and quantification of proteins. However, it remains unclear if high quality assay libraries with more accurate peptide ion coordinates can improve peptide target identification rates in DIA analysis. In this study, we systematically improved and evaluated the common algorithmic steps for assay library generation and demonstrate that increased assay quality results in substantially higher identification rates of peptide targets from mouse organ protein lysates measured by DIA-MS. The introduced changes are (1) a new spectrum interpretation algorithm, (2) reapplication of segmented retention time normalization, (3) a ppm fragment mass error matching threshold, (4) usage of internal peptide fragments, and (5) a multilevel false discovery rate calculation. Taken together, these changes yielded 14-36% more identified peptide targets at 1% assay false discovery rate and are implemented in three new open source tools, Fraggle, Tramler, and Franklin, available at https://github.com/fickludd/eviltools . The improved algorithms provide ways to better utilize discovery MS data, translating to substantially increased DIA performance and ultimately better foundations for drawing biological conclusions in DIA-based experiments.

  6. Single Cell Gel Electrophoresis in DNA Damage Detection (Comet Assay

    Directory of Open Access Journals (Sweden)

    Aysen Durmaz

    2010-08-01

    Full Text Available “Single cell gel electrophoresis (SCGE”, also called “Comet Assay”, is a sensitive, reliable and rapid technique for quantifying and analyzing DNA damage in individual cells. The comet assay is widely used in living cells, researches and the applications on comet assay is becoming broader day by day. To date, the comet assay has been used for a variety of applications, including genotoxic and cytotoxic agent analyses, environmental toxicology, cancer research, and radiation biology. Briefly, in comet assay, fully frosted microscope slides were first covered with 0.5% normal melting point agarose (NMA and air-dried at the room temperature then cells were mixed with 0.5% low melting point agarose (LMA at 37oC to form a cell suspension which was spread onto the slide surface and let it solidified. A third layer of 0.5% low melting point agarose was then added and again allowed to solidify. After preparing the three layer agarose the slides were immersed in lysing solution at least for an hour. The slides were then placed in an electrophoresis tank which contained neutral or alkaline buffer solution and kept in there for a short while prior to electric field application. After electrophoresis, the slides were washed with neutralization solution or PBS and stained with a DNA-specific fluorescent dye and analyzed using a fluorescent microscope. [Archives Medical Review Journal 2010; 19(4.000: 236-

  7. The use of comet assay in plant toxicology: recent advances.

    Science.gov (United States)

    Santos, Conceição L V; Pourrut, Bertrand; Ferreira de Oliveira, José M P

    2015-01-01

    The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

  8. A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.

    Science.gov (United States)

    Shin, Daniel; Frane, Nicole D; Brecht, Ryan M; Keeler, Jesse; Nagarajan, Rajesh

    2015-12-01

    Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-l-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well-documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA- and acyl-ACP-utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The use of comet assay in plant toxicology: recent advances

    Science.gov (United States)

    Santos, Conceição L. V.; Pourrut, Bertrand; Ferreira de Oliveira, José M. P.

    2015-01-01

    The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage. PMID:26175750

  10. Comet assay: an essential tool in toxicological research.

    Science.gov (United States)

    Glei, M; Schneider, T; Schlörmann, W

    2016-10-01

    The comet assay is a versatile, reliable, cost-efficient, and fast technique for detecting DNA damage and repair in any tissue. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. Instead of highlighting one of the numerous specific aspects of the comet assay, the present review aims at giving an overview about the evolution of this widely applicable method from the first description by Ostling and Johanson to the OECD Guideline 489 for the in vivo mammalian comet assay. In addition, methodical aspects and the influence of critical steps of the assay as well as the evaluation of results and improvements of the method are reviewed. Methodical aspects regarding oxidative DNA damage and repair are also addressed. An overview about the most recent works and relevant cutting-edge reviews based on the comet assay with special regard to, e.g., clinical applications, nanoparticles or environmental risk assessment concludes this review. Taken together, the presented overview raises expectations to further decades of successful applications and enhancements of this excellent method.

  11. Drosophila comet assay: insights, uses, and future perspectives

    Science.gov (United States)

    Gaivão, Isabel; Sierra, L. María

    2014-01-01

    The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

  12. A high-throughput biliverdin assay using infrared fluorescence.

    Science.gov (United States)

    Berlec, Aleš; Štrukelj, Borut

    2014-07-01

    Biliverdin is an intermediate of heme degradation with an established role in veterinary clinical diagnostics of liver-related diseases. The need for chromatographic assays has so far prevented its wider use in diagnostic laboratories. The current report describes a simple, fast, high-throughput, and inexpensive assay, based on the interaction of biliverdin with infrared fluorescent protein (iRFP) that yields functional protein exhibiting infrared fluorescence. The assay is linear in the range of 0-10 µmol/l of biliverdin, has a limit of detection of 0.02 μmol/l, and has a limit of quantification of 0.03 µmol/l. The assay is accurate with relative error less than 0.15, and precise, with coefficient of variation less than 5% in the concentration range of 2-9 µmol/l of biliverdin. More than 95% of biliverdin was recovered from biological samples by simple dimethyl sulfoxide extraction. There was almost no interference by hemin, although bilirubin caused an increase in the biliverdin concentration, probably due to spontaneous oxidation of bilirubin to biliverdin. The newly developed biliverdin assay is appropriate for reliable quantification of large numbers of samples in veterinary medicine.

  13. The use of comet assay in plant toxicology: recent advances

    Directory of Open Access Journals (Sweden)

    Conceição LV Santos

    2015-06-01

    Full Text Available The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g. Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

  14. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  15. Scintillation proximity assay for measurement of RNA methylation.

    Science.gov (United States)

    Baker, Matthew R; Zarubica, Tamara; Wright, H Tonie; Rife, Jason P

    2009-03-01

    Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC'. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC' most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA-protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format.

  16. Real-time assay for testing components of protein synthesis

    Science.gov (United States)

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Goldman, Yale E.; Cooperman, Barry S.

    2012-01-01

    We present a flexible, real-time-coupled transcription–translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore. PMID:22422844

  17. Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays.

