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Sample records for validated commercial assays

  1. Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

    Science.gov (United States)

    Wu, John T.Y.; Dreger, Sally; Chow, Eva Y.W.; Bowlby, Evelyn E.

    2002-01-01

    Abstract This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV−) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV− values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures. PMID:12418782

  2. Performance validation of commercially available mobile waste-assay systems: Preliminary report

    Energy Technology Data Exchange (ETDEWEB)

    Schanfein, M.; Bonner, C.; Maez, R. [Los Alamos National Lab., NM (United States)] [and others

    1997-11-01

    Prior to disposal, nuclear waste must be accurately characterized to identify and quantify the radioactive content to reduce the radioactive hazard to the public. Validation of the waste-assay systems` performance is critical for establishing the credibility of the assay results for storage and disposal purposes. Canberra Nuclear has evaluated regulations worldwide and identified standard, modular, neutron- and gamma-waste-assay systems that can be used to characterize a large portion of existing and newly generated transuranic (TRU) and low-level waste. Before making claims of guaranteeing any system`s performance for specific waste types, the standardized systems` performance be evaluated. 7 figs., 11 tabs.

  3. Performance validation of commercially available mobile waste-assay systems: Preliminary report

    International Nuclear Information System (INIS)

    Schanfein, M.; Bonner, C.; Maez, R.

    1997-01-01

    Prior to disposal, nuclear waste must be accurately characterized to identify and quantify the radioactive content to reduce the radioactive hazard to the public. Validation of the waste-assay systems' performance is critical for establishing the credibility of the assay results for storage and disposal purposes. Canberra Nuclear has evaluated regulations worldwide and identified standard, modular, neutron- and gamma-waste-assay systems that can be used to characterize a large portion of existing and newly generated transuranic (TRU) and low-level waste. Before making claims of guaranteeing any system's performance for specific waste types, the standardized systems' performance be evaluated. 7 figs., 11 tabs

  4. Validation of 2 commercially available enzyme-linked immunosorbent assays for adiponectin determination in canine serum samples.

    Science.gov (United States)

    Tvarijonaviciute, Asta; Martínez-Subiela, Silvia; Ceron, José J

    2010-10-01

    The aim of this study was to validate 2 commercially available enzyme-linked immunosorbent assays (ELISAs) for adiponectin in dogs, 1 canine-specific and 1 originally designed for measurements in humans. Intra-assay and interassay precision was evaluated by multiple measurements in canine serum samples, and assay accuracy was indirectly determined by linearity under dilution. Interference caused by hemolysis and lipemia was also studied. Both assays were subsequently used for measuring adiponectin concentrations in clinically healthy dogs and those with different grades of obesity. The intra-assay and inter-assay precision was less than 7.5% and 13.5% in serum samples with low and high adiponectin concentrations, respectively. Lipemia and hemolysis did not affect the results of any of the assays. Both assays were able to differentiate lean dogs from those that were overweight or obese on the basis of the measured adiponectin concentrations. From these results it can be concluded that canine adiponectin concentrations can be measured reliably by means of the 2 ELISAs evaluated in this study.

  5. Validation of a commercially available enzyme-linked immunoabsorbent assay for the quantification of human α-Synuclein in cerebrospinal fluid.

    Science.gov (United States)

    Kruse, Niels; Mollenhauer, Brit

    2015-11-01

    The quantification of α-Synuclein in cerebrospinal fluid (CSF) as a biomarker has gained tremendous interest in the last years. Several commercially available immunoassays are emerging. We here describe the full validation of one commercially available ELISA assay for the quantification of α-Synuclein in human CSF (Covance alpha-Synuclein ELISA kit). The study was conducted within the BIOMARKAPD project in the European initiative Joint Program for Neurodegenerative Diseases (JPND). We investigated the effect of several pre-analytical and analytical confounders: i.e. (1) need for centrifugation of freshly drawn CSF, (2) sample stability, (3) delay of freezing, (4) volume of storage aliquots, (5) freeze/thaw cycles, (6) thawing conditions, (7) dilution linearity, (8) parallelism, (9) spike recovery, and (10) precision. None of these confounders influenced the levels of α-Synuclein in CSF significantly. We found a very high intra-assay precision. The inter-assay precision was lower than expected due to different performances of kit lots used. Overall the validated immunoassay is useful for the quantification of α-Synuclein in human CSF. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species.

    Science.gov (United States)

    Bienboire-Frosini, Cecile; Chabaud, Camille; Cozzi, Alessandro; Codecasa, Elisa; Pageat, Patrick

    2017-01-01

    The neurohormone oxytocin (OT) has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA) for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat). The Oxytocin EIA kit (EnzoLifeSciences) was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml). To validate the method, the following performance characteristics were evaluated using Validation Samples (VS) at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was verified. Although

  7. Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species

    Directory of Open Access Journals (Sweden)

    Cecile Bienboire-Frosini

    2017-09-01

    Full Text Available The neurohormone oxytocin (OT has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat. The Oxytocin EIA kit (EnzoLifeSciences was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml. To validate the method, the following performance characteristics were evaluated using Validation Samples (VS at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was

  8. Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum.

    Science.gov (United States)

    Gresch, Sarah C; Mutch, Lucas A; Janecek, Jody L; Hegstad-Davies, Rebecca L; Graham, Melanie L

    2017-09-01

    C-peptide concentration is widely used as a marker of insulin secretion and is especially relevant in evaluating islet graft function following transplantation, because its measurement is not confounded by the presence of exogenous insulin. To address the shortage of human islet donors, the use of porcine islets has been proposed as a possible solution and the stringent pig-to-non-human primate (NHP) model is often the most relevant for pre-clinical evaluation of the potential for diabetes reversal resulting from an islet xenograft. The Millipore radioimmunoassay (RIA) was exclusively used to measure porcine C-peptide (PCP) until 2013 when the assay was discontinued and subsequently a commercially available enzyme-linked immunosorbent assay (ELISA) from Mercodia has been widely adopted. Both assays have been used in pre-clinical trials evaluating the therapeutic potential of xenograft products in reversing diabetes in the pig-to-NHP model, to interpret data in a comparable way it may be useful to perform a harmonization of C-peptide measurements. We performed a method comparison by determining the PCP concentration in 620 serum samples collected from 20 diabetic cynomolgus macaques transplanted with adult porcine islets. All analyses were performed according to manufacturer instructions. With both assays, we demonstrated an acceptable detection limit, precision, and recovery. Linearity of the ELISA met acceptance criteria at all concentrations tested while linearity of the RIA only met acceptance criteria at five of the eight concentrations tested. The RIA had a detection limit of 0.16 ng/mL, and recovery ranged from 82% to 96% and met linearity acceptance criteria at 0.35 ng/mL and from 0.78 to 2.33 ng/mL. The ELISA had a detection limit of 0.03 ng/mL, and recovery ranged from 81% to 115% and met linearity acceptance criteria from 0.08 to 0.85 ng/mL. Both assays had intra-assay precision assay precision ELISA demonstrated a significant correlation with RIA (R

  9. How valid are commercially available medical simulators?

    Science.gov (United States)

    Stunt, JJ; Wulms, PH; Kerkhoffs, GM; Dankelman, J; van Dijk, CN; Tuijthof, GJM

    2014-01-01

    Background Since simulators offer important advantages, they are increasingly used in medical education and medical skills training that require physical actions. A wide variety of simulators have become commercially available. It is of high importance that evidence is provided that training on these simulators can actually improve clinical performance on live patients. Therefore, the aim of this review is to determine the availability of different types of simulators and the evidence of their validation, to offer insight regarding which simulators are suitable to use in the clinical setting as a training modality. Summary Four hundred and thirty-three commercially available simulators were found, from which 405 (94%) were physical models. One hundred and thirty validation studies evaluated 35 (8%) commercially available medical simulators for levels of validity ranging from face to predictive validity. Solely simulators that are used for surgical skills training were validated for the highest validity level (predictive validity). Twenty-four (37%) simulators that give objective feedback had been validated. Studies that tested more powerful levels of validity (concurrent and predictive validity) were methodologically stronger than studies that tested more elementary levels of validity (face, content, and construct validity). Conclusion Ninety-three point five percent of the commercially available simulators are not known to be tested for validity. Although the importance of (a high level of) validation depends on the difficulty level of skills training and possible consequences when skills are insufficient, it is advisable for medical professionals, trainees, medical educators, and companies who manufacture medical simulators to critically judge the available medical simulators for proper validation. This way adequate, safe, and affordable medical psychomotor skills training can be achieved. PMID:25342926

  10. Phytochemical Assays of Commercial Botanical Dietary Supplements

    Directory of Open Access Journals (Sweden)

    Robert Krochmal

    2004-01-01

    Full Text Available The growing popularity of botanical dietary supplements (BDS has been accompanied by concerns regarding the quality of commercial products. Health care providers, in particular, have an interest in knowing about product quality, in view of the issues related to herb-drug interactions and potential side effects. This study assessed whether commercial formulations of saw palmetto, kava kava, echinacea, ginseng and St. John's wort had consistent labeling and whether quantities of marker compounds agreed with the amounts stated on the label. We purchased six bottles each of two lots of supplements from nine manufacturers and analyzed the contents using established commercial methodologies at an independent laboratory. Product labels were found to vary in the information provided, such as serving recommendations and information about the herb itself (species, part of the plant, marker compound, etc. With regard to marker compound content, little variability was observed between different lots of the same brand, while the content did vary widely between brands (e.g. total phenolic compounds in Echinacea ranged from 3.9–15.3 mg per serving; total ginsenosides in ginseng ranged from 5.3–18.2 mg per serving. Further, the amounts recommended for daily use also differed between brands, increasing the potential range of a consumer's daily dose. Echinacea and ginseng were the most variable, while St. John's wort and saw palmetto were the least variable. This study highlights some of the key issues in the botanical supplement market, including the importance of standardized manufacturing practices and reliable labeling information. In addition, health care providers should keep themselves informed regarding product quality in order to be able to appropriately advise patients utilizing both conventional and herbal medicines.

  11. Phytochemical Assays of Commercial Botanical Dietary Supplements

    Science.gov (United States)

    2004-01-01

    The growing popularity of botanical dietary supplements (BDS) has been accompanied by concerns regarding the quality of commercial products. Health care providers, in particular, have an interest in knowing about product quality, in view of the issues related to herb-drug interactions and potential side effects. This study assessed whether commercial formulations of saw palmetto, kava kava, echinacea, ginseng and St. John's wort had consistent labeling and whether quantities of marker compounds agreed with the amounts stated on the label. We purchased six bottles each of two lots of supplements from nine manufacturers and analyzed the contents using established commercial methodologies at an independent laboratory. Product labels were found to vary in the information provided, such as serving recommendations and information about the herb itself (species, part of the plant, marker compound, etc.) With regard to marker compound content, little variability was observed between different lots of the same brand, while the content did vary widely between brands (e.g. total phenolic compounds in Echinacea ranged from 3.9–15.3 mg per serving; total ginsenosides in ginseng ranged from 5.3–18.2 mg per serving). Further, the amounts recommended for daily use also differed between brands, increasing the potential range of a consumer's daily dose. Echinacea and ginseng were the most variable, while St. John's wort and saw palmetto were the least variable. This study highlights some of the key issues in the botanical supplement market, including the importance of standardized manufacturing practices and reliable labeling information. In addition, health care providers should keep themselves informed regarding product quality in order to be able to appropriately advise patients utilizing both conventional and herbal medicines. PMID:15841264

  12. Nano-immunosafety: issues in assay validation

    International Nuclear Information System (INIS)

    Boraschi, Diana; Italiani, Paola; Oostingh, Gertie J; Duschl, Albert; Casals, Eudald; Puntes, Victor F; Nelissen, Inge

    2011-01-01

    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.

  13. How valid are commercially available medical simulators?

    Directory of Open Access Journals (Sweden)

    Stunt JJ

    2014-10-01

    Full Text Available JJ Stunt,1 PH Wulms,2 GM Kerkhoffs,1 J Dankelman,2 CN van Dijk,1 GJM Tuijthof1,2 1Orthopedic Research Center Amsterdam, Department of Orthopedic Surgery, Academic Medical Centre, Amsterdam, the Netherlands; 2Department of Biomechanical Engineering, Faculty of Mechanical, Materials and Maritime Engineering, Delft University of Technology, Delft, the Netherlands Background: Since simulators offer important advantages, they are increasingly used in medical education and medical skills training that require physical actions. A wide variety of simulators have become commercially available. It is of high importance that evidence is provided that training on these simulators can actually improve clinical performance on live patients. Therefore, the aim of this review is to determine the availability of different types of simulators and the evidence of their validation, to offer insight regarding which simulators are suitable to use in the clinical setting as a training modality. Summary: Four hundred and thirty-three commercially available simulators were found, from which 405 (94% were physical models. One hundred and thirty validation studies evaluated 35 (8% commercially available medical simulators for levels of validity ranging from face to predictive validity. Solely simulators that are used for surgical skills training were validated for the highest validity level (predictive validity. Twenty-four (37% simulators that give objective feedback had been validated. Studies that tested more powerful levels of validity (concurrent and predictive validity were methodologically stronger than studies that tested more elementary levels of validity (face, content, and construct validity. Conclusion: Ninety-three point five percent of the commercially available simulators are not known to be tested for validity. Although the importance of (a high level of validation depends on the difficulty level of skills training and possible consequences when skills are

  14. Principles of validation of diagnostic assays for infectious diseases

    International Nuclear Information System (INIS)

    Jacobson, R.H.

    1998-01-01

    Assay validation requires a series of inter-related processes. Assay validation is an experimental process: reagents and protocols are optimized by experimentation to detect the analyte with accuracy and precision. Assay validation is a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. Assay validation is a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the target population; accurate predictions of the infection status of animals from test results (PV+ and PV-) are conditional upon the estimated prevalence of disease/infection in the target population. Assay validation is an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results; the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status. Assay validation is a continuous process: the assay remains valid only insofar as it continues to provide accurate and precise results as proven through statistical verification. Therefore, the work required for validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples rather, to assure valid test results from an assay requires constant vigilance and maintenance of the assay, along with reassessment of its performance characteristics for each unique population of animals to which it is applied. (author)

  15. Desomorphine Screening Using Commercial Enzyme-Linked Immunosorbent Assays.

    Science.gov (United States)

    Winborn, Jessica; Kerrigan, Sarah

    2017-06-01

    Desomorphine ("Krokodil") is a semi-synthetic opioid that has drawn attention as a recreational drug, particularly in Russia, neighboring former Soviet Republics, Eastern and Central Europe. It has no accepted medicinal uses and is currently a schedule I drug in the United States. In clandestine environments, desomorphine is synthesized from codeine using red phosphorous, hydroiodic acid and gasoline. Residual starting materials in illicit preparations have been associated with severe dermatological effects and extensive tissue necrosis. Desomorphine is not well studied, and there are limited reports concerning its pharmacology or detection in biological matrices. Immunoassays are widely relied upon for both antemortem and postmortem toxicology screening. Although desomorphine is an opioid of the phenanthrene-type, its ability to bind to conventional opioid antibodies has not been described. In this report we describe the cross-reactivity of desomorphine using six commercially available enzyme-linked immunosorbent assays (Immunalysis Opiates Direct ELISA, Immunalysis Oxycodone/Oxymorphone Direct ELISA, Randox Opiate ELISA, OraSure Technologies OTI Opiate Micro-plate EIA, Neogen Opiate Group ELISA and Neogen Oxycodone/Oxymorphone ELISA). Cross-reactivites were highly variable between assays, ranging from 77 to desomorphine than those directed towards oxycodone. The Immunalysis Opiates Direct ELISA produced the greatest cross-reactivity, although several of the assays evaluated produced cross-reactivity of a sufficient magnitude to be effective for desomorphine screening. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Clinical validation of the Tempus xO assay

    Science.gov (United States)

    Beaubier, Nike; Tell, Robert; Huether, Robert; Bontrager, Martin; Bush, Stephen; Parsons, Jerod; Shah, Kaanan; Baker, Tim; Selkov, Gene; Taxter, Tim; Thomas, Amber; Bettis, Sam; Khan, Aly; Lau, Denise; Lee, Christina; Barber, Matthew; Cieslik, Marcin; Frankenberger, Casey; Franzen, Amy; Weiner, Ali; Palmer, Gary; Lonigro, Robert; Robinson, Dan; Wu, Yi-Mi; Cao, Xuhong; Lefkofsky, Eric; Chinnaiyan, Arul; White, Kevin P.

    2018-01-01

    We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification. PMID:29899824

  17. How valid are commercially available medical simulators?

    NARCIS (Netherlands)

    Stunt, J.J.; Wulms, P.H.; Kerkhoffs, G.M.; Dankelman, J.; Van Dijk, C.N.; Tuijthof, G.J.M.

    2014-01-01

    Background: Since simulators offer important advantages, they are increasingly used in medical education and medical skills training that require physical actions. A wide variety of simulators have become commercially available. It is of high importance that evidence is provided that training on

  18. Targeted resequencing and variant validation using pxlence PCR assays

    Directory of Open Access Journals (Sweden)

    Frauke Coppieters

    2016-01-01

    Full Text Available The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  19. 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.

  20. Results with commercial radioassay kits compared with microbiological assay of folate in serum and whole-blood

    International Nuclear Information System (INIS)

    McGown, E.L.; Lewis, C.M.; Dong, M.H.; Sauberlich, H.E.

    1978-01-01

    Results with three commercial folate radioassay kits [Bio-Rad, New England Nuclear (NEN), and RIA Products] were compared with those by microbiological assay for more than 200 samples of human serum and whole blood. All but one kit (NEN) compared favorably with the microbiological assay for serum samples, although there were notable diagnostic discrepancies. Two kits (NEN and Bio-Rad) were tested on whole-blood samples; both yielded values significantly higher than those by microbiological assay. The frequency distributions of erythrocyte folate data differed strikingly between the two kits; the NEN method yielded a much narrower range of normal values than did either the Bio-Rad or the microbiological assay. Radioassay kits appear to be suitable diagnostic agents for serum folate, if the behavior of a particular kit is investigated thoroughly before its routine use. However, the diagnostic value of radioassays of erythrocyte folate needs to be validated

  1. Assay Validation For Quantitation of Sn 2+ In Radiopharmaceutical Kits

    International Nuclear Information System (INIS)

    Muthalib, A; Ramli, Martalena; Herlina; Sarmini, Endang; Suharmadi; Besari, Canti

    1998-01-01

    An assay validation for quantitation of Sn2+ in radiopharmaceutical kits based on indirect iodometric titration is described. The method is based on the oxidation of sn2+ using a known excess of iodine and the excess unreacted iodine titrated with thiosulphate. Typical analytical parameters considered in this assay validation are precision, accuracy, selectivity or specificity, range, and linearity. The precision of the analytical method is quit good represented by coefficient of variance in range of 1.0% to 6.9 %, for 10 runs of analysis except one analysis shows the coefficient of 10.2 %. The method has an accuracy of 95.6 % - 99 % as percent recoveries at theoretical Sn2+ amounts of 463 μg to 2318μg

  2. Validation and Application of a Dried Blood Spot Ceftriaxone Assay

    Science.gov (United States)

    Page-Sharp, Madhu; Nunn, Troy; Salman, Sam; Moore, Brioni R.; Batty, Kevin T.; Davis, Timothy M. E.

    2015-01-01

    Dried blood spot (DBS) antibiotic assays can facilitate pharmacokinetic/pharmacodynamic (PK/PD) studies in situations where venous blood sampling is logistically and/or ethically problematic. In this study, we aimed to develop, validate, and apply a DBS ceftriaxone assay. A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) DBS ceftriaxone assay was assessed for matrix effects, process efficiency, recovery, variability, and limits of quantification (LOQ) and detection (LOD). The effects of hematocrit, protein binding, red cell partitioning, and chad positioning were evaluated, and thermal stability was assessed. Plasma, DBS, and cell pellet ceftriaxone concentrations in 10 healthy adults were compared, and plasma concentration-time profiles of DBS and plasma ceftriaxone were incorporated into population PK models. The LOQ and LOD for ceftriaxone in DBS were 0.14 mg/liter and 0.05 mg/liter, respectively. Adjusting for hematocrit, red cell partitioning, and relative recovery, DBS-predicted plasma concentrations were comparable to measured plasma concentrations (r > 0.95, P 95% initial concentrations in DBS for 14 h, 35 h, 30 days, 21 weeks, and >11 months, respectively. The present DBS ceftriaxone assay is robust and can be used as a surrogate for plasma concentrations to provide valid PK and PK/PD data in a variety of clinical situations, including in studies of young children and of those in remote or resource-poor settings. PMID:26438505

  3. Evaluation of a new commercial assay for diagnosis of pulmonary and nonpulmonary tuberculosis

    DEFF Research Database (Denmark)

    Johansen, I S; Thomsen, V Ø; Johansen, A

    2002-01-01

    A new commercial assay for the diagnosis of tuberculosis, the BDProbeTec ET Direct Detection assay (Becton Dickinson, USA), was evaluated using 351 respiratory and 372 nonrespiratory specimens. The results were compared to detection of Mycobacterium tuberculosis complex (MTC) by conventional...

  4. 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....

  5. Total antioxidants in plasma of hemodialysed patients and healthy controls measured by two commercial assays

    OpenAIRE

    Ruskovska, Tatjana; Jansen, Eugene

    2012-01-01

    Introduction: As a result of an increased interest in oxidative stress research, in both basic and clinical studies, numerous commercial test kits became available. The aim of this study was to evaluate the results for total antioxidants measured by two commercial assays in a complex clinical condition such as single hemodialysis session in patients on chronic hemodialysis treatment, in comparison to healthy controls. Methods: The level of plasma total antioxidants was measured by BAP ...

  6. Detection and quantification of serum or plasma HCV RNA: mini review of commercially available assays.

    Science.gov (United States)

    Le Guillou-Guillemette, Helene; Lunel-Fabiani, Francoise

    2009-01-01

    The treatment schedule (combination of compounds, doses, and duration) and the virological follow-up for management of antiviral treatment in patients chronically infected by HCV is now well standardized, but to ensure good monitoring of the treated patients, physicians need rapid, reproducible, and sensitive molecular virological tools with a wide range of detection and quantification of HCV RNA in blood samples. Several assays for detection and/or quantification of HCV RNA are currently commercially available. Here, all these assays are detailed, and a brief description of each step of the assay is provided. They are divided into two categories by method: those based on signal amplification and those based on target amplification. These two categories are then divided into qualitative, quantitative, and quantitative detection assays. The real-time reverse-transcription polymerase chain reaction (RT-PCR)-based assays are the most promising strategy in the HCV virological area.

  7. Specificity and sensitivity of commercially available assays for glucagon-like peptide-1 (GLP-1)

    DEFF Research Database (Denmark)

    Bak, Monika Judyta; Albrechtsen, Nicolai Jacob Wewer; Pedersen, Jens

    2014-01-01

    assessed with 3 commercial kits. RESULTS: The USCN LIFE assay detected none of the GLP-1 isoforms. The active GLP-1 ELISAs from Millipore and DRG seemed identical and were specific for intact GLP-1 in buffer and plasma. The Meso Scale Discovery Total GLP-1 kit detected all six GLP-1 isoforms, although...

  8. SCALE Validation Experience Using an Expanded Isotopic Assay Database for Spent Nuclear Fuel

    International Nuclear Information System (INIS)

    Gauld, Ian C.; Radulescu, Georgeta; Ilas, Germina

    2009-01-01

    The availability of measured isotopic assay data to validate computer code predictions of spent fuel compositions applied in burnup-credit criticality calculations is an essential component for bias and uncertainty determination in safety and licensing analyses. In recent years, as many countries move closer to implementing or expanding the use of burnup credit in criticality safety for licensing, there has been growing interest in acquiring additional high-quality assay data. The well-known open sources of assay data are viewed as potentially limiting for validating depletion calculations for burnup credit due to the relatively small number of isotopes measured (primarily actinides with relatively few fission products), sometimes large measurement uncertainties, incomplete documentation, and the limited burnup and enrichment range of the fuel samples. Oak Ridge National Laboratory (ORNL) recently initiated an extensive isotopic validation study that includes most of the public data archived in the Organization for Economic Cooperation and Development/Nuclear Energy Agency (OECD/NEA) electronic database, SFCOMPO, and new datasets obtained through participation in commercial experimental programs. To date, ORNL has analyzed approximately 120 different spent fuel samples from pressurized-water reactors that span a wide enrichment and burnup range and represent a broad class of assembly designs. The validation studies, completed using SCALE 5.1, are being used to support a technical basis for expanded implementation of burnup credit for spent fuel storage facilities, and other spent fuel analyses including radiation source term, dose assessment, decay heat, and waste repository safety analyses. This paper summarizes the isotopic assay data selected for this study, presents validation results obtained with SCALE 5.1, and discusses some of the challenges and experience associated with evaluating the results. Preliminary results obtained using SCALE 6 and ENDF

  9. Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

    Directory of Open Access Journals (Sweden)

    Hampus Sunner

    2015-09-01

    Full Text Available Research on glucuronoyl esterases (GEs has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.

  10. Comparative Assessment of Anti-HLA Antibodies Using Two Commercially Available Luminex-Based Assays.

    Science.gov (United States)

    Clerkin, Kevin J; See, Sarah B; Farr, Maryjane A; Restaino, Susan W; Serban, Geo; Latif, Farhana; Li, Lingzhi; Colombo, Paolo C; Vlad, George; Ray, Bryan; Vasilescu, Elena R; Zorn, Emmanuel

    2017-11-01

    Allospecific anti-HLA antibodies (Abs) are associated with rejection of solid organ grafts. The 2 main kits to detect anti-HLA Ab in patient serum are commercialized by Immucor and One Lambda/ThermoFisher. We sought to compare the performance of both platforms. Background-adjusted mean fluorescence intensity (MFI) values were used from both platforms to compare sera collected from 125 pretransplant and posttransplant heart and lung transplant recipients. Most HLA class I (94.5%) and HLA class II (89%) Abs with moderate to high MFI titer (≥4000) were detected by both assays. A modest correlation was observed between MFI values obtained from the 2 assays for both class I ( r = 0.3, r 2 = 0.09, P < 0.0001) and class II Ab ( r = 0.707, r 2 = 0.5, P < 0.0001). Both assays detected anti-class I and II Ab that the other did not; however, no specific HLA allele was detected preferentially by either of the 2 assays. For a limited number of discrepant sera, dilution resulted in comparable reactivity profiles between the 2 platforms. Immucor and One Lambda/ThermoFisher assays have a similar, albeit nonidentical, ability to detect anti-HLA Ab. Although the correlation between the assays was present, significant variances exist, some of which can be explained by a dilution-sensitive "prozone" effect.

  11. GPM ground validation via commercial cellular networks: an exploratory approach

    Science.gov (United States)

    Rios Gaona, Manuel Felipe; Overeem, Aart; Leijnse, Hidde; Brasjen, Noud; Uijlenhoet, Remko

    2016-04-01

    The suitability of commercial microwave link networks for ground validation of GPM (Global Precipitation Measurement) data is evaluated here. Two state-of-the-art rainfall products are compared over the land surface of the Netherlands for a period of 7 months, i.e., rainfall maps from commercial cellular communication networks and Integrated Multi-satellite Retrievals for GPM (IMERG). Commercial microwave link networks are nowadays the core component in telecommunications worldwide. Rainfall rates can be retrieved from measurements of attenuation between transmitting and receiving antennas. If adequately set up, these networks enable rainfall monitoring tens of meters above the ground at high spatiotemporal resolutions (temporal sampling of seconds to tens of minutes, and spatial sampling of hundreds of meters to tens of kilometers). The GPM mission is the successor of TRMM (Tropical Rainfall Measurement Mission). For two years now, IMERG offers rainfall estimates across the globe (180°W - 180°E and 60°N - 60°S) at spatiotemporal resolutions of 0.1° x 0.1° every 30 min. These two data sets are compared against a Dutch gauge-adjusted radar data set, considered to be the ground truth given its accuracy, spatiotemporal resolution and availability. The suitability of microwave link networks in satellite rainfall evaluation is of special interest, given the independent character of this technique, its high spatiotemporal resolutions and availability. These are valuable assets for water management and modeling of floods, landslides, and weather extremes; especially in places where rain gauge networks are scarce or poorly maintained, or where weather radar networks are too expensive to acquire and/or maintain.

  12. Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

    Science.gov (United States)

    Rösner, Stephan; Gehlweiler, Kevin; Küsters, Uta; Kolbert, Mathias; Hübner, Kirsten; Pfennigwerth, Niels; Mack, Dietrich

    2018-01-01

    Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany). 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

  13. Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

    Directory of Open Access Journals (Sweden)

    Stephan Rösner

    Full Text Available Multidrug-resistant Gram-negative bacilli (MDR-GNB producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany. 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

  14. A comparative study in three commercial kits of radioligand assay for GAD-Ab

    International Nuclear Information System (INIS)

    Yang Yehua; Zhou Zhiguang; Huang Gan; Yang Feike; Li Zhangwei

    2006-01-01

    Objective: To provide data for perfect selection of glutamic acid decarboxylase antibodies (GAD-Ab) assay commercial kits. Methods: The sera of 88 titer-graded samples by radioligand assay (RLA) were measured with two types of enzyme-linked immunosorbent assay (ELISA) kits and one type of immunoradiometric assay (IRMA) kit for GAD-Ab in order to test their consistency with RLA. Another 140 diabetic sera were measured with RLA and screened by a type of suitable kit to search the cutoff point for discriminating patients who needed rechecking. Results: Compared with RLA, concordance ratio of 3 kits: Medizym kit(75%) > RSR kit(73.9%) > Biomerica kit(62.5%); Kappa value: Medizym kit (0.408, common) > RSR kit (0.405, common) > Biomerica kit (0.185, failing); correlation to RLA index: RSR kit (r= 0.992) > Medizym kit(r= 0. 791 ) > Biomerica kit (r = -0. 055); area under the receiver operating characteristic (ROC) curve: Medizym kit (0.812) > RSR kit (0. 727 ) > Biomerica kit (0.666). Conclusions: The efficiencies of RSR kit and Medizym kit are good. Serum concentration between 0.25-0.99 U/ml measured by RSR kit is suggested to further recheck by RLA. (authors)

  15. Validation and determination of a reference interval for canine HbA1c using an immunoturbidimetric assay.

    Science.gov (United States)

    Goemans, Anne F; Spence, Susanna J; Ramsey, Ian K

    2017-06-01

    Hemoglobin A1c (HbA1c) provides a reliable measure of glycemic control over 2-3 months in human diabetes mellitus. In dogs, presence of HbA1c has been demonstrated, but there are no validated commercial assays. The purpose of the study was to validate a commercially available automated immunoturbidimetric assay for canine HbA1c and determine an RI in a hospital population. The specificity of the assay was assessed by inducing glycosylation in vitro using isolated canine hemoglobin, repeatability by measuring canine samples 5 times in succession, long term inter-assay imprecision by measuring supplied control materials, stability using samples stored at 4°C over 5 days and -20°C over 8 weeks, linearity by mixing samples of known HbA1c in differing proportions, and the effect of anticoagulants with paired samples. An RI was determined using EDTA-anticoagulated blood samples from 60 nondiabetic hospitalized animals of various ages and breeds. Hemoglobin A1c was also measured in 10 diabetic dogs. The concentration of HbA1c increased proportionally with glucose concentration in vitro. For repeat measurements, the CV was 4.08% (range 1.16-6.10%). Samples were stable for 5 days at 4°C. The assay was linear within the assessed range. Heparin- and EDTA-anticoagulated blood provided comparable results. The RI for HbA1c was 9-18.5 mmol/mol. There was no apparent effect of age or breed on HbA1c. In diabetic dogs, HbA1c ranged from 14 to 48 mmol/mol. The assay provides a reliable method for canine HbA1c measurement with good analytic performance. © 2017 American Society for Veterinary Clinical Pathology.

  16. Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two-center study.

    Science.gov (United States)

    Bowyer, A E; Hillarp, A; Ezban, M; Persson, P; Kitchen, S

    2016-07-01

    Essentials Validated assays are required to precisely measure factor IX (FIX) activity in FIX products. N9-GP and two other FIX products were assessed in various coagulation assay systems at two sites. Large variations in FIX activity measurements were observed for N9-GP using some assays. One-stage and chromogenic assays accurately measuring FIX activity for N9-GP were identified. Background Measurement of factor IX activity (FIX:C) with activated partial thromboplastin time-based one-stage clotting assays is associated with a large degree of interlaboratory variation in samples containing glycoPEGylated recombinant FIX (rFIX), i.e. nonacog beta pegol (N9-GP). Validation and qualification of specific assays and conditions are necessary for the accurate assessment of FIX:C in samples containing N9-GP. Objectives To assess the accuracy of various one-stage clotting and chromogenic assays for measuring FIX:C in samples containing N9-GP as compared with samples containing rFIX or plasma-derived FIX (pdFIX) across two laboratory sites. Methods FIX:C, in severe hemophilia B plasma spiked with a range of concentrations (from very low, i.e. 0.03 IU mL(-1) , to high, i.e. 0.90 IU mL(-1) ) of N9-GP, rFIX (BeneFIX), and pdFIX (Mononine), was determined at two laboratory sites with 10 commercially available one-stage clotting assays and two chromogenic FIX:C assays. Assays were performed with a plasma calibrator and different analyzers. Results A high degree of variation in FIX:C measurement was observed for one-stage clotting assays for N9-GP as compared with rFIX or pdFIX. Acceptable N9-GP recovery was observed in the low-concentration to high-concentration samples tested with one-stage clotting assays using SynthAFax or DG Synth, or with chromogenic FIX:C assays. Similar patterns of FIX:C measurement were observed at both laboratory sites, with minor differences probably being attributable to the use of different analyzers. Conclusions These results suggest that, of the

  17. Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Schneider, Uffe Vest; Scheel, Troels

    2006-01-01

    BACKGROUND: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently...... triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor...

  18. Development and validation of a multiplex add-on assay of biomarkers related to sepsis using xMAP technology

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Vest Schneider, Uffe; Scheel, Troels

    2006-01-01

    BACKGROUND: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently...... triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor...

  19. Validation of honey-bee smelling profile by using a commercial electronic nose

    Directory of Open Access Journals (Sweden)

    Ana R. Correa

    2017-09-01

    Full Text Available Honey is a natural sweetener and its quality labels are associated to its botanical or geographical origin, which is being established by palynological and sensorial analysis. The use of fast and non-invasive techniques such as an electronic nose can become an alternative for honey classification. In this study, the operational parameters of a commercial electronic nose were validated to determine the honey odor profile. A central composite design with five factors, three levels and 28 assays was used, varying sample amounts (1, 2 and 3 g, incubation temperature (30, 40 and 50 °C, incubation time 30 min, gas flow (50, 150 and 250 mL/min and injection time (100, 200 and 300 s. The commercial nose had ten sensors. Repeatability was evaluated with a coefficient of variation of 10 %. The response surface methodology was used and the optimal operating conditions were: 3 g of sample, incubation at 50 °C for 17 min, gas flow of 100 mL/min and sampling time of 150 s. Finally, these parameters were used to analyze 19 samples of honey, which were classified according to their odor profiles, showing that it can be a useful tool to classify honey.

  20. Diagnostic performance of a commercial immunoblot assay for myositis antibody testing.

    Science.gov (United States)

    Bundell, Chris; Rojana-Udomsart, Arada; Mastaglia, Frank; Hollingsworth, Peter; McLean-Tooke, Andrew

    2016-06-01

    The objective of this study was to establish a population based reference range for a commercial immunoblot assay detecting myositis specific autoantibodies (MSAs) and myositis associated autoantibodies (MAAs), and to assess the diagnostic performance of this reference range against the manufacturer's recommended ranges in a myositis patient cohort. A total of 124 patients from a myositis cohort and 197 healthy controls were serologically assessed using a commercial immunoblot containing eleven autoantigens (Jo-1, EJ, OJ, PL7, PL12, Mi-2, SRP, Ku, PMScl75, PMScl100 and Ro52) according to the manufacturer's instructions. Use of the manufacturer's reference ranges resulted in detection of MSAs in 19.4% of myositis patients and 9.1% of controls; MAAs were detected in 41.1% of myositis patients and 14.2% of controls. Reference values derived from the healthy control population resulted in significant differences in cut-off values for some autoantibodies, particularly Ro52 and PMScl75. Use of local reference ranges reduced detection of MSAs to 16.9% of myositis patients and 3% of healthy controls, with MAAs 23.4% of patients and 2% of healthy controls. Application of population based reference ranges resulted in significant differences in detection of MSAs and MAAs compared to the manufacturer's recommended ranges. Cut-off levels should be assessed to ensure suitability for the population tested. Copyright © 2016. Published by Elsevier B.V.

  1. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy.

    Science.gov (United States)

    Radrizzani, Marina; Soncin, Sabrina; Bolis, Sara; Lo Cicero, Viviana; Andriolo, Gabriella; Turchetto, Lucia

    2016-01-01

    The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.

  2. Quantitative comparison of in house irma/ria methods using reagents supplied in bulk with assays based on commercial kits

    International Nuclear Information System (INIS)

    Sajid, K.M.; Jafri, S.R.A.

    1997-01-01

    Due to high cost of commercial kit assays, local trials were started to establish low cost immunoassay techniques at MINAR, Multan. First available alternate of commercial kits was the use of matched reagents supplied in bulk by NETRIA through INMOL, Lahore under IAEA assistance. As quality is crucial in RIA estimations this laboratory collected passive quality control data of 50, 51 and 52 in-house assay batches of T3,T4 and TSH to compare with the same number of last Amerlex RIAs(successive lots). A qualitative comparison based on computerized data analysis shows linear correlation between the results of two assay systems with low and acceptable precision in T3 and T4 assays. In TSH assays both systems show high imprecision although Amerlex RIA system is relatively more precise than in-house TSH- IRMA. T3 and T4 assays in both the systems show wide working ranges covering all clinical regions. In TSH, working ranges of both the techniques do not cover all clinical regions. In-house TSH assay excludes below 5.5 mulU/ml, whereas Amerlex excludes levels below 4.5 mulL/ml. This may reduce the clinical efficacy of these tests. Amerlex-M T3 and T4 assays show high negative drift with relatively less between variation, whereas in-house assays show low positive drift with high between batch variation. (author)

  3. Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

    Science.gov (United States)

    Ni, Yan G; Yuan, Xiling; Newitt, John A; Peterson, Jon E; Gleason, Carol R; Haulenbeek, Jonathan; Santockyte, Rasa; Lafont, Virginie; Marsilio, Frank; Neely, Robert J; DeSilva, Binodh; Piccoli, Steven P

    2015-07-01

    Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

  4. 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

    A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first...

  5. Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Bergmann, S M; Wang, Q; Zeng, W; Li, Y; Wang, Y; Matras, M; Reichert, M; Fichtner, D; Lenk, M; Morin, T; Olesen, N J; Skall, H F; Lee, P-Y; Zheng, S; Monaghan, S; Reiche, S; Fuchs, W; Kotler, M; Way, K; Bräuer, G; Böttcher, K; Kappe, A; Kielpinska, J

    2017-11-01

    Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs. © 2017 John Wiley & Sons Ltd.

  6. Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus

    NARCIS (Netherlands)

    Nodelijk, G.; Wensvoort, G.; Kroese, B.; Leengoed, van L.A.M.G.; Colijn, E.; Verheijden, J.H.M.