    Directory of Open Access Journals (Sweden)

    Kazutoyo Miura

    Full Text Available Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA. The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH Harmonised Tripartite Guideline Q2(R1 details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability. We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in

  18. Formalization, annotation and analysis of diverse drug and probe screening assay datasets using the BioAssay Ontology (BAO.

    Directory of Open Access Journals (Sweden)

    Uma D Vempati

    Full Text Available Huge amounts of high-throughput screening (HTS data for probe and drug development projects are being generated in the pharmaceutical industry and more recently in the public sector. The resulting experimental datasets are increasingly being disseminated via publically accessible repositories. However, existing repositories lack sufficient metadata to describe the experiments and are often difficult to navigate by non-experts. The lack of standardized descriptions and semantics of biological assays and screening results hinder targeted data retrieval, integration, aggregation, and analyses across different HTS datasets, for example to infer mechanisms of action of small molecule perturbagens. To address these limitations, we created the BioAssay Ontology (BAO. BAO has been developed with a focus on data integration and analysis enabling the classification of assays and screening results by concepts that relate to format, assay design, technology, target, and endpoint. Previously, we reported on the higher-level design of BAO and on the semantic querying capabilities offered by the ontology-indexed triple store of HTS data. Here, we report on our detailed design, annotation pipeline, substantially enlarged annotation knowledgebase, and analysis results. We used BAO to annotate assays from the largest public HTS data repository, PubChem, and demonstrate its utility to categorize and analyze diverse HTS results from numerous experiments. BAO is publically available from the NCBO BioPortal at http://bioportal.bioontology.org/ontologies/1533. BAO provides controlled terminology and uniform scope to report probe and drug discovery screening assays and results. BAO leverages description logic to formalize the domain knowledge and facilitate the semantic integration with diverse other resources. As a consequence, BAO offers the potential to infer new knowledge from a corpus of assay results, for example molecular mechanisms of action of perturbagens.

  19. Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays.

    Science.gov (United States)

    Miura, Kazutoyo; Deng, Bingbing; Tullo, Gregory; Diouf, Ababacar; Moretz, Samuel E; Locke, Emily; Morin, Merribeth; Fay, Michael P; Long, Carole A

    2013-01-01

    Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA). The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds) to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability). We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in preclinical and

  20. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Science.gov (United States)

    Chan, Grace Ka Yan; Kleinheinz, Tracy L; Peterson, David; Moffat, John G

    2013-01-01

    In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  1. Neutron Based Non-Destructive Assay (NDA) Measurement Systems for Safeguard

    Energy Technology Data Exchange (ETDEWEB)

    Swinhoe, Martyn Thomas [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-09-21

    The objectives of this project are to introduce the assay methods for plutonium measurements using the HLNC; introduce the assay method for bulk uranium measurements using the AWCC; and introduce the assay method for fuel assembly measurements using the UNCL.

  2. Water-soluble conjugated polymers for fluorescent-enzyme assays.

    Science.gov (United States)

    Feng, Fude; Liu, Libing; Yang, Qiong; Wang, Shu

    2010-08-17

    Enzyme assays are receiving more and more research and application interest because of the rapidly increasing demands of clinical diagnosis, environmental analysis, drug discovery, and molecular biology. Water-soluble light-harvesting conjugated polymers (CPs) coordinate the action of a large number of absorbing units to afford an amplified fluorescence signal, which makes them useful as optical platforms in highly sensitive chemical and biological sensors. This Feature Article highlights recent developments of water-soluble CPs for fluorescent assays of enzymes. Different signal transduction mechanisms, such as electron transfer, fluorescence resonance energy transfer (FRET), and aggregation or conformation changes of CPs, are employed in these assays according to the dissimilar nature of enzymes. Potential challenges and future research directions in these approaches based on CPs are also discussed. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Development of robotic plasma radiochemical assays for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Alexoff, D.L.; Shea, C.; Fowler, J.S.; Gatley, S.J.; Schlyer, D.J. [Brookhaven National Lab., Upton, NY (United States). Dept. of Chemistry

    1995-12-01

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [{sup 11}C]Benztropine, [{sup 11}C]cocaine, [{sup 11}C]clorgyline, [{sup 11}C]deprenyl, [{sup 11}C]methadone, [{sup 11}C]methylphenidate, [{sup 11}C]raclorpride, and [{sup 11}C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers.

  4. Kynetic resazurin assay (KRA) for bacterial quantification of foodborne pathogens

    Science.gov (United States)

    Arenas, Yaxal; Mandel, Arkady; Lilge, Lothar

    2012-03-01

    Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.

  5. Lipase assay in duodenal juice using a conductimetric method.

    Science.gov (United States)

    Ballot, C; Favre-Bonvin, G; Wallach, J M

    1984-11-15

    Lipase activity in duodenal juice is known to undergo important variations in pathologic states, especially in cases of chronic pancreatitis. Almost all of the current assay methods are based on the measurement of hydrolysis of olive oil or triolein, mainly by potentiometry. As we have developed a conductimetric method for enzyme activity measurements, we have applied it to lipase assay. A higher experimental conductimetric sensitivity is obtained when liberated acids have a short chain (higher limiting equivalent conductivity). We have therefore used triacetin as a substrate and compared out method with potentiometry (pH-stat) and spectrophotometry. The correlation coefficients of both methods with conductimetry were 0.94 and 0.97, respectively, indicating that the conductimetric method may be used for lipase assay in duodenal juice, using triacetin as a substrate.

  6. Application of statistical process control to qualitative molecular diagnostic assays

    LENUS (Irish Health Repository)

    O'Brien, Cathal P.

    2014-11-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  7. Optimization of protein samples for NMR using thermal shift assays.

    Science.gov (United States)

    Kozak, Sandra; Lercher, Lukas; Karanth, Megha N; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane

    2016-04-01

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

  8. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  9. Rapid electrochemiluminescence assays of polymerase chain reaction products.