    1996-01-01

    A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine respiratory and reproductive syndrome virus (PRRSV) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used were collected from pigs experimentally infected with

  7. Recommendations for adaptation and validation of commercial kits for biomarker quantification in drug development.

    Science.gov (United States)

    Khan, Masood U; Bowsher, Ronald R; Cameron, Mark; Devanarayan, Viswanath; Keller, Steve; King, Lindsay; Lee, Jean; Morimoto, Alyssa; Rhyne, Paul; Stephen, Laurie; Wu, Yuling; Wyant, Timothy; Lachno, D Richard

    2015-01-01

    Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

  8. Assessing the validity of commercial and municipal food environment data sets in Vancouver, Canada.

    Science.gov (United States)

    Daepp, Madeleine Ig; Black, Jennifer

    2017-10-01

    The present study assessed systematic bias and the effects of data set error on the validity of food environment measures in two municipal and two commercial secondary data sets. Sensitivity, positive predictive value (PPV) and concordance were calculated by comparing two municipal and two commercial secondary data sets with ground-truthed data collected within 800 m buffers surrounding twenty-six schools. Logistic regression examined associations of sensitivity and PPV with commercial density and neighbourhood socio-economic deprivation. Kendall's τ estimated correlations between density and proximity of food outlets near schools constructed with secondary data sets v. ground-truthed data. Vancouver, Canada. Food retailers located within 800 m of twenty-six schools RESULTS: All data sets scored relatively poorly across validity measures, although, overall, municipal data sets had higher levels of validity than did commercial data sets. Food outlets were more likely to be missing from municipal health inspections lists and commercial data sets in neighbourhoods with higher commercial density. Still, both proximity and density measures constructed from all secondary data sets were highly correlated (Kendall's τ>0·70) with measures constructed from ground-truthed data. Despite relatively low levels of validity in all secondary data sets examined, food environment measures constructed from secondary data sets remained highly correlated with ground-truthed data. Findings suggest that secondary data sets can be used to measure the food environment, although estimates should be treated with caution in areas with high commercial density.

  9. The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

    DEFF Research Database (Denmark)

    Hecker, Markus; Hollert, Henner; Cooper, Ralph

    2011-01-01

    In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebra......In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals...... in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization...... for Economic Cooperation and Development (OECD) test guideline.A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals...

  10. Validity of urinary monoamine assay sales under the "spot baseline urinary neurotransmitter testing marketing model".

    Science.gov (United States)

    Hinz, Marty; Stein, Alvin; Uncini, Thomas

    2011-01-01

    Spot baseline urinary monoamine assays have been used in medicine for over 50 years as a screening test for monoamine-secreting tumors, such as pheochromocytoma and carcinoid syndrome. In these disease states, when the result of a spot baseline monoamine assay is above the specific value set by the laboratory, it is an indication to obtain a 24-hour urine sample to make a definitive diagnosis. There are no defined applications where spot baseline urinary monoamine assays can be used to diagnose disease or other states directly. No peer-reviewed published original research exists which demonstrates that these assays are valid in the treatment of individual patients in the clinical setting. Since 2001, urinary monoamine assay sales have been promoted for numerous applications under the "spot baseline urinary neurotransmitter testing marketing model". There is no published peer-reviewed original research that defines the scientific foundation upon which the claims for these assays are made. On the contrary, several articles have been published that discredit various aspects of the model. To fill the void, this manuscript is a comprehensive review of the scientific foundation and claims put forth by laboratories selling urinary monoamine assays under the spot baseline urinary neurotransmitter testing marketing model.

  11. Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects

    Directory of Open Access Journals (Sweden)

    Rossi François

    2011-02-01

    Full Text Available Abstract Background With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines. To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated. Results In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin. Conclusion The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

  12. Comparative Assessment of Anti-HLA Antibodies Using Two Commercially Available Luminex-Based Assays

    Directory of Open Access Journals (Sweden)

    Kevin J. Clerkin, MD, MSc

    2017-11-01

    Conclusions. Immucor and One Lambda/ThermoFisher assays have a similar, albeit nonidentical, ability to detect anti-HLA Ab. Although the correlation between the assays was present, significant variances exist, some of which can be explained by a dilution-sensitive “prozone” effect.

  13. Validation of the Filovirus Plaque Assay for Use in Preclinical Studies

    Directory of Open Access Journals (Sweden)

    Amy C. Shurtleff

    2016-04-01

    Full Text Available A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4 studies. After standardization studies were completed, Good Laboratory Practices (GLP-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV variant and Ebola Virus Kikwit (EBOV variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID. The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay’s precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.

  14. Improving validation methods for molecular diagnostics: application of Bland-Altman, Deming and simple linear regression analyses in assay comparison and evaluation for next-generation sequencing.

    Science.gov (United States)

    Misyura, Maksym; Sukhai, Mahadeo A; Kulasignam, Vathany; Zhang, Tong; Kamel-Reid, Suzanne; Stockley, Tracy L

    2018-02-01

    A standard approach in test evaluation is to compare results of the assay in validation to results from previously validated methods. For quantitative molecular diagnostic assays, comparison of test values is often performed using simple linear regression and the coefficient of determination (R 2 ), using R 2 as the primary metric of assay agreement. However, the use of R 2 alone does not adequately quantify constant or proportional errors required for optimal test evaluation. More extensive statistical approaches, such as Bland-Altman and expanded interpretation of linear regression methods, can be used to more thoroughly compare data from quantitative molecular assays. We present the application of Bland-Altman and linear regression statistical methods to evaluate quantitative outputs from next-generation sequencing assays (NGS). NGS-derived data sets from assay validation experiments were used to demonstrate the utility of the statistical methods. Both Bland-Altman and linear regression were able to detect the presence and magnitude of constant and proportional error in quantitative values of NGS data. Deming linear regression was used in the context of assay comparison studies, while simple linear regression was used to analyse serial dilution data. Bland-Altman statistical approach was also adapted to quantify assay accuracy, including constant and proportional errors, and precision where theoretical and empirical values were known. The complementary application of the statistical methods described in this manuscript enables more extensive evaluation of performance characteristics of quantitative molecular assays, prior to implementation in the clinical molecular laboratory. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  15. Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

    Science.gov (United States)

    Reisinger, Kerstin; Blatz, Veronika; Brinkmann, Joep; Downs, Thomas R; Fischer, Anja; Henkler, Frank; Hoffmann, Sebastian; Krul, Cyrille; Liebsch, Manfred; Luch, Andreas; Pirow, Ralph; Reus, Astrid A; Schulz, Markus; Pfuhler, Stefan

    2018-03-01

    Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion ® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion ® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will

  16. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    DEFF Research Database (Denmark)

    Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

    2017-01-01

    to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

  17. Use of statistical analysis to validate ecogenotoxicology findings arising from various comet assay components.

    Science.gov (United States)

    Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, Khalid Abdullah; Masoud, Muhammad Shahreef; Mahboob, Shahid

    2018-04-01

    Cirrhinus mrigala, Labeo rohita, and Catla catla are economically important fish for human consumption in Pakistan, but industrial and sewage pollution has drastically reduced their population in the River Chenab. Statistics are an important tool to analyze and interpret comet assay results. The specific aims of the study were to determine the DNA damage in Cirrhinus mrigala, Labeo rohita, and Catla catla due to chemical pollution and to assess the validity of statistical analyses to determine the viability of the comet assay for a possible use with these freshwater fish species as a good indicator of pollution load and habitat degradation. Comet assay results indicated a significant (P comet head diameter, comet tail length, and % DNA damage. Regression analysis and correlation matrices conducted among the parameters of the comet assay affirmed the precision and the legitimacy of the results. The present study, therefore, strongly recommends that genotoxicological studies conduct appropriate analysis of the various components of comet assays to offer better interpretation of the assay data.

  18. Analytic Validation of Immunohistochemistry Assays: New Benchmark Data From a Survey of 1085 Laboratories.

    Science.gov (United States)

    Stuart, Lauren N; Volmar, Keith E; Nowak, Jan A; Fatheree, Lisa A; Souers, Rhona J; Fitzgibbons, Patrick L; Goldsmith, Jeffrey D; Astles, J Rex; Nakhleh, Raouf E

    2017-09-01

    - A cooperative agreement between the College of American Pathologists (CAP) and the United States Centers for Disease Control and Prevention was undertaken to measure laboratories' awareness and implementation of an evidence-based laboratory practice guideline (LPG) on immunohistochemical (IHC) validation practices published in 2014. - To establish new benchmark data on IHC laboratory practices. - A 2015 survey on IHC assay validation practices was sent to laboratories subscribed to specific CAP proficiency testing programs and to additional nonsubscribing laboratories that perform IHC testing. Specific questions were designed to capture laboratory practices not addressed in a 2010 survey. - The analysis was based on responses from 1085 laboratories that perform IHC staining. Ninety-six percent (809 of 844) always documented validation of IHC assays. Sixty percent (648 of 1078) had separate procedures for predictive and nonpredictive markers, 42.7% (220 of 515) had procedures for laboratory-developed tests, 50% (349 of 697) had procedures for testing cytologic specimens, and 46.2% (363 of 785) had procedures for testing decalcified specimens. Minimum case numbers were specified by 85.9% (720 of 838) of laboratories for nonpredictive markers and 76% (584 of 768) for predictive markers. Median concordance requirements were 95% for both types. For initial validation, 75.4% (538 of 714) of laboratories adopted the 20-case minimum for nonpredictive markers and 45.9% (266 of 579) adopted the 40-case minimum for predictive markers as outlined in the 2014 LPG. The most common method for validation was correlation with morphology and expected results. Laboratories also reported which assay changes necessitated revalidation and their minimum case requirements. - Benchmark data on current IHC validation practices and procedures may help laboratories understand the issues and influence further refinement of LPG recommendations.

  19. Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    Directory of Open Access Journals (Sweden)

    Kathleen Ingenhoven

    2017-07-01

    Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

  20. Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

  1. Application of commercial enzyme linked immunosorbent assays (ELISA for the detection of antibodies for foot-and-mouth disease virus in wild boar and red deer

    Directory of Open Access Journals (Sweden)

    Terzić Svjetlana

    2012-01-01

    Full Text Available For detecting antibodies towards foot and mouth (FMD virus in sera collected from red deer hinds (Cervus elaphus and wild boars (Sus scrofa, three commercially available enzyme-linked immunosorbent assays (ELISA were used. Two ELISA kits (PrioCHECK FMDV NS and CHEKIT FMD-3ABC were used for the detection of antibodies towards non-structural proteins of FMD virus and one assay was based on the detection of antibodies for serotype O (PrioCHECK FMDV type O. All of the sera tested in our study were negative for antibodies against FMD virus. The aim of this study was to investigate the usefulness of commercially available ELISA kits given for marketing authorization in Croatia in testing the prevalence of FMD antibodies in wild boar and red deer populations. Since the producers of ELISA kits used in our study did not declare wild animals as a target species, we hypothesised that the same kits could be used for serological diagnosis of FMD in red deer and wild boars. Our study confirmed that the kits used are acceptable for detecting antibodies in both species tested, however, the investigation highlighted the problem of validating the kits due to the absence of available positive sera originating from red deer, as well as other susceptible species, especially artiodactyls.

  2. Validation of an in vitro contractility assay using canine ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.; Pullen, G.F.; Valentin, J.P.; Pollard, C.E.

    2012-04-15

    Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of accuracy.

  3. 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

    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......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...... the same HPV infections. Here, we determined the characteristics of the concordant (all four assays returning a positive HPV test result) and discordant samples (all other HPV-positive samples) in primary cervical screening at 30-65 years (n=2859) and in a concurrent referral population from the same...

  4. Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.

    Science.gov (United States)

    Wyant, Tim; Estevam, Jose; Yang, Lili; Rosario, Maria

    2016-03-01

    Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society.

  5. Improved quantification of a commercial enzyme-linked immunosorbent assay kit for measuring anti-MDA5 antibody.

    Science.gov (United States)

    Gono, Takahisa; Okazaki, Yuka; Murakami, Akihiro; Kuwana, Masataka

    2018-04-09

    To compare the quantitative performance for measuring anti-MDA5 antibody titer of two enzyme-linked immunosorbent assay (ELISA) systems: an in-house ELISA and the commercial MESACUP TM anti-MDA5 test. Anti-MDA5 antibody titer was measured in sera from 70 patients with dermatomyositis using an in-house ELISA and the MESACUP TM anti-MDA5 test side-by-side. For the commercial ELISA kit, serum samples diluted 1:101 were used according to the manufacturer's protocol, but serial dilutions of sera were also examined to identify the optimal serum dilution for quantification. The anti-MDA5 antibody titers measured by the in-house and commercial ELISAs were positively correlated with each other (r = 0.53, p = .0001), but the antibody titer measured by the commercial ELISA was less sensitive to change after medical treatment, and 37 (80%) of 46 anti-MDA5-positive sera had antibody titer exceeding the quantification range specified by the manufacturer (≥150 index). Experiments using diluted serum samples revealed that diluting the sera 1:5050 improved the quantitative performance of the MESACUP TM anti-MDA5 test, including a better correlation with the in-house ELISA results and an increased sensitivity to change. We improved the ability of the commercial ELISA kit to quantify anti-MDA5 antibody titer by altering its protocol.

  6. The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens

    Directory of Open Access Journals (Sweden)

    Broukhanski George

    2008-09-01

    Full Text Available Abstract The Seeplex™ TB Detection-2 assay (Rockville, MD is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

  7. Commercial demand for energy: a disaggregated approach. [Model validation for 1970-1975; forecasting to 2000

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, J.R.; Cohn, S.; Cope, J.; Johnson, W.S.

    1978-04-01

    This report describes the structure and forecasting accuracy of a disaggregated model of commercial energy use recently developed at Oak Ridge National Laboratory. The model forecasts annual commercial energy use by ten building types, five end uses, and four fuel types. Both economic (utilization rate, fuel choice, capital-energy substitution) and technological factors (equipment efficiency, thermal characteristics of buildings) are explicitly represented in the model. Model parameters are derived from engineering and econometric analysis. The model is then validated by simulating commercial energy use over the 1970--1975 time period. The model performs well both with respect to size of forecast error and ability to predict turning points. The model is then used to evaluate the energy-use implications of national commercial buildings standards based on the ASHRAE 90-75 recommendations. 10 figs., 12 tables, 14 refs.

  8. Validation of a next-generation sequencing assay for clinical molecular oncology.

    Science.gov (United States)

    Cottrell, Catherine E; Al-Kateb, Hussam; Bredemeyer, Andrew J; Duncavage, Eric J; Spencer, David H; Abel, Haley J; Lockwood, Christina M; Hagemann, Ian S; O'Guin, Stephanie M; Burcea, Lauren C; Sawyer, Christopher S; Oschwald, Dayna M; Stratman, Jennifer L; Sher, Dorie A; Johnson, Mark R; Brown, Justin T; Cliften, Paul F; George, Bijoy; McIntosh, Leslie D; Shrivastava, Savita; Nguyen, Tudung T; Payton, Jacqueline E; Watson, Mark A; Crosby, Seth D; Head, Richard D; Mitra, Robi D; Nagarajan, Rakesh; Kulkarni, Shashikant; Seibert, Karen; Virgin, Herbert W; Milbrandt, Jeffrey; Pfeifer, John D

    2014-01-01

    Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  9. Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials

    NARCIS (Netherlands)

    Kabel, M.A.; Maarel, van der M.J.E.C.; Klip, G.; Voragen, A.G.J.; Schols, H.A.

    2006-01-01

    Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex

  10. Standard Assays Do Not Predict the Efficiency of Commercial Cellulase Preparations Towards Plant Materials

    NARCIS (Netherlands)

    Kabel, Mirjam A.; Maarel, Marc J.E.C. van der; Klip, Gert; Voragen, Alphons G.J.; Schols, Henk A.

    2006-01-01

    Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex

  11. Validation of a UV Spectrometric Method for the Assay of Tolfenamic Acid in Organic Solvents

    Directory of Open Access Journals (Sweden)

    Sofia Ahmed

    2015-01-01

    Full Text Available The present study has been carried out to validate a UV spectrometric method for the assay of tolfenamic acid (TA in organic solvents. TA is insoluble in water; therefore, a total of thirteen commonly used organic solvents have been selected in which the drug is soluble. Fresh stock solutions of TA in each solvent in a concentration of 1 × 10−4 M (2.62 mg% were prepared for the assay. The method has been validated according to the guideline of International Conference on Harmonization and parameters like linearity, range, accuracy, precision, sensitivity, and robustness have been studied. Although the method was found to be efficient for the determination of TA in all solvents on the basis of statistical data 1-octanol, followed by ethanol and methanol, was found to be comparatively better than the other studied solvents. No change in the stock solution stability of TA has been observed in each solvent for 24 hours stored either at room (25±1°C or at refrigerated temperature (2–8°C. A shift in the absorption maxima has been observed for TA in various solvents indicating drug-solvent interactions. The studied method is simple, rapid, economical, accurate, and precise for the assay of TA in different organic solvents.

  12. A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.

    Science.gov (United States)

    Slattery, Scott D; Hahn, Klaus M

    2014-12-01

    Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single-chain and dual-chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for effects of upstream molecules. Copyright © 2014 John Wiley & Sons, Inc.

  13. Evaluation of Two Commercial Enzyme-Linked Immunosorbent Assay Kits for the Detection of Human Circulating Metrnl.

    Science.gov (United States)

    Zheng, Si-Li; Li, Zhi-Yong; Zhang, Zheng; Wang, Dong-Sheng; Xu, Jian; Miao, Chao-Yu

    2018-04-01

    Metrnl is a newly discovered secreted protein with neurotrophic activity and metabolic effect, while in earlier studies its circulating level in human was not explored. We evaluated two commercial enzyme-linked immunosorbent assay kits (DY7867-05, R&D Systems and SK00478-02, Aviscera Bioscience) for the detection of human circulating Metrnl. The DY7867-05 kit showed superiority over the SK00478-02 kit since it generated better curve fitting degree, smaller variation among tests, higher inter-assay reproducibility and better specificity, and could effectively detect human Metrnl in six types of blood samples. Subsequent analysis was performed using the DY7867-05 kit. Sample storage conditions were investigated. No gender difference in circulating Metrnl levels was found, while people with newly diagnosed type 2 diabetes mellitus (T2DM) had significantly lower Metrnl levels compared to the healthy controls.

  14. Development and Validation of a Microbiological Agar Assay for Determination of Orbifloxacin in Pharmaceutical Preparations

    Directory of Open Access Journals (Sweden)

    Hérida R. N. Salgado

    2011-08-01

    Full Text Available Orbifloxacin is a fluoroquinolone with broad-spectrum antimicrobial activity, and belongs to the third generation of quinolones. Regarding the quality control of medicines, a validated microbiological assay for determination of orbifloxacin in pharmaceutical formulations has not as yet been reported. For this purpose, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method to quantify orbifloxacin in tablet formulations. The assay is based on the inhibitory effect of orbifloxacin upon the strain of Staphylococcus aureus ATCC 25923 used as test microorganism. The results were treated statistically by analysis of variance and were found to be linear (r = 0.9992 in the selected range of 16.0–64.0 μg/mL, precise with relative standard deviation (RSD of repeatability intraday = 2.88%, intermediate precision RSD = 3.33%, and accurate (100.31%. The results demonstrated the validity of the proposed bioassay, which allows reliable orbifloxacin quantitation in pharmaceutical samples and therefore can be used as a useful alternative methodology for the routine quality control of this medicine.

  15. Analysis of Two Commercially Available Bortezomib Products: Differences in Assay of Active Agent and Impurity Profile

    OpenAIRE

    Byrn, Stephen R.; Tishmack, Patrick A.; Milton, Mark J.; van de Velde, Helgi

    2011-01-01

    The analytical properties of two commercially available bortezomib products (VELCADE® and Bortenat) were compared using nuclear magnetic resonance, mass spectrometry, high-performance liquid chromatography, and gas chromatography. The data showed differences between the two products. Based on these data, Bortenat samples contained more active ingredients than indicated by the label (mean, 116.5% and 117.9% of label, in 2-mg and 3.5-mg vials, respectively). In comparison, VELCADE samples conta...

  16. Clinical validation of an epigenetic assay to predict negative histopathological results in repeat prostate biopsies.

    Science.gov (United States)

    Partin, Alan W; Van Neste, Leander; Klein, Eric A; Marks, Leonard S; Gee, Jason R; Troyer, Dean A; Rieger-Christ, Kimberly; Jones, J Stephen; Magi-Galluzzi, Cristina; Mangold, Leslie A; Trock, Bruce J; Lance, Raymond S; Bigley, Joseph W; Van Criekinge, Wim; Epstein, Jonathan I

    2014-10-01

    The DOCUMENT multicenter trial in the United States validated the performance of an epigenetic test as an independent predictor of prostate cancer risk to guide decision making for repeat biopsy. Confirming an increased negative predictive value could help avoid unnecessary repeat biopsies. We evaluated the archived, cancer negative prostate biopsy core tissue samples of 350 subjects from a total of 5 urological centers in the United States. All subjects underwent repeat biopsy within 24 months with a negative (controls) or positive (cases) histopathological result. Centralized blinded pathology evaluation of the 2 biopsy series was performed in all available subjects from each site. Biopsies were epigenetically profiled for GSTP1, APC and RASSF1 relative to the ACTB reference gene using quantitative methylation specific polymerase chain reaction. Predetermined analytical marker cutoffs were used to determine assay performance. Multivariate logistic regression was used to evaluate all risk factors. The epigenetic assay resulted in a negative predictive value of 88% (95% CI 85-91). In multivariate models correcting for age, prostate specific antigen, digital rectal examination, first biopsy histopathological characteristics and race the test proved to be the most significant independent predictor of patient outcome (OR 2.69, 95% CI 1.60-4.51). The DOCUMENT study validated that the epigenetic assay was a significant, independent predictor of prostate cancer detection in a repeat biopsy collected an average of 13 months after an initial negative result. Due to its 88% negative predictive value adding this epigenetic assay to other known risk factors may help decrease unnecessary repeat prostate biopsies. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  17. Use of monoclonal immunoradiometric assays for sensitive TSH measurements: evaluation of four commercially obtainable kits

    International Nuclear Information System (INIS)

    Smitz, J.; Schiettecatte, J.; Stierteghem, A.C. van; Jonckheer, M.H.

    1987-01-01

    Four commerically available monoclonal immunoradiometric methods for the assay of TSH are tested. These four kits are: RIA-GNOST TSH code 0CPL of Behring, SUCROSEP TSH IRMA of Boots, TSH-IRMA-CT-100 of Medgenix, TSH-RIA bead II Ultrasensitive of Abbott. The accuracy, sensitivity and reproducibility of the four kits are compared. The methods are also clinically evaluated. The authors concluded that the kits of Abbott, Behring and Boots are suitable for use in the routine laboratory. 4 refs.; 4 figs.; 7 tabs

  18. The validation of an invitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions

    International Nuclear Information System (INIS)

    Keating, Christopher; Martinez, Vicente; Ewart, Lorna; Gibbons, Stephen; Grundy, Luke; Valentin, Jean-Pierre; Grundy, David

    2010-01-01

    Motility-related gastrointestinal adverse drug reactions (GADRs), such as constipation and diarrhea, are some of the most frequently reported adverse events associated with the clinical development of new chemical entities, and for marketed drugs. However, biomarkers capable of detecting such GADRs are lacking. Here, we describe an in vitro assay developed to detect and quantify changes in intestinal motility as a surrogate biomarker for constipation/diarrhea-type GADRs. In vitro recordings of intraluminal pressure were used to monitor the presence of colonic peristaltic motor complexes (CPMCs) in mouse colonic segments. CPMC frequency, contractile and total mechanical activity were assessed. To validate the assay, two experimental protocols were conducted. Initially, five drugs with known gastrointestinal effects were tested to determine optimal parameters describing excitation and inhibition as markers for disturbances in colonic motility. This was followed by a 'blinded' evaluation of nine drugs associated with or without clinically identified constipation/diarrhea-type GADRs. Concentration-response relationships were determined for these drugs and the effects were compared with their maximal free therapeutic plasma concentration in humans. The assay detected stimulatory and inhibitory responses, likely correlating to the occurrence of diarrhea or constipation. Concentration-related effects were identified and potential mechanisms of action were inferred for several drugs. Based on the results from the fourteen drugs assessed, the sensitivity of the assay was calculated at 90%, with a specificity of 75% and predictive capacity of 86%. These results support the potential use of this assay in screening for motility-related GADRs during early discovery phase, safety pharmacology assessment.

  19. Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

    Directory of Open Access Journals (Sweden)

    Lyerly Herbert K

    2008-03-01

    Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

  20. Methodology for validating technical tools to assess customer Demand Response: Application to a commercial customer

    International Nuclear Information System (INIS)

    Alcazar-Ortega, Manuel; Escriva-Escriva, Guillermo; Segura-Heras, Isidoro

    2011-01-01

    The authors present a methodology, which is demonstrated with some applications to the commercial sector, in order to validate a Demand Response (DR) evaluation method previously developed and applied to a wide range of industrial and commercial segments, whose flexibility was evaluated by modeling. DR is playing a more and more important role in the framework of electricity systems management for the effective integration of other distributed energy resources. Consequently, customers must identify what they are using the energy for in order to use their flexible loads for management purposes. Modeling tools are used to predict the impact of flexibility on the behavior of customers, but this result needs to be validated since both customers and grid operators have to be confident in these flexibility predictions. An easy-to-use two-steps method to achieve this goal is presented in this paper.

  1. Development and Validation of HPLC-PDA Assay method of Frangula emodin

    Directory of Open Access Journals (Sweden)

    Deborah Duca

    2016-03-01

    Full Text Available Frangula emodin, (1,3,8-trihydroxy-6-methyl-anthraquinone, is one of the anthraquinone derivatives found abundantly in the roots and bark of a number of plant families traditionally used to treat constipation and haemorrhoids. The present study describes the development and subsequent validation of a specific Assay HPLC method for emodin. The separation was achieved on a Waters Symmetry C18, 4.6 × 250 mm, 5 μm particle size, column at a temperature of 35 °C, with UV detection at 287 and 436 nm. An isocratic elution mode consisting of 0.1% formic acid and 0.01% trifluoroacetic acid as the aqueous mobile phase, and methanol was used. The method was successfully and statistically validated for linearity, range, precision, accuracy, specificity and solution stability.

  2. Electrochemical mechanism and kinetics studies of haloperidol and its assay in commercial formulations

    Energy Technology Data Exchange (ETDEWEB)

    Ribeiro, Francisco W.P. [Departamento de Quimica Analitica e Fisico-Quimica, Centro de Ciencias, Universidade Federal do Ceara, Bloco 940 Campus do Pici 60455-970, Fortaleza, CE (Brazil); Soares, Janete E.S. [Departamento de Farmacia, Faculdade de Farmacia, Odontologia e Enfermagem, Universidade Federal do Ceara, Rua Capitao Francisco Pedro, 1210 Rodolfo Teofilo 60430-370, Fortaleza, CE (Brazil); Becker, Helena; De Souza, Djenaine; Lima-Neto, Pedro de [Departamento de Quimica Analitica e Fisico-Quimica, Centro de Ciencias, Universidade Federal do Ceara, Bloco 940 Campus do Pici 60455-970, Fortaleza, CE (Brazil); Correia, Adriana N., E-mail: adriana@ufc.b [Departamento de Quimica Analitica e Fisico-Quimica, Centro de Ciencias, Universidade Federal do Ceara, Bloco 940 Campus do Pici 60455-970, Fortaleza, CE (Brazil)

    2011-02-01

    The kinetics and mechanism for electrochemical reduction of haloperidol, a psychotherapeutic drug used in the treatment of schizophrenia, were studied using square wave and cyclic voltammetries allied to a hanging mercury drop electrode. The experimental and voltammetric parameters were optimized at 0.04 mol L{sup -1} Brinton-Robinson buffer (pH 10), with a pulse potential frequency of 100 s{sup -1}, a pulse amplitude of 30 mV and scan increment of 2 mV. Two well-defined peaks were observed, which exhibited properties of fast electron transfer with a strong adsorption process of reactants and products on the electrode surface. The first peak was related to a fast and reversible anion-radical formation originating from the reduction of the carbonyl group, and the second was related to the irreversible reduction of the anion-radical previously formed. Analytical parameters such as: linearity range, equation of the analytical curves, correlation coefficients, detection and quantification limits, recovery efficiency, and relative standard deviation for intraday and interday were compared to similar results obtained by use of the UV-vis spectrophotometry technique, and the analytical results obtained in commercial formulations show that the voltammetric procedure using a hanging mercury drop electrode is suitable for analyzing haloperidol in complex commercial formulation samples.

  3. Electrochemical mechanism and kinetics studies of haloperidol and its assay in commercial formulations

    International Nuclear Information System (INIS)

    Ribeiro, Francisco W.P.; Soares, Janete E.S.; Becker, Helena; De Souza, Djenaine; Lima-Neto, Pedro de; Correia, Adriana N.

    2011-01-01

    The kinetics and mechanism for electrochemical reduction of haloperidol, a psychotherapeutic drug used in the treatment of schizophrenia, were studied using square wave and cyclic voltammetries allied to a hanging mercury drop electrode. The experimental and voltammetric parameters were optimized at 0.04 mol L -1 Brinton-Robinson buffer (pH 10), with a pulse potential frequency of 100 s -1 , a pulse amplitude of 30 mV and scan increment of 2 mV. Two well-defined peaks were observed, which exhibited properties of fast electron transfer with a strong adsorption process of reactants and products on the electrode surface. The first peak was related to a fast and reversible anion-radical formation originating from the reduction of the carbonyl group, and the second was related to the irreversible reduction of the anion-radical previously formed. Analytical parameters such as: linearity range, equation of the analytical curves, correlation coefficients, detection and quantification limits, recovery efficiency, and relative standard deviation for intraday and interday were compared to similar results obtained by use of the UV-vis spectrophotometry technique, and the analytical results obtained in commercial formulations show that the voltammetric procedure using a hanging mercury drop electrode is suitable for analyzing haloperidol in complex commercial formulation samples.

  4. Evaluation and validation of a single-dilution potency assay based upon serology of vaccines containing diphtheria toxoid: statistical analysis

    NARCIS (Netherlands)

    Marsman FR; Akkermans AM; Hendriksen CFM; de Jong WH

    1993-01-01

    This document presents the results of a validation study to the use of a single dilution assay in potency testing of the diphtheria component of DPT-polio vaccines. Based on historical data of multi-dilution assays on 27 consecutive batches a simulation study was performed to test the actual

  5. The local lymph node assay and the assessment of relative potency: status of validation.

    Science.gov (United States)

    Basketter, David A; Gerberick, Frank; Kimber, Ian

    2007-08-01

    For the prediction of skin sensitization potential, the local lymph node assay (LLNA) is a fully validated alternative to guinea-pig tests. More recently, information from LLNA dose-response analyses has been used to assess the relative potency of skin sensitizing chemicals. These data are then deployed for risk assessment and risk management. In this commentary, the utility and validity of these relative potency measurements are reviewed. It is concluded that the LLNA does provide a valuable assessment of relative sensitizing potency in the form of the estimated concentration of a chemical required to produce a threefold stimulation of draining lymph node cell proliferation compared with concurrent controls (EC3 value) and that all reasonable validation requirements have been addressed successfully. EC3 measurements are reproducible in both intra- and interlaboratory evaluations and are stable over time. It has been shown also, by several independent groups, that EC3 values correlate closely with data on relative human skin sensitization potency. Consequently, the recommendation made here is that LLNA EC3 measurements should now be regarded as a validated method for the determination of the relative potency of skin sensitizing chemicals, a conclusion that has already been reached by a number of independent expert groups.

  6. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    Science.gov (United States)

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

  7. Validation of an immunodiagnostic assay for detection of 13 Streptococcus pneumoniae serotype-specific polysaccharides in human urine.

    Science.gov (United States)

    Pride, Michael W; Huijts, Susanne M; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C; Bonten, Marc J M; Jansen, Kathrin U

    2012-08-01

    To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults.

  8. Molecular identification of python species: development and validation of a novel assay for forensic investigations.

    Science.gov (United States)

    Ciavaglia, Sherryn A; Tobe, Shanan S; Donnellan, Stephen C; Henry, Julianne M; Linacre, Adrian M T

    2015-05-01

    Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. Large scale commercial fabrication of high quality graphene-based assays for biomolecule detection

    Science.gov (United States)

    Lerner, Mitchell; Gao, Yingning; Goldsmith, Brett; Barron, Francie

    Large numbers of high quality graphene transistors with mobility approximately 5000 cm2 / V * s were fabricated by chemical vapor deposition and packaged into ceramic carriers with an open cavity design. The ceramic carrier is compatible with standard electronics assembly, enabling the readout of graphene properties on the benchtop without large, expensive probing systems. After chemical functionalization, these sensors demonstrate sensitivity in the pM range and selectivity to many classes of biomolecules as a three terminal liquid-gated field effect transistor. High precision measurements of protein kinetics captured using this technology, commercially known as AGILE R100, are comparable and can exceed the capabilities of state-of-the-art biomolecule characterization tools. Recently published in Sensors and Actuators B

  10. A solid phase radioimmunoassay for free triiodothyronine in serum: assay development and validation

    International Nuclear Information System (INIS)

    Liu Fangyan; Zhou Meiying; Yin Linxiang; Yang Jianzhong; Hua Chenglin

    1999-01-01

    A solid phase radioimmunoassay for free triiodothyronine in serum was developed based on double-antibody coated tubes. The method was turned out to be reliable with good reproducibility, higher sensitivity and easy performance. The measurable range of FT 3 in serum was 1.2 to 38 pmol/L. The mean coefficients of variation within and between assays were 1.79%∼3.18% and 4.72%∼9.31%, respectively. The FT 3 concentrations in euthyroid serum as determined by this method were 2.8 to 7.8 pmol/L. The FT 3 values determined by this new method correlated well with those measured by a commercial radioimmunoassay (r = 0.853)

  11. Analytical validation of a new point-of-care assay for serum amyloid A in horses.

    Science.gov (United States)

    Schwartz, D; Pusterla, N; Jacobsen, S; Christopher, M M

    2018-01-17

    Serum amyloid A (SAA) is a major acute phase protein in horses. A new point-of-care (POC) test for SAA (Stablelab) is available, but studies evaluating its analytical accuracy are lacking. To evaluate the analytical performance of the SAA POC test by 1) determining linearity and precision, 2) comparing results in whole blood with those in serum or plasma, and 3) comparing POC results with those obtained using a previously validated turbidimetric immunoassay (TIA). Assay validation. Analytical validation of the POC test was done in accordance with American Society of Veterinary Clinical Pathology guidelines using residual equine serum/plasma and whole blood samples from the Clinical Pathology Laboratory at the University of California-Davis. A TIA was used as the reference method. We also evaluated the effect of haematocrit (HCT). The POC test was linear for SAA concentrations of up to at least 1000 μg/mL (r = 0.991). Intra-assay CVs were 13, 18 and 15% at high (782 μg/mL), intermediate (116 μg/mL) and low (64 μg/mL) concentrations. Inter-assay (inter-batch) CVs were 45, 14 and 15% at high (1372 μg/mL), intermediate (140 μg/mL) and low (56 μg/mL) concentrations. SAA results in whole blood were significantly lower than those in serum/plasma (P = 0.0002), but were positively correlated (r = 0.908) and not affected by HCT (P = 0.261); proportional negative bias was observed in samples with SAA>500 μg/mL. The difference between methods exceeded the 95% confidence interval of the combined imprecision of both methods (15%). Analytical validation could not be performed in whole blood, the sample most likely to be used stall side. The POC test has acceptable accuracy and precision in equine serum/plasma with SAA concentrations of up to at least 1000 μg/mL. Low inter-batch precision at high concentrations may affect serial measurements, and the use of the same test batch and sample type (serum/plasma or whole blood) is recommended. Comparison of results between the

  12. Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

    International Nuclear Information System (INIS)

    Maria Lina R; Andi Yasmon

    2011-01-01

    There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32 P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

  13. Analysis of two commercially available bortezomib products: differences in assay of active agent and impurity profile.

    Science.gov (United States)

    Byrn, Stephen R; Tishmack, Patrick A; Milton, Mark J; van de Velde, Helgi

    2011-06-01

    The analytical properties of two commercially available bortezomib products (VELCADE(®) and Bortenat) were compared using nuclear magnetic resonance, mass spectrometry, high-performance liquid chromatography, and gas chromatography. The data showed differences between the two products. Based on these data, Bortenat samples contained more active ingredients than indicated by the label (mean, 116.5% and 117.9% of label, in 2-mg and 3.5-mg vials, respectively). In comparison, VELCADE samples contained a mean of 99.3% of active ingredient, which was consistent with the approved specification range (US, 90-110%; EU, 95-105%). Clinical data demonstrate that patients exposed to higher than recommended doses of bortezomib on the standard twice-weekly dosing schedule are likely to have an increased risk of major toxicities. Bortenat 2-mg vials contained an isovaleraldehyde impurity; the origin of this is unknown. Additionally, the ratio of boronic acid to boronic ester differed between Bortenat 2 mg (0.27:1) and 3.5 mg (0.13:1) and VELCADE (0.10:1) samples reconstituted in saline indicating that the Bortenat product is not equivalent to the VELCADE product.

  14. Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.

    Science.gov (United States)

    Sarzotti-Kelsoe, Marcella; Daniell, Xiaoju; Todd, Christopher A; Bilska, Miroslawa; Martelli, Amanda; LaBranche, Celia; Perez, Lautaro G; Ochsenbauer, Christina; Kappes, John C; Rountree, Wes; Denny, Thomas N; Montefiori, David C

    2014-07-01

    A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Development and validation of stability indicating UPLC assay method for ziprasidone active pharma ingredient

    Directory of Open Access Journals (Sweden)

    Sonam Mittal

    2012-01-01

    Full Text Available Background: Ziprasidone, a novel antipsychotic, exhibits a potent highly selective antagonistic activity on D2 and 5HT2A receptors. Literature survey for ziprasidone revealed several analytical methods based on different techniques but no UPLC method has been reported so far. Aim: Aim of this research paper is to present a simple and rapid stability indicating isocratic, ultra performance liquid chromatographic (UPLC method which was developed and validated for the determination of ziprasidone active pharmaceutical ingredient. Forced degradation studies of ziprasidone were studied under acid, base, oxidative hydrolysis, thermal stress and photo stress conditions. Materials and Methods: The quantitative determination of ziprasidone drug was performed on a Supelco analytical column (100×2.1 mm i.d., 2.7 ΅m with 10 mM ammonium acetate buffer (pH: 6.7 and acetonitrile (ACN as mobile phase with the ratio (55:45-Buffer:ACN at a flow rate of 0.35 ml/ min. For UPLC method, UV detection was made at 318 nm and the run time was 3 min. Developed UPLC method was validated as per ICH guidelines. Results and Conclusion: Mild degradation of the drug substance was observed during oxidative hydrolysis and considerable degradation observed during basic hydrolysis. During method validation, parameters such as precision, linearity, ruggedness, stability, robustness, and specificity were evaluated, which remained within acceptable limits. Developed UPLC method was successfully applied for evaluating assay of Ziprasidone active Pharma ingredient.

  16. Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.