    Science.gov (United States)

    Kenten, J H; Casadei, J; Link, J; Lupold, S; Willey, J; Powell, M; Rees, A; Massey, R

    1991-09-01

    We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

  10. Smartphone instrument for portable enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Long, Kenneth D; Yu, Hojeong; Cunningham, Brian T

    2014-11-01

    We demonstrate the utilization of a smartphone camera as a spectrometer that is capable of measuring Enzyme Linked Immunosorbent Assays (ELISA) at biologically-relevant concentrations with the aid of a custom cradle that aligns a diffraction grating and a collimating lens between a light source and the imaging sensor. Two example biomarkers are assayed using conventional ELISA protocols: IL-6, a protein used diagnostically for several types of cancer, and Ara h 1, one of the principle peanut allergens. In addition to the demonstration of limits of detection at medically-relevant concentrations, a screening of various cookies was completed to measure levels of peanut cross-contamination in local bakeries. The results demonstrate the utility of the instrument for quantitatively performing broad classes of homogeneous colorimetric assays, in which the endpoint readout is the color change of a liquid sample.

  11. Accurate detection of BRAF p.V600E mutations in challenging melanoma specimens requires stringent immunohistochemistry scoring criteria or sensitive molecular assays.

    Science.gov (United States)

    Fisher, Kevin E; Cohen, Cynthia; Siddiqui, Momin T; Palma, John F; Lipford, Edward H; Longshore, John W

    2014-11-01

    Malignant melanoma patients require BRAF mutation testing prior to initiating BRAF inhibitor therapy. Molecular testing remains the diagnostic gold standard, but recent work suggests that BRAF immunohistochemistry (IHC) confers comparable results. Sample attributes and scoring criteria that may affect BRAF IHC interpretation, however, are poorly defined. We investigated formalin-fixed, paraffin-embedded samples with variable challenging interpretative attributes: metastases, core needle biopsies, sample tissues less than 60 mm(2), samples with greater than 50% necrosis, and/or samples with greater than 10% melanin pigmentation. Three pathologists independently scored 122 BRAF V600E IHC-labeled melanoma samples for percentage (0%-100%) of staining intensity (0-3+). Interscorer BRAF IHC discrepancies were resolved by consensus review. Lenient (≥1+, >0%) and stringent (≥2+, ≥10%) IHC scoring criteria were compared to BRAF V600 mutation (cobas) results (n = 118). Specimens with greater than 10% melanin pigmentation and metastatic samples produced the majority of interobserver IHC and IHC/cobas scoring discrepancies. Consensus review using stringent scoring criteria decreased the number of discrepant results, yielded very good interobserver reproducibility, and improved specificity and positive predictive value for BRAF p.V600E detection. BRAF p.V600K mutations accounted for 57.1% of false-negative IHC results when stringent, consensus criteria scoring were used. The cobas test detected 75.0% (8/12) of BRAF IHC-negative BRAF p.V600K mutations confirmed by next-generation sequencing. Molecular BRAF testing is the preferred screening test for BRAF inhibitor therapy eligibility because of superior sensitivity in challenging interpretative melanoma specimens. However, BRAF V600E IHC has excellent specificity and positive predictive value when stringent, consensus scoring criteria are implemented. To decrease IHC scoring discrepancies, pathologists should interpret

  12. Analytical and clinical sensitivity of the 3M rapid detection influenza A+B assay.

    Science.gov (United States)

    Dale, Suzanne E; Mayer, Christine; Mayer, Marie C; Menegus, Marilyn A

    2008-11-01

    The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems.

  13. Analytical and Clinical Sensitivity of the 3M Rapid Detection Influenza A+B Assay

    Science.gov (United States)

    Dale, Suzanne E.; Mayer, Christine; Mayer, Marie C.; Menegus, Marilyn A.

    2008-01-01

    The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems. PMID:18832133

  14. Characterizing Reactivity to Onchocerca volvulus Antigens in Multiplex Bead Assays.

    Science.gov (United States)

    Feeser, Karla R; Cama, Vitaliano; Priest, Jeffrey W; Thiele, Elizabeth A; Wiegand, Ryan E; Lakwo, Thomson; Feleke, Sindew M; Cantey, Paul T

    2017-09-01

    Multiplex bead assays (MBAs) may provide a powerful integrated tool for monitoring, evaluation, and post-elimination surveillance of onchocerciasis and co-endemic diseases; however, the specificity and sensitivity of Onchocerca volvulus antigens have not been characterized within this context. An MBA was developed to evaluate three antigens (OV-16, OV-17, and OV-33) for onchocerciasis. Receiver operating characteristics (ROC) analyses were used to characterize antigen performance using a panel of 610 specimens: 109 O. volvulus-positive specimens, 426 non-onchocerciasis controls with filarial and other confirmed parasitic infection, and 75 sera from patients with no other parasitic infection. The IgG and IgG4 assays for OV-16 demonstrated sensitivities of 95.4% and 96.3%, and specificities of 99.4% and 99.8%, respectively. The OV-17 IgG and IgG4 assays had sensitivities of 86.2% and 76.1% and specificities of 79.2% and 82.8%. For OV-33, the IgG and IgG4 assays had sensitivities of 90.8% and 96.3%, and specificities of 96.8% and 98.6%. The OV-16 IgG4-based MBA had the best assay characteristics, followed by OV-33 IgG4. The OV-16 IgG4 assay would be useful for monitoring and evaluation using the MBA platform. Further evaluations are needed to review the potential use of OV-33 as a confirmatory test in the context of program evaluations.

  15. Digital microfluidics for spheroid-based invasion assays.

    Science.gov (United States)

    Bender, Brian F; Aijian, Andrew P; Garrell, Robin L

    2016-04-21

    Cell invasion is a key process in tissue growth, wound healing, and tumor progression. Most invasion assays examine cells cultured in adherent monolayers, which fail to recapitulate the three-dimensional nuances of the tissue microenvironment. Multicellular cell spheroids have a three-dimensional (3D) morphology and mimic the intercellular interactions found in tissues in vivo, thus providing a more physiologically relevant model for studying the tissue microenvironment and processes such as cell invasion. Spheroid-based invasion assays often require tedious, manually intensive handling protocols or the use of robotic liquid handling systems, which can be expensive to acquire, operate, and maintain. Here we describe a digital microfluidic (DμF) platform that enables formation of spheroids by the hanging drop method, encapsulation of the spheroids in collagen, and the exposure of spheroids to migration-modulating agents. Collagen sol-gel solutions up to 4 mg mL(-1), which form gels with elastic moduli up to ∼50 kPa, can be manipulated on the device. In situ spheroid migration assays show that cells from human fibroblast spheroids exhibit invasion into collagen gels, which can be either enhanced or inhibited by the delivery of exogenous migration modulating agents. Exposing fibroblast spheroids to spheroid secretions from colon cancer spheroids resulted in a >100% increase in fibroblast invasion into the collagen gel, consistent with the cancer-associated fibroblast phenotype. These data show that DμF can be used to automate the liquid handling protocols for spheroid-based invasion assays and create a cell invasion model that mimics the tissue microenvironment more closely than two-dimensional culturing techniques do. A DμF platform that facilitates the creation and assaying of 3D in vitro tissue models has the potential to make automated 3D cell-based assays more accessible to researchers in the life sciences.