    Science.gov (United States)

    Domesle, Kelly J; Yang, Qianru; Hammack, Thomas S; Ge, Beilei

    2018-01-02

    Loop-mediated isothermal amplification (LAMP) has emerged as a promising alternative to PCR for pathogen detection in food testing and clinical diagnostics. This study aimed to validate a Salmonella LAMP method run on both turbidimetry (LAMP I) and fluorescence (LAMP II) platforms in representative animal food commodities. The U.S. Food and Drug Administration (FDA)'s culture-based Bacteriological Analytical Manual (BAM) method was used as the reference method and a real-time quantitative PCR (qPCR) assay was also performed. The method comparison study followed the FDA's microbiological methods validation guidelines, which align well with those from the AOAC International and ISO. Both LAMP assays were 100% specific among 300 strains (247 Salmonella of 185 serovars and 53 non-Salmonella) tested. The detection limits ranged from 1.3 to 28 cells for six Salmonella strains of various serovars. Six commodities consisting of four animal feed items (cattle feed, chicken feed, horse feed, and swine feed) and two pet food items (dry cat food and dry dog food) all yielded satisfactory results. Compared to the BAM method, the relative levels of detection (RLODs) for LAMP I ranged from 0.317 to 1 with a combined value of 0.610, while those for LAMP II ranged from 0.394 to 1.152 with a combined value of 0.783, which all fell within the acceptability limit (2.5) for an unpaired study. This also suggests that LAMP was more sensitive than the BAM method at detecting low-level Salmonella contamination in animal food and results were available 3days sooner. The performance of LAMP on both platforms was comparable to that of qPCR but notably faster, particularly LAMP II. Given the importance of Salmonella in animal food safety, the LAMP assays validated in this study holds great promise as a rapid, reliable, and robust method for routine screening of Salmonella in these commodities. Published by Elsevier B.V.

  17. Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange.

    Science.gov (United States)

    Curtis, C F

    2001-02-24

    This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable.

  18. Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2.

    Science.gov (United States)

    Jönsson, K H; Daas, A; Buchheit, K H; Terao, E

    2016-01-01

    The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable

  19. Biological dosimetry by the triage dicentric chromosome assay - Further validation of international networking

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, Ruth C., E-mail: Ruth.Wilkins@hc-sc.gc.ca [Health Canada, Ottawa, ON K1A 0K9 (Canada); Romm, Horst; Oestreicher, Ursula [Bundesamt fur Strahlenschutz, 38226 Salzgitter (Germany); Marro, Leonora [Health Canada, Ottawa, ON K1A 0K9 (Canada); Yoshida, Mitsuaki A. [Biological Dosimetry Section, Dept. of Dose Assessment, Research Center for Radiation Emergency Medicine, NIRS, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Department Radiation Biology, Institute of Radiation Emergency Medicine, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564 (Japan); Suto, Y. [Biological Dosimetry Section, Dept. of Dose Assessment, Research Center for Radiation Emergency Medicine, NIRS, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Prasanna, Pataje G.S. [National Cancer Institute, Division of Cancer Treatment and Diagnosis, Radiation Research Program, 6130 Executive Blvd., MSC 7440, Bethesda, MD 20892-7440 (United States)

    2011-09-15

    Biological dosimetry is an essential tool for estimating radiation doses received to personnel when physical dosimetry is not available or inadequate. The current preferred biodosimetry method is based on the measurement of radiation-specific dicentric chromosomes in exposed individuals' peripheral blood lymphocytes. However, this method is labor-, time- and expertise-demanding. Consequently, for mass casualty applications, strategies have been developed to increase its throughput. One such strategy is to develop validated cytogenetic biodosimetry laboratory networks, both national and international. In a previous study, the dicentric chromosome assay (DCA) was validated in our cytogenetic biodosimetry network involving five geographically dispersed laboratories. A complementary strategy to further enhance the throughput of the DCA among inter-laboratory networks is to use a triage DCA where dose assessments are made by truncating the labor-demanding and time-consuming metaphase spread analysis to 20 - 50 metaphase spreads instead of routine 500 - 1000 metaphase spread analysis. Our laboratory network also validated this triage DCA, however, these dose estimates were made using calibration curves generated in each laboratory from the blood samples irradiated in a single laboratory. In an emergency situation, dose estimates made using pre-existing calibration curves which may vary according to radiation type and dose rate and therefore influence the assessed dose. Here, we analyze the effect of using a pre-existing calibration curve on assessed dose among our network laboratories. The dose estimates were made by analyzing 1000 metaphase spreads as well as triage quality scoring and compared to actual physical doses applied to the samples for validation. The dose estimates in the laboratory partners were in good agreement with the applied physical doses and determined to be adequate for guidance in the treatment of acute radiation syndrome.

  20. Clinical evaluation of a helicase-dependant amplification (HDA)-based commercial assay for the simultaneous detection of HSV-1 and HSV-2.

    Science.gov (United States)

    Teo, Jeanette W P; Chiang, Donald; Jureen, Roland; Lin, Raymond T P

    2015-11-01

    In this study, we evaluate the performance of a commercial assay, the AmpliVue HSV 1+2 Assay (Quidel), which employs HDA for the detection of both HSV-1 and HSV-2. The assay was tested on 307 clinical specimens (genital, oral, and dermal). When compared to shell vial virus culture and immunofluorescence typing of HSV, the positive percent agreement and negative percent agreement values were 98.2% and 90.9%, respectively. Excellent assay performance was demonstrated. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay

    International Nuclear Information System (INIS)

    Ehling, G.; Hecht, M.; Heusener, A.; Huesler, J.; Gamer, A.O.; Loveren, H. van; Maurer, Th.; Riecke, K.; Ullmann, L.; Ulrich, P.; Vandebriel, R.; Vohr, H.-W.

    2005-01-01

    The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern. Similar to the EC 3 values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme

  2. Collaborative study for the validation of alternative in vitro potency assays for human tetanus immunoglobulins.

    Science.gov (United States)

    Gross, S; Janssen, S W J; de Vries, B; Terao, E; Daas, A; Buchheit, K-H

    2010-07-01

    An international collaborative study to validate 2 alternative in vitro methods for the potency testing of human tetanus immunoglobulin products was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). The study, run in the framework of the Biological Standardisation Programme (BSP) under the aegis of the European Commission and the Council of Europe, involved 21 official medicines control and industry laboratories from 15 countries. Both methods, an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA), showed good reproducibility, repeatability and precision. EIA and TIA discriminated between low, medium and high potency samples. Potency estimates correlated well and both values were in close agreement with those obtained by in vivo methods. Moreover, these alternative methods allowed to resolve discrepant results between laboratories that were due to product potency loss and reporting errors. The study demonstrated that EIA and TIA are suitable quality control methods for tetanus immunoglobulin, which can be standardised in a control laboratory using a quality assurance system. Consequently, the Group of Experts on Human Blood and Blood Products of the European Pharmacopoeia revised the monograph on human tetanus immunoglobulins to include both the methods as compendial alternatives to the in vivo mouse challenge assay. 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

  3. Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

    Science.gov (United States)

    Ivens, Katherine O; Baumert, Joseph L; Taylor, Steve L

    2016-07-01

    Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions. © 2016 Institute of Food Technologists®

  4. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  5. Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications.

    Directory of Open Access Journals (Sweden)

    Erin P Price

    Full Text Available The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ, limit of detection (LoD, linearity, ruggedness and robustness to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

  6. Complete validation of a unique digestion assay to detect Trichinella larvae in horse meat demonstrates the reliability of this assay for meeting food safety and trade requirements.

    Science.gov (United States)

    Forbes, L B; Hill, D E; Parker, S; Tessaro, S V; Gamble, H R; Gajadhar, A A

    2008-03-01

    A tissue digestion assay using a double separatory funnel procedure for the detection of Trichinella larvae in horse meat was validated for application in food safety programs and trade. The assay consisted of a pepsin-HCl digestion step to release larvae from muscle tissue and two sequential sedimentation steps in separatory funnels to recover and concentrate larvae for detection with a stereomicroscope. With defined critical control points, the assay was conducted within a quality assurance system compliant with International Organization for Standardization-International Electrotechnical Commission (ISO/IEC) 17025 guidelines. Samples used in the validation were obtained from horses experimentally infected with Trichinella spiralis to obtain a range of muscle larvae densities. One-, 5-, and 10-g samples of infected tissue were combined with 99, 95, and 90 g, respectively, of known negative horse tissue to create a 100-g sample for testing. Samples of 5 and 10 g were more likely to be positive than were 1-g samples when larval densities were less than three larvae per gram (lpg). This difference is important because ingested meat with 1 lpg is considered the threshold for clinical disease in humans. Using a 5-g sample size, all samples containing 1.3 to 2 lpg were detected, and 60 to 100% of samples with infected horse meat containing 0.1 to 0.7 lpg were detected. In this study, the double separatory funnel digestion assay was efficient and reliable for its intended use in food safety and trade. This procedure is the only digestion assay for Trichinella in horse meat that has been validated as consistent and effective at critical levels of sensitivity.

  7. Evaluation of five commercially available assays and measurement of serum total protein concentration via refractometry for the diagnosis of failure of passive transfer of immunity in foals.

    Science.gov (United States)

    Davis, Rachel; Giguère, Steeve

    2005-11-15

    To determine and compare sensitivity, specificity, accuracy, and predictive values of measurement of serum total protein concentration by refractometry as well as 5 commercially available kits for the diagnosis of failure of passive transfer (FPT) of immunity in foals. Prospective study. 65 foals with various medical problems and 35 clinically normal foals. IgG concentration in serum was assessed by use of zinc sulfate turbidity (assay C), glutaraldehyde coagulation (assay D), 2 semiquantitative immunoassays (assays F and G), and a quantitative immunoassay (assay H). Serum total protein concentration was assessed by refractometry. Radial immunodiffusion (assays A and B) was used as the reference method. For detection of IgG or = 6.0 g/dL indicated adequate IgG concentrations. Most assays were adequate as initial screening tests. However, their use as a definitive test would result in unnecessary treatment of foals with adequate IgG concentrations.

  8. Validation of secondary commercial data sources for physical activity facilities in urban and nonurban settings.

    Science.gov (United States)

    Han, Euna; Powell, Lisa; Slater, Sandy; Quinn, Christopher

    2012-11-01

    Secondary data are often necessary to assess the availability of commercial physical activity (PA) facilities and examine its association with individual behaviors and outcomes, yet the validity of such sources has been explored only in a limited number of studies. Field data were collected on the presence and attributes of commercial PA facilities in a random sample of 30 urban, 15 suburban, and 15 rural Census tracts in the Chicago metropolitan statistical area and surrounding area. Approximately 40% of PA establishments in the field data were listed for both urban and nonurban tracts in both lists except for nonurban tracts in D&B (35%), which was significantly improved in the combined list of D&B and InfoUSA. Approximately one-quarter of the PA facilities listed in D&B were found on the ground, whereas 40% to 50% of PA facilities listed in InfoUSA were found on the ground. PA establishments that offered instruction programs or lessons or that had a court or pool were less likely to be listed, particularly in the nonurban tracts. Secondary commercial business lists on PA facilities should be used with caution in assessing the built environment.

  9. Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed.

    Science.gov (United States)

    Bahrdt, C; Krech, A B; Wurz, A; Wulff, D

    2010-03-01

    For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD(abs)) GMOs in processed and unprocessed food, feed and seed samples with high efficiency.

  10. The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay.

    Directory of Open Access Journals (Sweden)

    Hansook Kim Chong

    Full Text Available Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs.

  11. Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

    Science.gov (United States)

    Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2010-06-01

    Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

  12. Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy

    Science.gov (United States)

    Lucas, Julie L.; Tacheny, Erin A.; Ferris, Allison; Galusha, Michelle; Srivastava, Apurva K.; Ganguly, Aniruddha; Williams, P. Mickey; Sachs, Michael C.; Thurin, Magdalena; Tricoli, James V.; Ricker, Winnie; Gildersleeve, Jeffrey C.

    2017-01-01

    Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-Atri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-Atri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-Atri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-Atri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93–0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay. PMID:28771597

  13. Validated high-performance liquid chromatographic method for the standardisation of Ptychopetalum olacoides Benth., Olacaceae, commercial extracts

    Directory of Open Access Journals (Sweden)

    Renata Colombo

    2010-10-01

    Full Text Available Ptychopetalum olacoides Benth., Olacaceae, popularly known as marapuama or muirapuama or miriantã, is a species native to the Amazonian region of Brazil. Extracts of the bark of the plant have been used traditionally for its stimulating and aphrodisiac properties and currently commercialised by the herbal industry as constituents in a wide range of phytomedicines. Fractionation by open column chromatography followed by preparative HPLC-UV/PAD of the stem bark and of three commercial extracts of P. olacoides allowed the isolation of three components that were common to all extracts analysed, and these were identified by NMR to be vanillic acid, protocatechuic acid and theobromine. Vanillic acid, which has been proposed as a phytochemical marker for P. olacoides, was employed as an external standard in the development and validation of a rapid qualitative and quantitative HPLC assay for the analyte. The recoveries values of the developed method were 99.02% and the LOD and LOQ values were 0.033 and 0.11 mg.L-1, respectively. The described method may be applied to the standardisation of herbs, extracts or phytomedicines commercialised as marapuama.

  14. Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Cristiana Lalli

    2015-01-01

    Full Text Available Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.

  15. Development and validation of bonded composite doubler repairs for commercial aircraft.

    Energy Technology Data Exchange (ETDEWEB)

    Roach, Dennis Patrick; Rackow, Kirk A.

    2007-07-01

    A typical aircraft can experience over 2,000 fatigue cycles (cabin pressurizations) and even greater flight hours in a single year. An unavoidable by-product of aircraft use is that crack, impact, and corrosion flaws develop throughout the aircraft's skin and substructure elements. Economic barriers to the purchase of new aircraft have placed even greater demands on efficient and safe repair methods. The use of bonded composite doublers offers the airframe manufacturers and aircraft maintenance facilities a cost effective method to safely extend the lives of their aircraft. Instead of riveting multiple steel or aluminum plates to facilitate an aircraft repair, it is now possible to bond a single Boron-Epoxy composite doubler to the damaged structure. The FAA's Airworthiness Assurance Center at Sandia National Labs (AANC), Boeing, and Federal Express completed a pilot program to validate and introduce composite doubler repair technology to the U.S. commercial aircraft industry. This project focused on repair of DC-10 fuselage structure and its primary goal was to demonstrate routine use of this repair technology using niche applications that streamline the design-to-installation process. As composite doubler repairs gradually appear in the commercial aircraft arena, successful flight operation data is being accumulated. These commercial aircraft repairs are not only demonstrating the engineering and economic advantages of composite doubler technology but they are also establishing the ability of commercial maintenance depots to safely adopt this repair technique. This report presents the array of engineering activities that were completed in order to make this technology available for widespread commercial aircraft use. Focused laboratory testing was conducted to compliment the field data and to address specific issues regarding damage tolerance and flaw growth in composite doubler repairs. Fatigue and strength tests were performed on a simulated wing

  16. Comparison and validation of ELISA assays for plasma insulin-like ...

    African Journals Online (AJOL)

    1 in the horse. ... accurate and precise of the three kits; the other two assays gave apparently much lower concentrations, with poor recovery of spiked recombinant human IGF-1 and unacceptably poor intra-assay coefficients of variation (CV).

  17. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    Science.gov (United States)

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  18. Quantifying the foodscape: A systematic review and meta-analysis of the validity of commercially available business data

    OpenAIRE

    Lebel, Alexandre; Daepp, Madeleine I. G.; Block, Jason P.; Walker, Renée; Lalonde, Benoît; Kestens, Yan; Subramanian, S. V.

    2017-01-01

    This paper reviews studies of the validity of commercially available business (CAB) data on food establishments (?the foodscape?), offering a meta-analysis of characteristics associated with CAB quality and a case study evaluating the performance of commonly-used validity indicators describing the foodscape. Existing validation studies report a broad range in CAB data quality, although most studies conclude that CAB quality is ?moderate? to ?substantial?. We conclude that current studies may ...

  19. Statistical inference for the within-device precision of quantitative measurements in assay validation.

    Science.gov (United States)

    Liu, Jen-Pei; Lu, Li-Tien; Liao, C T

    2009-09-01

    Intermediate precision is one of the most important characteristics for evaluation of precision in assay validation. The current methods for evaluation of within-device precision recommended by the Clinical Laboratory Standard Institute (CLSI) guideline EP5-A2 are based on the point estimator. On the other hand, in addition to point estimators, confidence intervals can provide a range for the within-device precision with a probability statement. Therefore, we suggest a confidence interval approach for assessment of the within-device precision. Furthermore, under the two-stage nested random-effects model recommended by the approved CLSI guideline EP5-A2, in addition to the current Satterthwaite's approximation and the modified large sample (MLS) methods, we apply the technique of generalized pivotal quantities (GPQ) to derive the confidence interval for the within-device precision. The data from the approved CLSI guideline EP5-A2 illustrate the applications of the confidence interval approach and comparison of results between the three methods. Results of a simulation study on the coverage probability and expected length of the three methods are reported. The proposed method of the GPQ-based confidence intervals is also extended to consider the between-laboratories variation for precision assessment.

  20. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation.

    Science.gov (United States)

    Onoue, Satomi; Hosoi, Kazuhiro; Wakuri, Shinobu; Iwase, Yumiko; Yamamoto, Toshinobu; Matsuoka, Naoko; Nakamura, Kazuichi; Toda, Tsuguto; Takagi, Hironori; Osaki, Naoto; Matsumoto, Yasuhiro; Kawakami, Satoru; Seto, Yoshiki; Kato, Masashi; Yamada, Shizuo; Ohno, Yasuo; Kojima, Hajime

    2013-11-01

    A reactive oxygen species (ROS) assay was previously developed for photosafety evaluation of pharmaceuticals, and the present multi-center study aimed to establish and validate a standard protocol for ROS assay. In three participating laboratories, two standards and 42 coded chemicals, including 23 phototoxins and 19 nonphototoxic drugs/chemicals, were assessed by the ROS assay according to the standardized protocol. Most phototoxins tended to generate singlet oxygen and/or superoxide under UV-vis exposure, but nonphototoxic chemicals were less photoreactive. In the ROS assay on quinine (200 µm), a typical phototoxic drug, the intra- and inter-day precisions (coefficient of variation; CV) were found to be 1.5-7.4% and 1.7-9.3%, respectively. The inter-laboratory CV for quinine averaged 15.4% for singlet oxygen and 17.0% for superoxide. The ROS assay on 42 coded chemicals (200 µm) provided no false negative predictions upon previously defined criteria as compared with the in vitro/in vivo phototoxicity, although several false positives appeared. Outcomes from the validation study were indicative of satisfactory transferability, intra- and inter-laboratory variability, and predictive capacity of the ROS assay. Copyright © 2012 John Wiley & Sons, Ltd.

  1. Validation of commercial business lists as a proxy for licensed alcohol outlets.

    Science.gov (United States)

    Carlos, Heather A; Gabrielli, Joy; Sargent, James D

    2017-05-19

    Studies of retail alcohol outlets are restricted to regions due to lack of U.S. national data. Commercial business lists (BL) offer a possible solution, but no data exists to determine if BLs could serve as an adequate proxy for license data. This paper compares geospatial measures of alcohol outlets derived from a commercial BL with license data for a large US state. We validated BL data as a measure of off-premise alcohol outlet density and proximity compared to license data for 5528 randomly selected California residential addresses. We calculated three proximity measures (Euclidean distance, road network travel time and distance) and two density measures (kernel density estimation and the count within a 2-mile radius) for each dataset. The data was acquired in 2015 and processed and analyzed in 2015 and 2016. Correlations and reliabilities between density (correlation 0.98; Cronbach's α 0.97-0.99) and proximity (correlations 0.77-0.86; α 0.87-0.92) measures were high. For proximity, BL data matched license in 55-57% of addresses, overstated distance in 19%, and understated in 24-26%. BL data can serve as a reliable proxy for licensed alcohol outlets, thus extending the work that can be performed in studies on associations between retail alcohol outlets and drinking outcomes.

  2. Validation of an optical surface plasmon resonance biosensor assay for screening (fluoro)quinolones in egg, fish and poultry

    NARCIS (Netherlands)

    Huet, A.C.; Charlier, C.; Weigel, S.; Benrejeb Godefroy, S.; Delahaut, P.

    2009-01-01

    A surface plasmon resonance biosensor immunoassay has been developed for multi-residue determination of 13 (fluoro)quinolone antibiotics in poultry meat, eggs and fish. The following performance characteristics were determined according to the guidelines laid down for screening assay validation in

  3. Validation and Optimization of an Ex Vivo Assay of Intestinal Mucosal Biopsies in Crohn's Disease

    DEFF Research Database (Denmark)

    Vadstrup, Kasper; Galsgaard, Elisabeth Douglas; Gerwien, Jens

    2016-01-01

    human explant method to test drug candidates and pathophysiological conditions in CD intestinal biopsies. Mucosal biopsies from 27 CD patients and 6 healthy individuals were collected to validate an explant assay test where the polarized tissue was cultured on a novel metal mesh disk, slightly immersed...

  4. Comparison and validation of ELISA assays for plasma insulin-like growth factor-1 in the horse

    Directory of Open Access Journals (Sweden)

    Courtnay L. Baskerville

    2017-03-01

    Full Text Available Insulin-like growth factor-1 (IGF-1 plays several important physiological roles, and IGF-related pathways have been implicated in developmental osteochondral disease and endocrinopathic laminitis. This factor is also a downstream marker of growth hormone activity and its peptide mimetics. Unfortunately, previously used assays for measuring equine IGF-1 (radioimmunoassays and ELISAs are no longer commercially available, and many of the kits on the market give poor results when used on horse samples. The aim of the present study was to compare three different ELISA assays (two human and one horse-specific. Plasma samples from six Standardbreds, six ponies and six Andalusians were used. The human IGF-1 ELISA kit from Immunodiagnostic Systems (IDS proved to be the most accurate and precise of the three kits; the other two assays gave apparently much lower concentrations, with poor recovery of spiked recombinant human IGF-1 and unacceptably poor intra-assay coefficients of variation (CV. The IDS assay gave an intra-assay CV of 3.59 % and inter-assay CV of 7.31%. Mean percentage recovery of spiked IGF-1 was 88.82%, and linearity and dilutional parallelism were satisfied. The IGF-1 plasma concentrations were 123.21 ±8.24 ng/mL for Standardbreds, 124.95 ±3.69 ng/mL for Andalusians and 174.26 ±1.94 ng/mL for ponies. Therefore of the three assays assessed, the IGF-1 ELISA manufactured by IDS was the most suitable for use with equine plasma samples and may have many useful applications in several different research areas. However, caution should be used when comparing equine studies where different analytical techniques and assays may have been used to measure this growth factor.

  5. OECD validation of the Hershberger assay in Japan: phase 2 dose response of methyltestosterone, vinclozolin, and p,p'-DDE.

    Science.gov (United States)

    Yamasaki, Kanji; Sawaki, Masakuni; Ohta, Ryo; Okuda, Hirokazu; Katayama, Seiichi; Yamada, Tomoya; Ohta, Takafumi; Kosaka, Tadashi; Owens, William

    2003-01-01

    The Organisation for Economic Co-operation and Development has initiated the development of new guidelines for the screening and testing of potential endocrine disruptors. The Hershberger assay is one of the assays selected for validation based on the need for in vivo screening to detect androgen agonists or antagonists by measuring the response of five sex accessory organs and tissues of castrated juvenile male rats: the ventral prostate, the seminal vesicles with coagulating glands, the levator ani and bulbocavernosus muscle complex, the Cowper's glands, and the glans penis. The phase 1 feasibility demonstration stage of the Hershberger validation program has been successfully completed with a single androgen agonist and a single antagonist as reference substances. The phase 2 validation program employs a range of additional androgen agonists and antagonists as well as 5alpha-reductase inhibitors. Seven Japanese laboratories have contributed phase 2 validation studies of the Hershberger assay using methyltestosterone, vinclozolin, and 2,2-bis (4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE). The methyltestosterone doses were 0, 0.05, 0.5, 5, and 50 mg/kg/day, and the vinclozolin and p,p'-DDE doses were 0, 3, 10, 30, and 100 mg/kg/day. All chemicals were orally administered by gavage for 10 consecutive days. In the antagonist version of the assay using vinclozolin and p,p'-DDE, 0.2 mg/kg/day of testosterone propionate was coadministered by subcutaneous injection. All five accessory sex preproductive organs and tissues consistently responded with statistically significant changes in weight within a narrow window. Therefore, the Japanese studies support the Hershberger assay as a reliable and reproducible screening assay for the detection of androgen agonistic and antagonistic effects. PMID:14644666

  6. Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators.

    Science.gov (United States)

    Onoue, Satomi; Hosoi, Kazuhiro; Toda, Tsuguto; Takagi, Hironori; Osaki, Naoto; Matsumoto, Yasuhiro; Kawakami, Satoru; Wakuri, Shinobu; Iwase, Yumiko; Yamamoto, Toshinobu; Nakamura, Kazuichi; Ohno, Yasuo; Kojima, Hajime

    2014-06-01

    A previous multi-center validation study demonstrated high transferability and reliability of reactive oxygen species (ROS) assay for photosafety evaluation. The present validation study was undertaken to verify further the applicability of different solar simulators and assay performance. In 7 participating laboratories, 2 standards and 42 coded chemicals, including 23 phototoxins and 19 non-phototoxic drugs/chemicals, were assessed by the ROS assay using two different solar simulators (Atlas Suntest CPS series, 3 labs; and Seric SXL-2500V2, 4 labs). Irradiation conditions could be optimized using quinine and sulisobenzone as positive and negative standards to offer consistent assay outcomes. In both solar simulators, the intra- and inter-day precisions (coefficient of variation; CV) for quinine were found to be below 10%. The inter-laboratory CV for quinine averaged 15.4% (Atlas Suntest CPS) and 13.2% (Seric SXL-2500V2) for singlet oxygen and 17.0% (Atlas Suntest CPS) and 7.1% (Seric SXL-2500V2) for superoxide, suggesting high inter-laboratory reproducibility even though different solar simulators were employed for the ROS assay. In the ROS assay on 42 coded chemicals, some chemicals (ca. 19-29%) were unevaluable because of limited solubility and spectral interference. Although several false positives appeared with positive predictivity of ca. 76-92% (Atlas Suntest CPS) and ca. 75-84% (Seric SXL-2500V2), there were no false negative predictions in both solar simulators. A multi-center validation study on the ROS assay demonstrated satisfactory transferability, accuracy, precision, and predictivity, as well as the availability of other solar simulators. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Prospective Validation of a 21-Gene Expression Assay in Breast Cancer.

    Science.gov (United States)

    Sparano, Joseph A; Gray, Robert J; Makower, Della F; Pritchard, Kathleen I; Albain, Kathy S; Hayes, Daniel F; Geyer, Charles E; Dees, Elizabeth C; Perez, Edith A; Olson, John A; Zujewski, JoAnne; Lively, Tracy; Badve, Sunil S; Saphner, Thomas J; Wagner, Lynne I; Whelan, Timothy J; Ellis, Matthew J; Paik, Soonmyung; Wood, William C; Ravdin, Peter; Keane, Maccon M; Gomez Moreno, Henry L; Reddy, Pavan S; Goggins, Timothy F; Mayer, Ingrid A; Brufsky, Adam M; Toppmeyer, Deborah L; Kaklamani, Virginia G; Atkins, James N; Berenberg, Jeffrey L; Sledge, George W

    2015-11-19

    Prior studies with the use of a prospective-retrospective design including archival tumor samples have shown that gene-expression assays provide clinically useful prognostic information. However, a prospectively conducted study in a uniformly treated population provides the highest level of evidence supporting the clinical validity and usefulness of a biomarker. We performed a prospective trial involving women with hormone-receptor-positive, human epidermal growth factor receptor type 2 (HER2)-negative, axillary node-negative breast cancer with tumors of 1.1 to 5.0 cm in the greatest dimension (or 0.6 to 1.0 cm in the greatest dimension and intermediate or high tumor grade) who met established guidelines for the consideration of adjuvant chemotherapy on the basis of clinicopathologic features. A reverse-transcriptase-polymerase-chain-reaction assay of 21 genes was performed on the paraffin-embedded tumor tissue, and the results were used to calculate a score indicating the risk of breast-cancer recurrence; patients were assigned to receive endocrine therapy without chemotherapy if they had a recurrence score of 0 to 10, indicating a very low risk of recurrence (on a scale of 0 to 100, with higher scores indicating a greater risk of recurrence). Of the 10,253 eligible women enrolled, 1626 women (15.9%) who had a recurrence score of 0 to 10 were assigned to receive endocrine therapy alone without chemotherapy. At 5 years, in this patient population, the rate of invasive disease-free survival was 93.8% (95% confidence interval [CI], 92.4 to 94.9), the rate of freedom from recurrence of breast cancer at a distant site was 99.3% (95% CI, 98.7 to 99.6), the rate of freedom from recurrence of breast cancer at a distant or local-regional site was 98.7% (95% CI, 97.9 to 99.2), and the rate of overall survival was 98.0% (95% CI, 97.1 to 98.6). Among patients with hormone-receptor-positive, HER2-negative, axillary node-negative breast cancer who met established guidelines for

  8. Field validation of food service listings: a comparison of commercial and online geographic information system databases.

    Science.gov (United States)

    Seliske, Laura; Pickett, William; Bates, Rebecca; Janssen, Ian

    2012-08-01

    Many studies examining the food retail environment rely on geographic information system (GIS) databases for location information. The purpose of this study was to validate information provided by two GIS databases, comparing the positional accuracy of food service places within a 1 km circular buffer surrounding 34 schools in Ontario, Canada. A commercial database (InfoCanada) and an online database (Yellow Pages) provided the addresses of food service places. Actual locations were measured using a global positioning system (GPS) device. The InfoCanada and Yellow Pages GIS databases provided the locations for 973 and 675 food service places, respectively. Overall, 749 (77.1%) and 595 (88.2%) of these were located in the field. The online database had a higher proportion of food service places found in the field. The GIS locations of 25% of the food service places were located within approximately 15 m of their actual location, 50% were within 25 m, and 75% were within 50 m. This validation study provided a detailed assessment of errors in the measurement of the location of food service places in the two databases. The location information was more accurate for the online database, however, when matching criteria were more conservative, there were no observed differences in error between the databases.

  9. In Vitro Dissolution Profile of Dapagliflozin: Development, Method Validation, and Analysis of Commercial Tablets

    Directory of Open Access Journals (Sweden)

    Rafaela Zielinski Cavalheiro de Meira

    2017-01-01

    Full Text Available Dapagliflozin was the first of its class (inhibitors of sodium-glucose cotransporter to be approved in Europe, USA, and Brazil. As the drug was recently approved, there is the need for research on analytical methods, including dissolution studies for the quality evaluation and assurance of tablets. The dissolution methodology was developed with apparatus II (paddle in 900 mL of medium (simulated gastric fluid, pH 1.2, temperature set at 37±0.5°C, and stirring speed of 50 rpm. For the quantification, a spectrophotometric (λ=224 nm method was developed and validated. In validation studies, the method proved to be specific and linear in the range from 0.5 to 15 μg·mL−1 (r2=0.998. The precision showed results with RSD values lower than 2%. The recovery of 80.72, 98.47, and 119.41% proved the accuracy of the method. Through a systematic approach by applying Factorial 23, the robustness of the method was confirmed (p>0.05. The studies of commercial tablets containing 5 or 10 mg demonstrated that they could be considered similar through f1, f2, and dissolution efficiency analyses. Also, the developed method can be used for the quality evaluation of dapagliflozin tablets and can be considered as a scientific basis for future official pharmacopoeial methods.

  10. Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

    Science.gov (United States)

    Omori, Takashi; Idehara, Kenji; Kojima, Hajime; Sozu, Takashi; Arima, Kazunori; Goto, Hirohiko; Hanada, Tomohiko; Ikarashi, Yoshiaki; Inoda, Taketo; Kanazawa, Yukiko; Kosaka, Tadashi; Maki, Eiji; Morimoto, Takashi; Shinoda, Shinsuke; Shinoda, Naoki; Takeyoshi, Masahiro; Tanaka, Masashi; Uratani, Mamoru; Usami, Masahito; Yamanaka, Atsushi; Yoneda, Tomofumi; Yoshimura, Isao; Yuasa, Atsuko

    2008-01-01

    The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

  11. A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

    Science.gov (United States)

    Chiaravalli, Jeanne; Glickman, J Fraser

    2017-08-01

    We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

  12. Real-time polymerase chain reaction detection of parvovirus B19 DNA in blood donations using a commercial and an in-house assay.

    Science.gov (United States)

    Koppelman, M H G M; van Swieten, P; Cuijpers, H T M

    2011-06-01

    European regulations require testing of manufacturing plasma for parvovirus B19 (B19) DNA to limit the load of this virus to a maximum acceptable level of 10 IU/µL. To meet this requirement, most manufacturers introduced a test algorithm to identify and eliminate high-load donations before making large manufacturing pools of plasma units. Sanquin screens all donations using a commercial assay from Roche and an in-house assay. Between 2006 and 2009, 6.2 million donations were screened using two different polymerase chain reaction (PCR) assays targeting B19 DNA. Donations with B19 DNA loads of greater than 1 × 10(6) IU/mL showing significant differences in viral load between the two assays were further analyzed by sequencing analysis. A total of 396 donations with B19 DNA loads of greater than 1 × 10(6) IU/mL were identified. Fifteen samples (3.8%) had discordant test results; 10 samples (2.5%) were underquantified by the Roche assay, two samples (0.5%) were underquantified by the in-house assay, and three samples (0.8%) were not detected by the Roche assay. Sequencing analysis revealed mismatches in primer and probe-binding regions. Phylogenetic analysis showed that 12 samples were B19 Genotype 1. The three samples not detected by the Roche assay were B19 Genotype 2. This study shows that 3.8% of the viremic B19 DNA-positive donations are not quantified correctly by the Roche or in-house B19 DNA assays. B19 Genotype 1 isolates showing incorrect test results are more common than B19 Genotype 2 or 3 isolates. Newly designed B19 PCR assays for blood screening should preferably have multiplexed formats targeting multiple regions of the B19 genome. © 2010 American Association of Blood Banks.

  13. Performance of 2 commercial real-time polymerase chain reaction assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients.

    Science.gov (United States)

    Orsi, Carlotta Francesca; Gennari, William; Venturelli, Claudia; La Regina, Annunziata; Pecorari, Monica; Righi, Elena; Machetti, Marco; Blasi, Elisabetta

    2012-06-01

    This article investigates the performance of 2 commercial real-time polymerase chain reaction (PCR) assays, MycAssay™ Aspergillus (Myc(Asp)Assay) and MycAssay™ Pneumocystis (Myc(PCP)Assay), on the ABI 7300 platform for the detection of Aspergillus (Asp) or Pneumocystis jirovecii (Pj) DNA in bronchoalveolar lavage (BAL) samples from 20 patients. Operationally, patients enrolled were clustered into 3 groups: invasive aspergillosis group (IA, 7 patients), Pj pneumonia group (PCP, 8 patients), and negative control group (5 patients). All the IA patients were Myc(Asp)Assay positive, whereas 12 non-IA patients returned negative PCR results. Furthermore, 7 of 8 PCP patients were Myc(PCP)Assay positive, while 9 non-PCP patients were PCR negative. In conclusion, these data provide an early indication of the effectiveness of both the Myc(Asp)Assay and Myc(PCP)Assay on the ABI 7300 platform for the detection of either Asp or Pj DNA in BAL from patients with deep fungal infections. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

    Science.gov (United States)

    Harvey, John J; Chester, Stephanie; Burke, Stephen A; Ansbro, Marisela; Aden, Tricia; Gose, Remedios; Sciulli, Rebecca; Bai, Jing; DesJardin, Lucy; Benfer, Jeffrey L; Hall, Joshua; Smole, Sandra; Doan, Kimberly; Popowich, Michael D; St George, Kirsten; Quinlan, Tammy; Halse, Tanya A; Li, Zhen; Pérez-Osorio, Ailyn C; Glover, William A; Russell, Denny; Reisdorf, Erik; Whyte, Thomas; Whitaker, Brett; Hatcher, Cynthia; Srinivasan, Velusamy; Tatti, Kathleen; Tondella, Maria Lucia; Wang, Xin; Winchell, Jonas M; Mayer, Leonard W; Jernigan, Daniel; Mawle, Alison C

    2016-02-01

    In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. An Improved Neutral a-Glucosidase Assay for Assessment of Epididymal Function—Validation and Comparison to the WHO Method

    Directory of Open Access Journals (Sweden)

    Frank Eertmans

    2014-01-01

    Full Text Available Neutral a-glucosidase (NAG activity in human seminal plasma is an important indicator for epididymis functionality. In the present study, the classic World Health Organization (WHO method has been adapted to enhance assay robustness. Changes include modified enzyme reaction buffer composition and usage of an alternative enzyme inhibitor for background correction (glucose instead of castanospermine. Both methods have been tested in parallel on 144 semen samples, obtained from 94 patients/donors and 50 vasectomized men (negative control, respectively. Passing-Bablok regression analysis demonstrated equal assay performance. In terms of assay validation, analytical specificity, detection limit, measuring range, precision, and cut-off values have been calculated. These data confirm that the adapted method is a reliable, improved tool for NAG analysis in human semen.

  16. Further Development and Validation of the Frog Embryo Teratogenesis Assay - Xenopus (FETAX). Phase III

    National Research Council Canada - National Science Library

    Bantle, John

    1996-01-01

    This interlaboratory study of the Frog Embryo Teratogenesis Assay (FETAX) was undertaken in order to assess the repeatability and reliability of data collected under the guide published by the American Society for Testing and Materials...

  17. Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine. Part 3: Technical Validation of Immunohistochemistry (IHC) Assays in Clinical IHC Laboratories.

    Science.gov (United States)

    Torlakovic, Emina E; Cheung, Carol C; D'Arrigo, Corrado; Dietel, Manfred; Francis, Glenn D; Gilks, C Blake; Hall, Jacqueline A; Hornick, Jason L; Ibrahim, Merdol; Marchetti, Antonio; Miller, Keith; van Krieken, J Han; Nielsen, Soren; Swanson, Paul E; Vyberg, Mogens; Zhou, Xiaoge; Taylor, Clive R

    2017-03-01

    Validation of immunohistochemistry (IHC) assays is a subject that is of great importance to clinical practice as well as basic research and clinical trials. When applied to clinical practice and focused on patient safety, validation of IHC assays creates objective evidence that IHC assays used for patient care are "fit-for-purpose." Validation of IHC assays needs to be properly informed by and modeled to assess the purpose of the IHC assay, which will further determine what sphere of validation is required, as well as the scope, type, and tier of technical validation. These concepts will be defined in this review, part 3 of the 4-part series "Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine."

  18. Evaluation of the specificity of antigen assays for plasminogen activator inhibitor 1 : Comparison of two new commercial kits

    NARCIS (Netherlands)

    Huisman, L.G.M.; Meijer, P.; Griensven, J. van; Kluft, C.

    1992-01-01

    t-PA depleted citrated plasma was used to prepare standards of different molecular forms of plasminogen activator inhibitor 1 (PAI-1). These standards were used to evaluate the specificity of two new PAI-1 antigen assays: the TintElize PAI-1 antigen assay (cat. no. 210221) and the Innotest PAI-1.