  16. Ecotoxicological applications and significance of the comet assay.

    Science.gov (United States)

    Jha, Awadhesh N

    2008-05-01

    Application of the single-cell gel electrophoresis or comet assay has revolutionized the field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively inexpensive method providing the opportunity to study DNA damage (including oxidative damage), repair and cell death (apoptosis) in different cell types without prior knowledge of karyotype and cell turnover rate. The assay has, however, often attracted criticism for its lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid technical progress has been made to improve cell- and tissue-specific adoption of the assay, only limited advancement has been made to transfer the methodologies to ecotoxicological studies. While reviewing the recent information available in the literature and underscoring the importance of induced genetic damage in natural species, the aims of this article are to (i) highlight and judiciously analyse the ecotoxicological relevance of the assay; (ii) attempt to correlate the comet response with other relevant biological responses or biomarkers; (iii) identify the technical challenges and various factors affecting its application in order to make it reliable, reproducible and robust; (iv) critically compare the technical developments in genetic toxicology and genetic ecotoxicology and (v) evaluate the future developments with respect to applications of the assay. It is suggested that while complementing other ecotoxicological parameters and further improving the methodologies, the comet assay will continue to play an important role in genetic ecotoxicology to determine induced genetic damage, which has significant consequences for short- and long-term survival of the natural or wild species. Information obtained through integrated studies using simultaneous applications of multiple biomarkers on different wild organisms could also provide an holistic dimension of toxicological impact of environmental contaminants for the

  17. Reference cells and ploidy in the comet assay

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg

    2015-02-01

    Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

  18. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

    Directory of Open Access Journals (Sweden)

    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  19. Detection and identification of aquatic birnaviruses by PCR assay.

    Science.gov (United States)

    Blake, S L; Schill, W B; McAllister, P E; Lee, M K; Singer, J T; Nicholson, B L

    1995-04-01

    A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.

  20. Methods of theophylline assay and therapeutic monitoring of this drug.

    Science.gov (United States)

    Mounié, J; Richard, L; Ribon, B; Hersant, J; Sarmini, H; Houin, G; Mouine, J

    1990-01-01

    The purpose of this article is to review various analytical methods of monitoring plasma theophylline. This article was investigated by the "Drug Commission" of SFBC (Société Française de Biologie Clinique). The primary objective is to provide the "know-how", particular for this analysis, which allows the choice between various analytical methods available: immunochemical or physiochemical ones. The techniques described are not necessarily the best, they are approved and tested methods which are the most frequently used in routine practice. The proposed immunochemical methods are: absorption spectroscopy methods: Enzyme ImmunoAssay (EIA), Enzyme Multiplied ImmunoAssay Technique (EMIT); Reflectance spectroscopy method: Apoenzyme Reactivation Immunoassay System (ARIS); Fluorometry spectroscopy method: Substrate Labeled FluoroImmunoAssay (SLFIA); Fluorometry spectroscopy on solid base; Polarization fluorescence spectroscopy ImmunoAssay (FPIA); Turbidimetric measurements: Particle Enhanced Turbidimetric Inhibition ImmunoAssay (PETINIA); Nephelometric measurement: Nephelometric Inhibition ImmunoAssay (NIIA). And the proposed physicochemical methods are: High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC). The second objective is a review of pharmacological properties of theophylline, necessary for a good understanding of therapeutic drug monitoring: intestinal resorption, distribution, metabolism and elimination, drug interactions, dose/response relationship, physiopathological variations and proposed "predictive" "theophylline test". The authors conclude that because of the multiplicity of methodologies used in theophylline therapeutic monitoring the choice of one of them is not easy. The best way to compare different techniques available would be the use of a "reference material" for theophylline monitoring and a quality control network between different clinical pharmacological laboratories.

  1. Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis.

    Science.gov (United States)

    Saito, Kazutoshi; Nukada, Yuko; Takenouchi, Osamu; Miyazawa, Masaaki; Sakaguchi, Hitoshi; Nishiyama, Naohiro

    2013-12-01

    Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  3. Potassium Bromate Assay by Redox Titrimetry Using Arsenic Trioxide

    OpenAIRE

    Smeller, Johanna M.; Leigh, Stefan D.

    2003-01-01

    Bromate, a disinfectant, is one of the analytes of interest in wastewater analysis. Environmental laboratories have a regulatory need for their measurements to be traceable to NIST standards. Bromate is not currently certified as a NIST Standard Reference Material (SRM). Therefore, a traceable assay of potassium bromate (KBrO3) is needed. KBrO3 was dissolved in water and assayed by redox titrimetry using arsenic trioxide (As2O3). A nominal (0.1 g) sample of As2O3 was dissolved in 10 mL of 5 m...

  4. Detection of high-risk human papillomavirus infection in tonsillar specimens using 2 commercially available assays.

    Science.gov (United States)

    Cockerill, Cara C; Orvidas, Laura J; Moore, Eric J; Binnicker, Matthew J; Duresko, Brian J; Espy, Mark J; Cockerill, Franklin R; Tombers, Nicole M; Pritt, Bobbi S

    2016-12-01

    THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

    Science.gov (United States)

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2015-09-15

    Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. A 3-plex methylation assay combined with the FGFR3 mutation assay sensitively detects recurrent bladder cancer in voided urine

    DEFF Research Database (Denmark)

    Kandimalla, Raju; Masius, Roy; Beukers, Willemien

    2013-01-01

    Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non–muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study......, and nonmalignant urines (n = 130). Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57......%, respectively. Conclusion: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range...