  19. Validation of commercially available automated canine-specific immunoturbidimetric method for measuring canine C-reactive protein

    DEFF Research Database (Denmark)

    Hillström, Anna; Hagman, Ragnvi; Tvedten, Harold

    2014-01-01

    BACKGROUND: Measurement of C-reactive protein (CRP) is used for diagnosing and monitoring systemic inflammatory disease in canine patients. An automated human immunoturbidimetric assay has been validated for measuring canine CRP, but cross-reactivity with canine CRP is unpredictable. OBJECTIVE......: The purpose of the study was to validate a new automated canine-specific immunoturbidimetric CRP method (Gentian cCRP). METHODS: Studies of imprecision, accuracy, prozone effect, interference, limit of quantification, and stability under different storage conditions were performed. The new method was compared...... with a human CRP assay previously validated for canine CRP determination. Samples from 40 healthy dogs were analyzed to establish a reference interval. RESULTS: Total imprecision was

  20. Transcriptome-Wide Single Nucleotide Polymorphisms (SNPs for Abalone (Haliotis midae: Validation and Application Using GoldenGate Medium-Throughput Genotyping Assays

    Directory of Open Access Journals (Sweden)

    Rouvay Roodt-Wilding

    2013-09-01

    Full Text Available Haliotis midae is one of the most valuable commercial abalone species in the world, but is highly vulnerable, due to exploitation, habitat destruction and predation. In order to preserve wild and cultured stocks, genetic management and improvement of the species has become crucial. Fundamental to this is the availability and employment of molecular markers, such as microsatellites and Single Nucleotide Polymorphisms (SNPs . Transcriptome sequences generated through sequencing-by-synthesis technology were utilized for the in vitro and in silico identification of 505 putative SNPs from a total of 316 selected contigs. A subset of 234 SNPs were further validated and characterized in wild and cultured abalone using two Illumina GoldenGate genotyping assays. Combined with VeraCode technology, this genotyping platform yielded a 65%−69% conversion rate (percentage polymorphic markers with a global genotyping success rate of 76%−85% and provided a viable means for validating SNP markers in a non-model species. The utility of 31 of the validated SNPs in population structure analysis was confirmed, while a large number of SNPs (174 were shown to be informative and are, thus, good candidates for linkage map construction. The non-synonymous SNPs (50 located in coding regions of genes that showed similarities with known proteins will also be useful for genetic applications, such as the marker-assisted selection of genes of relevance to abalone aquaculture.

  1. Transcriptome-wide single nucleotide polymorphisms (SNPs) for abalone (Haliotis midae): validation and application using GoldenGate medium-throughput genotyping assays.

    Science.gov (United States)

    Bester-Van Der Merwe, Aletta; Blaauw, Sonja; Du Plessis, Jana; Roodt-Wilding, Rouvay

    2013-09-23

    Haliotis midae is one of the most valuable commercial abalone species in the world, but is highly vulnerable, due to exploitation, habitat destruction and predation. In order to preserve wild and cultured stocks, genetic management and improvement of the species has become crucial. Fundamental to this is the availability and employment of molecular markers, such as microsatellites and single nucleotide (SNPs). Transcriptome sequences generated through sequencing-by-synthesis technology were utilized for the in vitro and in silico identification of 505 putative SNPs from a total of 316 selected contigs. A subset of 234 SNPs were further validated and characterized in wild and cultured abalone using two Illumina GoldenGate genotyping assays. Combined with VeraCode technology, this genotyping platform yielded a 65%-69% conversion rate (percentage polymorphic markers) with a global genotyping success rate of 76%-85% and provided a viable means for validating SNP markers in a non-model species. The utility of 31 of the validated SNPs in population structure analysis was confirmed, while a large number of SNPs (174) were shown to be informative and are, thus, good candidates for linkage map construction. The non-synonymous SNPs (50) located in coding regions of genes that showed similarities with known proteins will also be useful for genetic applications, such as the marker-assisted selection of genes of relevance to abalone aquaculture.

  2. Comparison of IgM Capture Enzyme- Linked Immunosorbent Assay (ELISA) using Inhouse method and commercially available MRL kit for serological confirmation of dengue infection

    International Nuclear Information System (INIS)

    Hapugoda, D.M.; De Silva R, Nilanthi; Abeywickreme, W.; Gunasena, Sunethra; Prithimala, L.D.; Jayawardene, S.L.G.J.; Kumari, Thamara

    2003-01-01

    Laboratory diagnosis of dengue infection is important for the management of the patients. In this study igM capture ELISA using an inhouse method and commercially available kit (MRL diagnostics,USA) was compared to detect diagnostic capability of Inhouse IgM ELISA for provision of diagnostic facilities to the public at an affordable cost. Eighty acute and convalescent serum samples were collected from serologically confirmed dengue patients. Serological confirmation of patients were performed by Haemagglutination Inhibition (HI) assay, gold standard assay for dengue on paired serum samples. All collected acute and convalescent sera were tested by IgM ELISA using the inhouse method and MRL kit. Antigen and conjugate for the inhouse IgM method were prepared in the laboratory. A cocktail of four dengue antigens containing 25 Antigen ELISA units of each type was prepared and used as the assay antigen. Conjugate was prepared using a serum sample with high dengue Anti flavi IgG antibody titre conjugated with Horseradish peroxidase. A prospective study of both IgM ELISA assays were performed using 113 acute sera collected from dengue suspected cases. Overall results showed that 46% and 52% acute sera collected from dengue confirmed patients were positive by inhouse ELISA assay and MRL kits respectively. In the prospective study done using acute sera collected from dengue suspected patients showed that 44% and 52% were positive by inhouse ELISA assay and MRL kits. There was no significant difference in positivity between these two assays. (P=0.18). Inhouse IgM ELISA can be used for provision of laboratory diagnosis of dengue virus infection more than 5 days. The assay is 10 times less costly than using MRL kits as assay antigen and conjugate can be prepared easily in the laboratory

  3. ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

    Science.gov (United States)

    Oldaker, Teri; Whitby, Liam; Saber, Maryam; Holden, Jeannine; Wallace, Paul K; Litwin, Virginia

    2018-01-01

    Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  4. Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.

    Science.gov (United States)

    Vardakou, Maria; Salmon, Melissa; Faraldos, Juan A; O'Maille, Paul E

    2014-01-01

    Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography-mass spectrometry (GC-MS). In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.•We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs.•We validate the method by direct comparison with established assays. The agreement of k cat/K M among methods makes this adaptation optimal for rapid evaluation of TPSs.•We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries.

  5. Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

    Science.gov (United States)

    van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

    2016-01-01

    Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels. © 2015 John Wiley & Sons Ltd.

  6. Analytic validation and comparison of three commercial immunoassays for measurement of plasma atrial/A-type natriuretic peptide concentration in horses

    DEFF Research Database (Denmark)

    Trachsel, D S; Schwarzwald, C C; Grenacher, B

    2014-01-01

    Measurement of atrial/A-type natriuretic peptide (ANP) concentrations may be of use for assessment of cardiac disease, and reliable data on the analytic performance of available assays are needed. To assess the suitability for clinical use of commercially available ANP assays, intra-assay and inter......-Altman analyses. For all assays, precision was moderate but acceptable and dilution parallelism was good. All assays showed analytic performance similar to other immunoassays used in veterinary medicine. However, the results from the three assays were poorly comparable. Our study highlights the need...

  7. Comparison of one commercial and two in-house TaqMan multiplex real-time PCR assays for detection of enteropathogenic, enterotoxigenic and enteroaggregative Escherichia coli.

    Science.gov (United States)

    Hahn, Andreas; Luetgehetmann, Marc; Landt, Olfert; Schwarz, Norbert Georg; Frickmann, Hagen

    2017-11-01

    Enteropathogenic, enterotoxigenic and enteroaggregative Escherichia coli (EPEC, ETEC, EAEC) are among the most frequent causes of diarrhoea during travel or on military deployments. Cost-efficient and reliable real-time multiplex PCR (mPCR) assays are desirable for surveillance or point prevalence studies in remote and resource-limited tropical settings. We compared one commercial PCR kit and two in-house assays without using a gold standard to estimate sensitivity and specificity of each assay. Residual materials from nucleic acid extractions of stool samples from two groups with presumably different prevalences and increased likelihood of being infected or colonised by diarrhoeagenic E. coli were included in the assessment. One group comprised samples from returnees from tropical deployments, the second group was of migrants and study participants from high-endemicity settings. Each sample was assessed with all of the PCR assays. Cycle threshold (Ct) values were descriptively compared. The calculated sensitivities for the commercial test vs. the in-house tests were for EPEC 0.84 vs. 0.89 and 0.96, for ETEC 0.83 vs. 0.76 and 0.61, and for EAEC 0.69 vs. 0.54 and 0.69. False positive results were rare - specificity was 0.94 and 0.97 for two EPEC tests and 1.0 for all other tests. Most positive samples had late Ct values corresponding to low quantities of pathogens. Discordant test results were associated with late Ct values. As commercial and in-house assays showed comparable results, in-house tests can be assumed to be safe while affording considerable savings, making them a valuable alternative for surveillance testing in resource-limited tropical areas. © 2017 John Wiley & Sons Ltd.

  8. Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

    2017-10-01

    A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection almond, limit of quantification almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers. © 2017 Institute of Food Technologists®.

  9. Development and validation of a quantitative PCR assay for Ichthyophonus spp.

    Science.gov (United States)

    White, Vanessa C; Morado, J Frank; Crosson, Lisa M; Vadopalas, Brent; Friedman, Carolyn S

    2013-04-29

    Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

  10. Evaluation of 4,4'-diaminodiphenyl ether in the rat comet assay: Part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay.

    Science.gov (United States)

    Priestley, Catherine C; Walker, Joanne S; O'Donovan, Michael R; Doherty, Ann T

    2015-07-01

    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, 4,4'-diaminodiphenyl ether (DPE), a known rodent genotoxic carcinogen, was tested in this laboratory. Sprague Dawley rats (7-9 weeks of age) were given three oral doses of DPE, 24 and 21 h apart and liver or stomach sampled 3h after the final dose. Under the conditions of the test, no increases in DNA damage in liver and stomach were observed with DPE (up to 200 mg/kg/day). A dose-dependent decrease in DNA migration, compared to vehicle controls, was noted for DPE in rat stomach. Further analysis is required to elucidate fully whether this decrease is a consequence of the mode of action or due to the toxicity of DPE. What is perhaps surprising is the inability of the comet assay to detect a known rat genotoxic carcinogen in liver. Further investigation is needed to clarify whether this apparent lack of response results from limited tissue exposure or metabolic differences between species. This finding highlights a need for careful consideration of study design when evaluating assay performance as a measure of in vivo genotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Lessons Learned: Transfer of the High-Definition Circulating Tumor Cell Assay Platform to Development as a Commercialized Clinical Assay Platform.

    Science.gov (United States)

    Kuhn, P; Keating, S M; Baxter, G T; Thomas, K; Kolatkar, A; Sigman, C C

    2017-11-01

    Planning and transfer of a new technology platform developed in an academic setting to a start-up company for medical diagnostic product development may appear daunting and costly in terms of complexity, time, and resources. In this review we outline the key steps taken and lessons learned when a technology platform developed in an academic setting was transferred to a start-up company for medical diagnostic product development in the interest of elucidating development toolkits for academic groups and small start-up companies starting on the path to commercialization and regulatory approval. © 2017, The American Society for Clinical Pharmacology and Therapeutics.

  12. Limited diagnostic capacities of two commercial assays for the detection of Leptospira immunoglobulin M antibodies in Laos

    NARCIS (Netherlands)

    Blacksell, Stuart D.; Smythe, Lee; Phetsouvanh, Rattanaphone; Dohnt, Michael; Hartskeerl, Rudy; SymondS, Meegan; Slack, Andrew; Vongsouvath, Manivanh; Davong, Viengmone; Lattana, Olay; Phongmany, Simmaly; Keolouangkot, Valy; White, Nicholas J.; Day, Nicholas P. J.; Newton, Paul N.

    2006-01-01

    The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using

  13. The validation of waste assay systems during active test at Rokkasho Reprocessing Plant

    International Nuclear Information System (INIS)

    Tamura, Takayuki; Miura, Yasushi; Iwamoto, Tomonori

    2007-01-01

    In order to implement accurate material accountancy at Rokkasho Reprocessing Plant (RRP) as a large scale reprocessing plant, it is necessary to introduce accurate measurement systems not only for mainstream material, but also appropriate measurement systems for solid waste materials. In this sense, the generated wastes by the active test operation have been measured with the Non-Destructive Assay Systems, such as Rokkasho Hulls Measurement System (RHMS) and Waste Crate Assay System (WCAS) for accountancy. This paper describes the experience of the NDA operation and the evaluation results for accountancy. (author)

  14. What is required for the validation of in vitro assays for predicting contaminant relative bioavailability? Considerations and criteria

    International Nuclear Information System (INIS)

    Juhasz, Albert L.; Basta, Nicholas T.; Smith, Euan

    2013-01-01

    A number of studies have shown the potential of in vitro assays to predict contaminant in vivo relative bioavailability in order to refine human health exposure assessment. Although the term ‘validated’ has been used to describe the goodness of fit between in vivo and in vitro observations, its misuse has arisen from semantic considerations in addition to the lack of defined criteria for establishing performance validation. While several internal validation methods may be utilised, performance validation should preferably focus on assessing the agreement of model predictions with a set of data which are independent of those used to construct the model. In order to achieve robust validated predictive models, a number of parameters (e.g. size of data set, source of independent soils, contaminant concentration range, animal model, relative bioavailability endpoint) need to be considered in addition to defined criteria for establishing performance validation which are currently lacking. -- Defined criteria for establishing in vivo–in vitro performance validation are required in order to ensure robust, defensible predictive models for human health exposure assessment

  15. Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

    Directory of Open Access Journals (Sweden)

    Alexandra Ibáñez Escribano

    2012-08-01

    Full Text Available A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL. The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

  16. Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)

    Science.gov (United States)

    Ferrante, Jason; Hunter, Margaret; Wellehan, James F.X.

    2018-01-01

    Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher (Puse as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

  17. Validation and modification of dried blood spot-based glycosylated hemoglobin assay for the longitudinal aging study in India.

    Science.gov (United States)

    Hu, Peifeng; Edenfield, Michael; Potter, Alan; Kale, Varsha; Risbud, Arun; Williams, Sharon; Lee, Jinkook; Bloom, David E; Crimmins, Eileen; Seeman, Teresa

    2015-01-01

    This study aims to validate a modified dried blood spot (DBS)-based glycosylated hemoglobin (HbA1c) assay protocol, after a pretest in India showed poor correlation between the original DBS-based protocol and venous results. The original protocol was tested on different chemistry analyzers and then simplified at the University of Washington (UW). A second pretest was conducted in India to validate the modified assay protocol, using 44 quality control specimens. Data from UW indicated that, using the original protocol, the correlation coefficients between DBS and venous results were above 0.98 on both Bio-Rad and Olympus chemistry analyzers. The protocol worked equally well on filter paper, with or without pre-treatment, and when the recommended amount of blood spot material, or less, was used. A second pretest of the modified protocol confirmed that DBS-based levels from both Olympus and Roche chemistry analyzers were well correlated with DBS results from UW (correlation coefficients were above 0.96), as well as with venous values (correlation coefficients were above 0.94). The DBS-based HbA1c values are highly correlated with venous results. The pre-treatment of filter paper does not appear to be necessary. The poor results from the first pretest are probably due to factors unrelated to the protocol, such as problems with the chemistry analyzer or assay reagents. © 2015 Wiley Periodicals, Inc.

  18. Selecting postoperative adjuvant systemic therapy for early stage breast cancer: A critical assessment of commercially available gene expression assays

    Science.gov (United States)

    Schuur, Eric; Angel Aristizabal, Javier; Bargallo Rocha, Juan Enrique; Cabello, Cesar; Elizalde, Roberto; García‐Estévez, Laura; Gomez, Henry L.; Katz, Artur; Nuñez De Pierro, Aníbal

    2017-01-01

    Risk stratification of patients with early stage breast cancer may support adjuvant chemotherapy decision‐making. This review details the development and validation of six multi‐gene classifiers, each of which claims to provide useful prognostic and possibly predictive information for early stage breast cancer patients. A careful assessment is presented of each test's analytical validity, clinical validity, and clinical utility, as well as the quality of evidence supporting its use. PMID:28211064

  19. Validation of Non-Invasive Waste Assay System (Gamma Box Counter) Performance at AECL Whiteshell Laboratories - 13136

    International Nuclear Information System (INIS)

    Attas, E.M.; Bialas, E.; Rhodes, M.J.

    2013-01-01

    Low-level radioactive waste (LLW) in solid form, resulting from decommissioning and operations activities at AECL's Whiteshell Laboratories (WL), is packaged in B-25 and B-1000 standard waste containers and characterized before it is shipped to an on-site interim storage facility, pending AECL decisions on long term management of its LLW. Assay of the waste packages before shipment contributes to an inventory of the interim storage facility and provides data to support acceptance at a future repository. A key characterization step is a gamma spectrometric measurement carried out under standard conditions using an automated, multi-detector Waste Assay System (WAS), purchased from Antech Corporation. A combination of ORTEC gamma acquisition software and custom software is used in this system to incorporate multiple measurements from two collimated high-resolution detectors. The software corrects the intensities of the gamma spectral lines for geometry and attenuation, and generates a table of calculated activities or limits of detection for a user-defined list of radioisotopes that may potentially be present. Validation of WAS performance was a prerequisite to routine operation. Documentation of the validation process provides assurance of the quality of the results produced, which may be needed one or two decades after they were generated. Aspects of the validation included setting up a quality control routine, measurements of standard point sources in reproducible positions, study of the gamma background, optimization of user-selectable software parameters, investigation of the effect of non-uniform distribution of materials and radionuclides, and comparison of results with measurements made using other gamma detector systems designed to assay bulk materials. The following key components of the validation process have been established. A daily quality control routine has been instituted, to verify stability of the gamma detector operation and the background levels

  20. Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples.

    Science.gov (United States)

    Leach, L; Zhu, Y; Chaturvedi, S

    2018-02-01

    Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 ( ITS 2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities. Copyright © 2018 Leach et al.

  1. Validation of an improved enzyme-linked immunosorbent assay for the diagnosis of trypanosomal antibodies in Ghanaian cattle

    International Nuclear Information System (INIS)

    Doku, C.K.; Seidu, I.B.M.

    2000-01-01

    The validation of an enzyme-linked immunosorbent assay (Ab-ELISA) for the detection of antibodies to pathogenic trypanosomes in cattle is described. Two hundred known negative sera obtained from the tsetse-free zone of Dori (Burkina Faso) were analyzed using microtitre plates pre-coated with crude antigen lysates of Trypanosoma congolense and T. vivax. A pre-test optimization was carried out and a percent positivity (PP) of 50% was chosen (specificity: >82%) for assaying field sera. A total of 440 serum samples collected from cattle in areas of known and unknown disease prevalence were assayed. For all animals the packed red cell volume (PCV) was determined and the buffy coat technique (BCT) and blood smears were examined to detect trypanosomes at the species level. A comparison of the BCT and Ab-ELISA results showed there was a much higher prevalence of antibodies to both species than the parasite prevalence as shown by the BCT (10 fold). The rate of agreement between BCT-positive and Ab-ELISA-positive samples for both species was low (<10%). No conclusion could be drawn from this finding because of the low number of known BCT positive cases that were identified. There was a better, albeit highly variable, agreement between BCT-negative and Ab-ELISA-negative samples (30-70%). Proposals for further improvement of the Ab-ELISA and prospects for the use of the assay in the monitoring of trypanosomosis control in Ghana are discussed. (author)

  2. Fuzzy-logic based strategy for validation of multiplex methods: example with qualitative GMO assays.

    Science.gov (United States)

    Bellocchi, Gianni; Bertholet, Vincent; Hamels, Sandrine; Moens, W; Remacle, José; Van den Eede, Guy

    2010-02-01

    This paper illustrates the advantages that a fuzzy-based aggregation method could bring into the validation of a multiplex method for GMO detection (DualChip GMO kit, Eppendorf). Guidelines for validation of chemical, bio-chemical, pharmaceutical and genetic methods have been developed and ad hoc validation statistics are available and routinely used, for in-house and inter-laboratory testing, and decision-making. Fuzzy logic allows summarising the information obtained by independent validation statistics into one synthetic indicator of overall method performance. The microarray technology, introduced for simultaneous identification of multiple GMOs, poses specific validation issues (patterns of performance for a variety of GMOs at different concentrations). A fuzzy-based indicator for overall evaluation is illustrated in this paper, and applied to validation data for different genetically modified elements. Remarks were drawn on the analytical results. The fuzzy-logic based rules were shown to be applicable to improve interpretation of results and facilitate overall evaluation of the multiplex method.