  7. Aptamers as the Agent in Decontamination Assays (Apta-Decontamination Assays: From the Environment to the Potential Application In Vivo

    Directory of Open Access Journals (Sweden)

    Mawethu Pascoe Bilibana

    2017-01-01

    Full Text Available The binding specificity and affinity of aptamers have long been harnessed as the key elements in the development of aptamer-based assays, particularly aptasensing application. One promising avenue that is currently explored based on the specificity and affinity of aptamers is the application of aptamers in the decontamination assays. Aptamers have been successfully harnessed as the decontamination agents to remove contaminants from the environment and to decontaminate infectious elements. The reversible denaturation property inherent in aptamers enables the repeated usage of aptamers, which can immensely save the cost of decontamination. Analogous to the point-of-care diagnostics, there is no doubt that aptamers can also be deployed in the point-of-care aptamer-based decontamination assay, whereby decontamination can be performed anywhere and anytime for instantaneous decision-making. It is also prophesied that aptamers can also serve more than as a decontaminant, probably as a tool to capture and kill hazardous elements, particularly pathogenic agents.

  8. Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays

    DEFF Research Database (Denmark)

    Engle, Ronald E; Russell, Rodney S; Purcell, Robert H

    2008-01-01

    international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and commercially available HCV RNA Nucleic Acid Technology kits...... (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples...... of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology...

  9. Highly Promiscuous Small Molecules from Biological Screening Assays Include Many Pan-Assay Interference Compounds but Also Candidates for Polypharmacology.

    Science.gov (United States)

    Gilberg, Erik; Jasial, Swarit; Stumpfe, Dagmar; Dimova, Dilyana; Bajorath, Jürgen

    2016-11-23

    In PubChem screening assays, 466 highly promiscuous compounds were identified that were examined for known pan-assay interference compounds (PAINS) and aggregators using publicly available filters. These filters detected 210 PAINS and 67 aggregators. Compounds passing the filters included additional PAINS that were not detected, mostly due to tautomerism, and a variety of other potentially reactive compounds currently not encoded as PAINS. For a subset of compounds passing the filters, there was no evidence of potential artifacts. These compounds are considered candidates for further exploring multitarget activities and the molecular basis of polypharmacology.

  10. Development of a slide agglutination assay for detection of blastomycosis.

    Science.gov (United States)

    Hatch, Wayne O; Scalarone, Gene M

    2013-11-01

    Blastomycosis, caused by the thermally dimorphic fungus Blastomyces dermatitides, which is endemic to eastern regions of the USA, is commonly misdiagnosed as a viral or bacterial infection and therefore treated improperly. Over the years, many immunodiagnostic assays to aid in the diagnosis of blastomycosis have been developed; however, a reliable assay for use in local clinics still remains elusive. Procedures for a slide agglutination assay for detection of antibody in serum from rabbits immunized with B. dermatitidis were evaluated with antigenic preparations from B. dermatitidis adsorbed to polystyrene microparticles. Yeast-phase lysates from five isolates of B. dermatitides: namely ER-593 (Eagle River, WI, USA), ER-598 (Eagle River, WI, USA), 48938 (India), B5896 (Mt. Iron, MN, USA), and T-58 (TN, USA) were evaluated for their sensitivity and specificity. Sensitivities of the lysates ranged from 29% to 83% whereas specificities ranged from 13% to 100%. Lysate ER-593 provided the most promising results with a sensitivity of 82% and specificity of 100%. This study provides suggests that a simple rapid slide agglutination assay for detecting blastomycosis may be used for screening patients with suspected B. dermatitidis infection. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

  11. Comparison of automated von Willebrand factor activity assays

    DEFF Research Database (Denmark)

    Timm, Annette; Hillarp, Andreas; Philips, Malou

    2015-01-01

    INTRODUCTION: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder. Measurement of von Willebrand factor (VWF) activity in plasma is often based on platelet agglutination stimulated by the ristocetin cofactor activity. Novel assays, based on latex beads with recombinant...

  12. Preliminary in vitro cytotoxic assay of human liver carcinoma cells ...

    African Journals Online (AJOL)

    From the preliminary in vitro cytotoxic assay study, triorganotin(IV) complexes (2 and 5) were found to exhibit better activity as compared to diorganotin(IV) complexes (1, 3 and 4) but lower activity as compared to the reference drug. In addition, within the diorganotin(IV) complexes, monomeric type (3) exhibited a slightly ...

  13. 40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

    Science.gov (United States)

    2010-07-01

    ... mutagen means an agent which causes a base change in DNA. In a reversion assay, this change may occur at... agent which causes the addition or deletion of single or multiple base pairs in the DNA molecule... nitrated organic compounds, i.e., nitroarenes, contribute to the overall mutagenic activity of a test...

  14. IP-10 release assays in the diagnosis of tuberculosis infection

    DEFF Research Database (Denmark)

    Ruhwald, Morten; Aabye, Martine G; Ravn, Pernille

    2012-01-01

    The current state-of-the-art tests for infection with Mycobacterium tuberculosis - the IFN-γ release assays - rely on accurate measurement of the cytokine IFN-γ. Many other potential biomarkers are expressed in concert with IFN-γ, and IP-10 in particular has shown promising results. IP-10...

  15. [The comet assay as a method of identifying chromosomes instability].

    Science.gov (United States)

    Czubaszek, Magdalena; Szostek, Małgorzata; Wójcik, Ewa; Andraszek, Katarzyna

    2014-06-02

    The basic method for analyzing the degree of DNA fragmentation caused by genotoxic factors is gel electrophoresis of single cells (single cell gel electrophoresis), also called the comet assay. The comet assay enables the analysis of the level of several different DNA modifications. The basic testing procedure has been only slightly modified. This method helps identify single-strand and double-strand DNA cracks, as well as any chemical and enzymatic modifications that can potentially turn into cracks in DNA or chromatids. The comet assay makes it possible to detect DNA damage at the level of single cells. It can be employed in analyses of any tissues which provide cellular suspensions. Analysed cells are submerged in agarose on a microscope slide. DNA is what is left after proteins have been broken down. The slide is then subjected to electrophoresis and stained with a fluorescent dye. A "comet-like" image is obtained. The "head" is the cell fixation site prior to lysis; the "tail" represents damaged DNA fragments. The extent of DNA damage is reflected in the length of the tail and the amount of DNA contained in it. The assay finds research applications in the following fields: genetic toxicology, monitoring of DNA repair following chemotherapy and radiotherapy, ecotoxicology, animal and human nourishment, biomonitoring of genotoxicity, epidemiology and assessment of material deposited in sperm and blood banks.

  16. Development of a VHH-Based Erythropoietin Quantification Assay

    DEFF Research Database (Denmark)

    Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon

    2015-01-01

    human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...

  17. Restriction Inhibition Assay: A Qualitative and Quantitative Method to ...

    African Journals Online (AJOL)

    rich fractions (PRFs) with high affinity for EcoRI and HindIII restriction .... DNA along with each restriction enzyme was kept to analyze the results of the ..... Puvvada MS, Hartley JA, Jenkins TC, Thurston DE. A quantitative assay to measure the ...

  18. A fluorescence sedimentation assay for dsDNA antibodies

    DEFF Research Database (Denmark)

    Duus, K; Draborg, A H; Güven, E

    2017-01-01

    on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic...

  19. Parasite lactate dehydrogenase assay for the determination of ...

    African Journals Online (AJOL)

    Results: The p(LDH) enzyme assay was successfully used to measure the IC50 of six antimalarial drugs, chloroquine, quinine, mefloquine, dehydroartemisinin, atovaquone and halofantrine but was not successful with the four other antimalarial drugs, doxycycline, azithromycin, pyrimethamine and sulphadoxine.

  20. New simple spectrophotometric assay of total carotenes in margarines

    NARCIS (Netherlands)

    Luterotti, S.; Bicanic, D.D.; Pozgaj, R.

    2006-01-01

    Direct and reliable spectrophotometric method for assaying total carotenes (TC) in margarines with the minimum of sample manipulation is proposed. For the first time saponification step used in determination of carotenes in margarines was omitted leading to a substantial cost saving and reduction of

  1. Yeast Protein–Protein Interaction Assays and Screens

    NARCIS (Netherlands)

    Folter, de S.; Immink, G.H.

    2011-01-01

    Most transcription factors fulfill their role in protein complexes. As a consequence, information about their interaction capacity sheds light on a protein’s function and the molecular mechanism underlying this activity. The yeast two-hybrid GAL4 (Y2H) assay is a powerful method to unravel and

  2. Earthworm dispersal assay for rapidly evaluating soil quality.

    Science.gov (United States)

    Kim, Shin Woong; Kim, Dokyung; Moon, Jongmin; Chae, Yooeun; Kwak, Jin Il; Park, Younsu; Jeong, Seung-Woo; An, Youn-Joo

    2017-10-01

    Earthworms enhance soil functioning and are therefore key species in the soil. Their presence is generally a positive sign for a terrestrial ecosystem, because these species serve as important biomarkers in soil quality evaluations. We describe a novel bioassay, the "dispersal assay," that is a simple and rapid technique for field-based soil quality evaluations. It is based on the premise that earthworms prefer optimal soils if given the choice. Thus, assay tubes containing a reference soil were inserted in target sites, and earthworms were placed into these tubes. According to their soil preference, the earthworms dispersed into the surrounding soil, remained in the initial soil within the tubes, avoided both by crawling up the tube, or died. Furthermore, sensitivity responses to metal concentrations, electrical conductivity, and soil pH were observed in field tests. Although the dispersal assay did not completely match traditional toxicity endpoints such as earthworm survival, we found that it can serve as an in situ screening test for assessing soil quality. Overall, our dispersal assay was relatively rapid (within 24 h), had low levels of variation, and showed high correlations between earthworm behavior and soil physicochemical properties. Environ Toxicol Chem 2017;36:2766-2772. © 2017 SETAC. © 2017 SETAC.

  3. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Andersen, Helene; Moghimi, Seyed Moien

    2013-01-01

    cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early...

  4. Flow Cytometric Bead Sandwich Assay Based on a Split Aptamer.

    Science.gov (United States)

    Shen, Luyao; Bing, Tao; Liu, Xiangjun; Wang, Junyan; Wang, Linlin; Zhang, Nan; Shangguan, Dihua

    2018-01-24

    A few aptamers still bind their targets after being split into two moieties. Split aptamers have shown great potential in the development of aptameric sensors. However, only a few split aptamers have been generated because of lack of knowledge on the binding structure of their parent aptamers. Here, we report the design of a new split aptamer and a flow cytometric bead sandwich assay using a split aptamer instead of double antibodies. Through DMS footprinting and mutation assay, we figured out the target-binding moiety and the structure-stabilizing moiety of the l-selectin aptamer, Sgc-3b. By separating the duplex strand in the structure-stabilizing moiety, we obtained a split aptamer that bound l-selectin. After optimization of one part of the split sequence to eliminate the nonspecific binding of the split sequence pair, we developed a split-aptamer-based cytometric bead assay (SACBA) for the detection of soluble l-selectin. SACBA showed good sensitivity and selectivity to l-selectin and was successfully applied for the detection of spiked l-selectin in the human serum. The strategies for generating split aptamers and designing the split-aptamer-based sandwich assay are simple and efficient and show good practicability in aptamer engineering.

  5. A Sensitive Cyclic Nucleotide Phosphodiesterase Assay for Transient Enzyme Kinetics

    NARCIS (Netherlands)

    Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van

    1983-01-01

    A new assay for cyclic nucleotide phosphodiesterase has been developed by using reverse-phase column chromatography for the separation of product and substrate of the enzymatic reaction. The polar 5'-nucleotides are not retarded by the column, while the more lipophilic cyclic nucleotides bind to the

  6. The comet assay: Reflections on its development, evolution and applications.

    Science.gov (United States)

    Singh, Narendra P

    2016-01-01

    The study of DNA damage and its repair is critical to our understanding of human aging and cancer. This review reflects on the development of a simple technique, now known as the comet assay, to study the accumulation of DNA damage and its repair. It describes my journey into aging research and the need for a method that sensitively quantifies DNA damage on a cell-by-cell basis and on a day-by-day basis. My inspirations, obstacles and successes on the path to developing this assay and improving its reliability and sensitivity are discussed. Recent modifications, applications, and the process of standardizing the technique are also described. What was once untried and unknown has become a technique used around the world for understanding and monitoring DNA damage. The comet assay's use has grown exponentially in the new millennium, as emphasis on studying biological phenomena at the single-cell level has increased. I and others have applied the technique across cell types (including germ cells) and species (including bacteria). As it enters new realms and gains clinical relevance, the comet assay may very well illuminate human aging and its prevention. Copyright © 2016. Published by Elsevier B.V.

  7. Designing of enzyme linked immunosorbent assay (ELISA) kit for ...

    African Journals Online (AJOL)

    The sensitivity of microscopic examination of fecal samples to recognize Giardia parasites is low. In the methods based on antigen scanning of parasites such as enzyme linked immunosorbent assay (ELISA), copro-antigens of parasite will be traced and diagnosed even if the live parasite is absent in the fecal samples.

  8. Recommendations for treated-area choice assays with termites (Isoptera)

    Science.gov (United States)

    Chris J. Peterson; Patrick D. Gerard; Janice Ems-Wilson

    2005-01-01

    The repellency of catnip (Nepeta cataria) essential oil was evaluated in a treated-area choice assay with subterranean termites (Reticulitermes spp.). It appeared that fewer R. virginicus were found on the treated portion of a petri dish within a period of about 7d; R. flavipes was not...

  9. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    Science.gov (United States)

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Assay of heavy water in drums for safeguards purposes

    Energy Technology Data Exchange (ETDEWEB)

    Fainberg, A.; Zucker, M.S.; Lemley, J.R.; Weinstock, E.V.

    1981-01-01

    This paper examines several techniques for rapid nondestructive assay of drums of D/sub 2/O. Five methods have been examined: neutron capture in hydrogen, photodisintegration of deuterium, neutron transmission, neutron die-away time measurements, and acoustic velocity measurements.

  11. 21 CFR 866.2350 - Microbiological assay culture medium.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Microbiological assay culture medium. 866.2350 Section 866.2350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2350...

  12. Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

    Science.gov (United States)

    Stocker, P; Ricquebourg, E; Vidal, N; Villard, C; Lafitte, D; Sellami, L; Pietri, S

    2015-08-01

    Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Lucigenin chemiluminescence assay as an adjunctive tool for ...

    Indian Academy of Sciences (India)

    A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have distinct advantages due to its cost benefits and relative savings in time. Furthermore, a bioassay to differentiate acute and chronic in vivo pulmonary reactions could have potential value as predictors of fibrogenicity ...

  14. PCR Assay for Species-Specific Identification of Bacteroides thetaiotaomicron

    Science.gov (United States)

    Teng, Lee-Jene; Hsueh, Po-Ren; Tsai, Jui-Chang; Chiang, Feng-Lin; Chen, Ching-Yi; Ho, Shen-Wu; Luh, Kwen-Tay

    2000-01-01

    Bacteroides thetaiotaomicron is the second most frequently encountered species of the anaerobes isolated from clinical specimens. We developed a PCR-based assay for the rapid identification of B. thetaiotaomicron. Specific primers were based on shared amplicons of about 1.2 kb generated from B. thetaiotaomicron by randomly amplified polymorphic DNA. This 1.2-kb fragment was sequenced and then used to design a set of PCR amplification primers. This PCR generated an amplification product of 721 bp, which was unique to all 65 isolates of B. thetaiotaomicron tested. There was no amplification with isolates of other bacterial species. Restriction enzyme digestion of the amplification product and dot blot hybridization further verified the specificity of the assay. These results suggest that this PCR assay targets a nucleotide sequence that is strongly conserved in B. thetaiotaomicron. This simple and rapid PCR assay provides a rapid and accurate method for identification of B. thetaiotaomicron and shows promise for the detection of B. thetaiotaomicron in clinical samples. PMID:10747167

  15. Assays without Borders | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    CPTAC researchers, partner with international labs to demonstrate the ability of targeted mass spectrometry–based assays to reproducibly quantify human proteins across labs, countries and continents in a recently published journal article. In a landmark paper appearing in the Dec.

  16. Species specific polymerase chain reaction (PCR) assay for ...

    African Journals Online (AJOL)

    A highly specific single step polymerase chain reaction (PCR) is described for the detection of pig (Sus domesticus) meat. A PCR assay was successfully optimized for amplification of 629 and 322-bp DNA fragment extracted from pig meat using designed species-specific primer pairs based on mitochondrial D-loop and 12S ...

  17. Phytochemical And In Vitro Antimicrobial Assay Of The Leaf Extract ...

    African Journals Online (AJOL)

    The methanolic leaf extract of Newbouldia laevis was subjected to preliminary phytochemical screening and In Vitro antimicrobial tests. The extract revealed the presence of flavonoids, tannins, terpenes, steroidal and cardiac glycosides. The antimicrobial activity of the plant extract was assayed by the agar plate disc ...

  18. Transuranic storage and assay facility interim safety basis

    Energy Technology Data Exchange (ETDEWEB)

    Porten, D.R., Fluor Daniel Hanford

    1997-02-12

    The Transuranic Waste Storage and Assay Facility (TRUSAF) Interim Safety Basis document provides the authorization basis for the interim operation and restriction on interim operations for the TRUSAF. The TRUSAF ISB demonstrates that the TRUSAF can be operated safely, protecting the workers, the public, and the environment. The previous safety analysis document TRUSAF Hazards Identification and Evaluation (WHC 1987) is superseded by this document.

  19. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  20. Defining cell culture conditions to improve human norovirus infectivity assays

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Bartholomew, Rachel A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Valdez, Catherine O. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Valentine, Nancy B. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Dohnalkova, Alice [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ozanich, Richard M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Bruckner-Lea, Cindy J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2013-01-10

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

  1. Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

    Science.gov (United States)

    Jose, D Amilan; Elstner, Martin; Schiller, Alexander

    2013-10-18

    Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Microbiological Assay Of Ingredients, Contact Surfaces And Stages ...

    African Journals Online (AJOL)

    Microbiological Assay Of Ingredients, Contact Surfaces And Stages In Akara Processing At Three Locations In Maiduguri, Nigeria. ... These microorganisms are pathogenic and therefore their presence during akara processing could cause a serious health problem. The need for advising akara producers and vendors of the ...

  3. An assay for lateral line regeneration in adult zebrafish.

    Science.gov (United States)

    Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

    2014-04-08

    Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

  4. An indicator cell assay for blood-based diagnostics.

    Directory of Open Access Journals (Sweden)

    Samuel A Danziger

    Full Text Available We have established proof of principle for the Indicator Cell Assay Platform™ (iCAP™, a broadly applicable tool for blood-based diagnostics that uses specifically-selected, standardized cells as biosensors, relying on their innate ability to integrate and respond to diverse signals present in patients' blood. To develop an assay, indicator cells are exposed in vitro to serum from case or control subjects and their global differential response patterns are used to train reliable, disease classifiers based on a small number of features. In a feasibility study, the iCAP detected pre-symptomatic disease in a murine model of amyotrophic lateral sclerosis (ALS with 94% accuracy (p-Value = 3.81E-6 and correctly identified samples from a murine Huntington's disease model as non-carriers of ALS. Beyond the mouse model, in a preliminary human disease study, the iCAP detected early stage Alzheimer's disease with 72% cross-validated accuracy (p-Value = 3.10E-3. For both assays, iCAP features were enriched for disease-related genes, supporting the assay's relevance for disease research.

  5. A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

    Science.gov (United States)

    Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

    2017-08-01

    Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  6. Accelerated Colorimetric Micro-assay for Screening Mold Inhibitors

    Science.gov (United States)

    Carol A. Clausen; Vina W. Yang

    2014-01-01

    Rapid quantitative laboratory test methods are needed to screen potential antifungal agents. Existing laboratory test methods are relatively time consuming, may require specialized test equipment and rely on subjective visual ratings. A quantitative, colorimetric micro-assay has been developed that uses XTT tetrazolium salt to metabolically assess mold spore...

  7. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

  8. Mass spectrometry-based biochemical assays for enzymeinhibitor screening

    NARCIS (Netherlands)

    de Boer, A.R.; Lingeman, H.; Niessen, W.M.A.; Irth, H.

    2007-01-01

    Screening for inhibitors of pharmacologically-relevant enzymes is in many cases an important starting point in drug discovery. While fluorescence-based detection techniques play an important role in high-throughput screening, mass spectrometry (MS)-based assays have gained in importance in recent

  9. Cell-based lipid flippase assay employing fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Günther-Pomorski, Thomas

    2016-01-01

    , studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid...

  10. An immunochemical assay to detect DNA damage in bovine sperm

    NARCIS (Netherlands)

    Schans, G.P. van der; Haring, R.; Dijk- Knijnenburg, H.C.M. van; Bruijnzeel, P.L.B.; Daas, N.H.G. den

    2000-01-01

    An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of

  11. Monoclonal antibodies in immunodiagnostic assays: a review of ...

    African Journals Online (AJOL)

    Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the immunodiagnostic assays. This is due to their binding accuracy to many pathogens, thus, making them valuable research tools. Since the discovery of hybridoma technology by Kohler and Milstein, the use of monoclonal antibodies ...

  12. Colorimetric Assay Of Naproxen Tablets by Derivatization Using 4 ...

    African Journals Online (AJOL)

    A colorimetric assay of naproxen tablets based on chemical derivatization with the chromogenic reagent 4-carboxyl-2,6-dinitrobenzene diazonium ion was developed. The optimal reaction time was found to be 5 min at 30 oC after vortex mixing of the drug/reagent mixture for 10 s. The optimal analytical wavelength was ...

  13. A greenhouse screening assay for Botrytis tulipae resistance in tulips

    NARCIS (Netherlands)

    Straathof, T.P.; Mes, J.J.; Eikelboom, W.; Tuyl, van J.M.

    2002-01-01

    As a leaf pathogen, Botrytis tulipae severely affects tulip bulb production. Chemical control is not desired for environmental reasons. Thus, resistant cultivars can play an important role in the control of this disease. To select genotypes resistant to B. tulipae, a reliable screening assay is

  14. Spectrophotometric assay of catalase with perborate as substrate

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, J.R.; Nance, S.L.; Tollaksen, S.L.

    1978-01-01

    A spectrophotometric assay for catalase in mouse liver fractions has been developed in which sodium perborate, rather than hydrogen peroxide, is used as the substrate. The method is convenient, rapid, and readily adapted to the measurement of catalase in subcellular fractions.

  15. Assay of Serum Antibodies against Newcastle Disease Virus in ...

    African Journals Online (AJOL)

    Assay of Serum Antibodies against Newcastle Disease Virus in Local Chickens at Kaduna, Nigeria. ... NDV-HI Geometric mean titre of 18.4 was recorded showing a low level of antibody protectiveness to NDV attack due to natural infection. The result highlights the epizootic nature of the disease among local chickens in the ...

  16. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to determine apoptosis induced by siRNA in Colo 320 cells. When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a ...

  17. Lucigenin chemiluminescence assay as an adjunctive tool for ...

    Indian Academy of Sciences (India)

    Unknown

    *Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health,. Morgantown, WV 26505, USA. †Corresponding author (Fax, 304-293-5449; Email, kvandyke@hsc.wvu.edu). A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have.

  18. Cytokine release assays: current practices and future directions.

    Science.gov (United States)

    Finco, D; Grimaldi, C; Fort, M; Walker, M; Kiessling, A; Wolf, B; Salcedo, T; Faggioni, R; Schneider, A; Ibraghimov, A; Scesney, S; Serna, D; Prell, R; Stebbings, R; Narayanan, P K

    2014-04-01

    As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Comparisons of competitive enzyme-linked immunosorbent assay ...

    African Journals Online (AJOL)

    The aim of this study was to comprise competitive enzyme-linked immunosorbent assay (C-ELISA) with one step RT-PCR test for the detection of BTV in sheep. A total of 770 blood samples were obtained from sheep (265 serum positive samples and 505 serum negative samples in C-ELISA). According to our data, out of ...

  20. Use of laminar flow patterning for miniaturised biochemical assays

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Krühne, Ulrich; Beyer, M.

    2004-01-01

    Laminar flow in microfluidic chambers was used to construct low (one dimensional) density arrays suitable for miniaturized biochemical assays. By varying the ratio of flows of two guiding streams flanking a sample stream, precise focusing and positioning of the latter was achieved, and reactive s...