  3. Validity of Thermal Ramping Assays Used to Assess Thermal Tolerance in Arthropods

    DEFF Research Database (Denmark)

    Overgaard, Johannes; Kristensen, Torsten Nygård; Sørensen, Jesper Givskov

    2012-01-01

    are useful assays for small insects because they incorporate an ecologically relevant gradual temperature change. However, recent model-based papers have suggested that estimates of thermal resistance may be strongly confounded by simultaneous starvation and dehydration stress. In the present study we...... empirically test these model predictions using two sets of independent experiments. We clearly demonstrate that results from ramping assays of small insects (Drosophila melanogaster) are not compromised by starvation- or dehydration-stress. Firstly we show that the mild disturbance of water and energy balance...... of D. melanogaster experienced during the ramping tests does not confound heat tolerance estimates. Secondly we show that flies pre-exposed to starvation and dehydration have ‘‘normal’’ heat tolerance and that resistance to heat stress is independent of the energetic and water status of the flies...

  4. Quantifying the foodscape: A systematic review and meta-analysis of the validity of commercially available business data

    Science.gov (United States)

    Lebel, Alexandre; Daepp, Madeleine I. G.; Block, Jason P.; Walker, Renée; Lalonde, Benoît; Kestens, Yan; Subramanian, S. V.

    2017-01-01

    This paper reviews studies of the validity of commercially available business (CAB) data on food establishments (“the foodscape”), offering a meta-analysis of characteristics associated with CAB quality and a case study evaluating the performance of commonly-used validity indicators describing the foodscape. Existing validation studies report a broad range in CAB data quality, although most studies conclude that CAB quality is “moderate” to “substantial”. We conclude that current studies may underestimate the quality of CAB data. We recommend that future validation studies use density-adjusted and exposure measures to offer a more meaningful characterization of the relationship of data error with spatial exposure. PMID:28358819

  5. Analytical validation of the Roche 25-OH Vitamin D Total assay

    DEFF Research Database (Denmark)

    Knudsen, Cindy Soendersoe; Nexo, Ebba; Højskov, Carsten Schriver

    2012-01-01

    ) showed Vitamin D Total nmol/L=1.07×(LC-MS/MS) nmol/L+4.7 nmol/L, whereas comparison of 25OHD2 using 23 patient samples showed Vitamin D Total nmol/L=0.55×(LC-MS/MS) nmol/L–2.38 nmol/L (Demings regression). Conclusions: The Roche Vitamin D Total assay is judged suitable for measurement of 25OHD in serum...

  6. TCV software test and validation tools and technique. [Terminal Configured Vehicle program for commercial transport aircraft operation

    Science.gov (United States)

    Straeter, T. A.; Williams, J. R.

    1976-01-01

    The paper describes techniques for testing and validating software for the TCV (Terminal Configured Vehicle) program which is intended to solve problems associated with operating a commercial transport aircraft in the terminal area. The TCV research test bed is a Boeing 737 specially configured with digital computer systems to carry out automatic navigation, guidance, flight controls, and electronic displays research. The techniques developed for time and cost reduction include automatic documentation aids, an automatic software configuration, and an all software generation and validation system.

  7. DBS-LC-MS/MS assay for caffeine: validation and neonatal application.

    Science.gov (United States)

    Bruschettini, Matteo; Barco, Sebastiano; Romantsik, Olga; Risso, Francesco; Gennai, Iulian; Chinea, Benito; Ramenghi, Luca A; Tripodi, Gino; Cangemi, Giuliana

    2016-09-01

    DBS might be an appropriate microsampling technique for therapeutic drug monitoring of caffeine in infants. Nevertheless, its application presents several issues that still limit its use. This paper describes a validated DBS-LC-MS/MS method for caffeine. The results of the method validation showed an hematocrit dependence. In the analysis of 96 paired plasma and DBS clinical samples, caffeine levels measured in DBS were statistically significantly lower than in plasma but the observed differences were independent from hematocrit. These results clearly showed the need for extensive validation with real-life samples for DBS-based methods. DBS-LC-MS/MS can be considered to be a good alternative to traditional methods for therapeutic drug monitoring or PK studies in preterm infants.

  8. MUTAGENICITY EVALUATION OF THE COMMERCIAL PRODUCT CI DISPERSE BLUE 291 USING DIFFERENT PROTOCOLS OF THE SALMONELLA ASSAY

    Science.gov (United States)

    Textile dyes can enter the water ecosystem through wastewater discharges potentially exposing humans through the consumption of water and food. The commercial disperse dye product C.I. Disperse Blue 291 containing the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5(diethylam...

  9. ISO 17025 validation of a next-generation sequencing assay for relationship testing

    DEFF Research Database (Denmark)

    Buchard, Anders; Kampmann, Marie-Louise; Poulsen, Lena

    2016-01-01

    17025 accredited laboratory in 2015. Here, the essential parts of the validation report submitted to the Danish Accreditation Fund are presented. A total of 100 unrelated Danes were typed in duplicates and the locus balance, heterozygote balance (Hb) and noise levels were analysed in detail. Two loci...

  10. Validation of the FDG (18F) radiochemical purity assay by thin layer chromatography

    International Nuclear Information System (INIS)

    Leao, R.L.C.; Oliveira, M.L.; Nascimento, J.E.; Nascimento, N.C.E.S.

    2013-01-01

    All methodologies utilized in radiopharmaceutical industry should be validated in order to prove that they meet the requirements of analytical applications, ensuring the reliability of the results. At a radiopharmaceutical industry there is one challenge aspect: sometimes it is not possible use a stable standard to perform the validation analysis. In order to overcome this difficulty, the objective of this study was to suggest a validation protocol for these methodologies, based on the recommendations of RE n° 899/Agencia Nacional de Vigilancia Sanitaria (ANVISA), and prove its efficiency, performing the radiochemical purity validation test of FDG (18F), by TLC. To obtain the calibration curve, we suggested that the theoretical activity values should be determined using a dose calibrator, simultaneously of each analysis performed by TLC, for 5 hours. The method was linear (R 2 of 0.996), precise (CV% <5%) and accurate (96.85% < accuracy < 102.56%). In relation to the robustness test, our experiments evaluated the influence of the distance travelled by mobile phase, variations at mobile phase concentration and type of chromatographic plate (silica gel on glass or aluminium plates). The detection and quantification limits were determined (321.9 and 1065.6 kBq, respectively). As expected, this methodology was nonspecific, showing a slight spot corresponding to the FDM. The proposed protocol was efficient and the methodology tested was effective to determine the radiochemical purity of FDG (18F), in accordance to the limits recommended by ANVISA. (author)

  11. Sensitivity and specificity of dried blood spots for HIV-1 viral load quantification: A laboratory assessment of 3 commercial assays.

    Science.gov (United States)

    Pannus, Pieter; Claus, Maarten; Gonzalez, Maria Mercedes Perez; Ford, Nathan; Fransen, Katrien

    2016-11-01

    The use of dried blood spots (DBS) instead of plasma as a specimen type for HIV-1 viral load (VL) testing facilitates the decentralization of specimen collection and can increase access to VL testing in resource-limited settings. The performance of DBS for VL testing is lower, however, when compared to the gold standard sample type plasma. In this diagnostic accuracy study, we evaluated 3 VL assays with DBS.Participants were recruited between August 2012 and April 2015. Both plasma and DBS specimens were prepared and tested for HIV-1 VL with the Roche CAP/CTM HIV-1 test v2.0, the Abbott RealTime HIV-1, and the bioMérieux NucliSENS EasyQ HIV-1 v2.0. Sensitivity and specificity to detect treatment failure at a threshold of 1000 cps/mL with DBS were determined.A total of 272 HIV-positive patients and 51 HIV-negative people were recruited in the study. The mean difference or bias between plasma and DBS VL was 25% of the specimens differed by >0.5 log cps/mL.All 3 assays had comparable sensitivities around 80% and specificities around 90%. Upward misclassification rates were around 10%, but downward misclassification rates ranged from 20.3% to 23.6%. Differences in between assays were not statistically significant (P > 0.1).The 3 VL assays evaluated had suboptimal performance with DBS but still performed better than immunological or clinical monitoring. Even after the introduction of the much-anticipated point-of-care VL devices, it is expected that DBS will remain important as a complementary option for supporting access to VL monitoring, particularly in rural, resource-limited settings. Manufacturers should accelerate efforts to develop more reliable, sensitive and specific methods to test VL on DBS specimens.

  12. Toward an international standard for PCR-based detection of Escherichia coli O157 - Part 1. Assay development and multi-center validation

    DEFF Research Database (Denmark)

    Abdulmawjood, A.; Bulte, M.; Cook, N.

    2003-01-01

    As part of a major European research project, a diagnostic PCR assay, including an internal amplification control, was developed and validated in a collaborative trial for the detection of Escherichia coli O157. The assay is based on amplification of sequences of the rJbE O157 gene. The collabora...

  13. Detection of LGI1 and CASPR2 antibodies with a commercial cell-based assay in patients with very high VGKC-complex antibody levels.

    Science.gov (United States)

    Yeo, T; Chen, Z; Chai, J Y H; Tan, K

    2017-07-15

    The presence of VGKC-complex antibodies, without LGI1/CASPR2 antibodies, as a standalone marker for neurological autoimmunity remains controversial. Additionally, the lack of an unequivocal VGKC-complex antibody cut-off level defining neurological autoimmunity makes it important to test for monospecific antibodies. We aim to determine the performance characteristics of a commercial assay (Euroimmun, Lübeck, Germany) for LGI1/CASPR2 antibody detection in patients with very high VGKC-complex antibody levels and report their clinico-serological associations. We identified 8 patients in our cohort with the highest VGKC-complex antibody levels (median 2663.5pM, range 933-6730pM) with VGKC-complex antibody related syndromes (Group A). Two other groups were identified; 1 group with suspected neuronal surface antibody syndromes and negative for VGKC-complex antibodies (Group B, n=8), and another group with cerebellar ataxia and negative for onconeuronal antibodies (Group C, n=8). Seven out of 8 patients (87.5%) in Group A had LGI1 and/or CASPR2 antibodies. One Group B patient had LGI1 antibodies but was negative on re-testing with a live cell assay. No Group C patients had monospecific antibodies. Inter-rater reliability was high; combining Groups A and B patients, the kappa statistic was 0.87 and 1.0 for LGI1 and CASPR2 antibodies respectively. We demonstrated that a high proportion of patients with very high VGKC-complex antibody levels and relevant clinical syndromes have LGI1 and/or CASPR2 antibodies detected by the commercial assay. Our findings lend support to the use of the assay for rapid and reliable detection of LGI1 and CASPR2 antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Quantitative 177Lu-SPECT/CT imaging and validation of a commercial dosimetry software

    International Nuclear Information System (INIS)

    D'Ambrosio, L.; Aloj, L.; Morisco, A.; Aurilio, M.; Prisco, A.; Di Gennaro, F.; Lastoria, S.; Madesani, D.

    2015-01-01

    Full text of publication follows. Aim: 3D dosimetry is an appealing yet complex application of SPECT/CT in patients undergoing radionuclide therapy. In this study we have developed a quantitative imaging protocol and we have validated commercially available dosimetry software (Dosimetry Tool-kit Package, GE Heathcare) in patients undergoing 177 Lu-DOTATATE therapy. Materials and methods: dosimetry tool-kit uses multi SPECT/CT and/or WB planar datasets for quantifying changes in radiopharmaceutical uptake over time to determine residence times. This software includes tools for performing reconstruction of SPECT/CT data, registration of all scans to a common reference, segmentation of the different organs, creating time activity curves, curve fitting and calculation of residence times. All acquisitions were performed using a hybrid dual-head SPECT-CT camera (Discovery 670, GE Heathcare) equipped with medium energy collimator using a triple-energy window. SPECT images were reconstructed using an iterative reconstruction algorithm with attenuation, scatter and collimator depth-dependent three-dimensional resolution recovery correction. Camera sensitivity and dead time were evaluated. Accuracy of activity quantification was performed on a large homogeneous source with addition of attenuating/scattering medium. A NEMA/IEC body phantom was utilized to measure the recovery coefficient that the software does not take into account. The residence times for organs at risk were calculated in five patients. OLINDA-EXM software was used to calculate absorbed doses. Results: 177 Lu-sensitivity factor was 13 counts/MBq/s. Dead time was <3% with 1.11 GBq in the field of view. The measured activity was consistent with the decay-corrected calibrated activity for large volumes (>100 cc). The recovery coefficient varied from 0.71 (26.5 ml) to 0.16 (2.5 ml) in the absence of background activity and from 0.58 to 0.13 with a source to background activity concentration ratio 20:1. The

  15. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    Science.gov (United States)

    van der Ploeg, René; Goudelis, Spyridon Theodoros; den Blaauwen, Tanneke

    2015-01-01

    The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. PMID:26263980

  16. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    Directory of Open Access Journals (Sweden)

    René van der Ploeg

    2015-07-01

    Full Text Available The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl isothiourea (A22 or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors.

  17. Calculations for Adjusting Endogenous Biomarker Levels During Analytical Recovery Assessments for Ligand-Binding Assay Bioanalytical Method Validation.

    Science.gov (United States)

    Marcelletti, John F; Evans, Cindy L; Saxena, Manju; Lopez, Adriana E

    2015-07-01

    It is often necessary to adjust for detectable endogenous biomarker levels in spiked validation samples (VS) and in selectivity determinations during bioanalytical method validation for ligand-binding assays (LBA) with a matrix like normal human serum (NHS). Described herein are case studies of biomarker analyses using multiplex LBA which highlight the challenges associated with such adjustments when calculating percent analytical recovery (%AR). The LBA test methods were the Meso Scale Discovery V-PLEX® proinflammatory and cytokine panels with NHS as test matrix. The NHS matrix blank exhibited varied endogenous content of the 20 individual cytokines before spiking, ranging from undetectable to readily quantifiable. Addition and subtraction methods for adjusting endogenous cytokine levels in %AR calculations are both used in the bioanalytical field. The two methods were compared in %AR calculations following spiking and analysis of VS for cytokines having detectable endogenous levels in NHS. Calculations for %AR obtained by subtracting quantifiable endogenous biomarker concentrations from the respective total analytical VS values yielded reproducible and credible conclusions. The addition method, in contrast, yielded %AR conclusions that were frequently unreliable and discordant with values obtained with the subtraction adjustment method. It is shown that subtraction of assay signal attributable to matrix is a feasible alternative when endogenous biomarkers levels are below the limit of quantitation, but above the limit of detection. These analyses confirm that the subtraction method is preferable over that using addition to adjust for detectable endogenous biomarker levels when calculating %AR for biomarker LBA.

  18. Development and validation of a quick easily used biochemical assay for evaluating the viability of small immobile arthropods.

    Science.gov (United States)

    Phillips, Craig B; Iline, Ilia I; Richards, Nicola K; Novoselov, Max; McNeill, Mark R

    2013-10-01

    Quickly, accurately, and easily assessing the efficacy of treatments to control sessile arthropods (e.g., scale insects) and stationary immature life stages (e.g., eggs and pupae) is problematic because it is difficult to tell whether treated organisms are alive or dead. Current approaches usually involve either maintaining organisms in the laboratory to observe them for development, gauging their response to physical stimulation, or assessing morphological characters such as turgidity and color. These can be slow, technically difficult, or subjective, and the validity of methods other than laboratory rearing has seldom been tested. Here, we describe development and validation of a quick easily used biochemical colorimetric assay for measuring the viability of arthropods that is sufficiently sensitive to test even very small organisms such as white fly eggs. The assay was adapted from a technique for staining the enzyme hexokinase to signal the presence of adenosine triphosphate in viable specimens by reducing a tetrazolium salt to formazan. Basic laboratory facilities and skills are required for production of the stain, but no specialist equipment, expertise, or facilities are needed for its use.

  19. Evaluation of Different Estimation Methods for Accuracy and Precision in Biological Assay Validation.

    Science.gov (United States)

    Yu, Binbing; Yang, Harry

    2017-01-01

    Biological assays ( bioassays ) are procedures to estimate the potency of a substance by studying its effects on living organisms, tissues, and cells. Bioassays are essential tools for gaining insight into biologic systems and processes including, for example, the development of new drugs and monitoring environmental pollutants. Two of the most important parameters of bioassay performance are relative accuracy (bias) and precision. Although general strategies and formulas are provided in USP, a comprehensive understanding of the definitions of bias and precision remain elusive. Additionally, whether there is a beneficial use of data transformation in estimating intermediate precision remains unclear. Finally, there are various statistical estimation methods available that often pose a dilemma for the analyst who must choose the most appropriate method. To address these issues, we provide both a rigorous definition of bias and precision as well as three alternative methods for calculating relative standard deviation (RSD). All methods perform similarly when the RSD ≤10%. However, the USP estimates result in larger bias and root-mean-square error (RMSE) compared to the three proposed methods when the actual variation was large. Therefore, the USP method should not be used for routine analysis. For data with moderate skewness and deviation from normality, the estimates based on the original scale perform well. The original scale method is preferred, and the method based on log-transformation may be used for noticeably skewed data. LAY ABSTRACT: Biological assays, or bioassays, are essential in the development and manufacture of biopharmaceutical products for potency testing and quality monitoring. Two important parameters of assay performance are relative accuracy (bias) and precision. The definitions of bias and precision in USP 〈1033〉 are elusive and confusing. Another complicating issue is whether log-transformation should be used for calculating the

  20. Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays

    DEFF Research Database (Denmark)

    Møller, H J; Møller-Pedersen, T; Damsgaard, T E

    1995-01-01

    The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA...... inhibition studies, immunoblotting and HPLC analyses confirmed the presence of substantial amounts of KS, probably as a large proteoglycan (> 120 kDa). Commercial and heterogenic glycosaminoglycan preparations therefore must be used with great caution in immunological analyses. On the other hand the shark...... cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate...

  1. Validation of an accelerated high-sensitivity troponin T assay protocol in an Australian cohort with chest pain.

    Science.gov (United States)

    Parsonage, William A; Greenslade, Jaimi H; Hammett, Christopher J; Lamanna, Arvin; Tate, Jillian R; Ungerer, Jacobus P; Chu, Kevin; Than, Martin; Brown, Anthony F T; Cullen, Louise

    2014-02-17

    To validate an accelerated biomarker strategy using a high-sensitivity cardiac troponin T (hs-cTnT) assay for diagnosing acute myocardial infarction (AMI) in patients presenting to the emergency department with chest pain; and to validate this strategy in combination with the National Heart Foundation of Australia/Cardiac Society of Australia and New Zealand risk stratification model. Single-centre, prospective, observational cohort study of 764 adults presenting to a tertiary hospital with symptoms of possible acute coronary syndrome between November 2008 and February 2011. AMI or cardiac death within 24 hours of presentation (primary), and major adverse cardiac events within 30 days (secondary). An elevated hs-cTnT assay result above the 99th percentile at either the 0 h or 2 h time points had sensitivity of 96.4% (95% CI, 87.9%-99.0%), specificity of 82.6% (95% CI, 79.7%-85.2%), negative predictive value of 99.7% (95% CI, 98.8%-99.9%) and positive predictive value of 30.5% (95% CI, 24.2%-37.6%) for diagnosing AMI. Compared with a traditional 6 h cardiac troponin testing strategy, the accelerated strategy led to reclassification of risk in only two patients with adverse cardiac outcomes, with no net effect on appropriate management. In patients presenting with chest pain, an accelerated biomarker strategy using the hs-cTnT assay performed well in the initial diagnosis of AMI. The accelerated strategy was also effective when incorporated into a comprehensive strategy of risk stratification that included clinical and demographic factors. The time saved by this approach could have a major impact on health service delivery. Australian New Zealand Clinical Trials Registry ACTRN12610000053022.

  2. Performance evaluation of four type-specific commercial assays for detection of herpes simplex virus type 1 antibodies in a Middle East and North Africa population.

    Science.gov (United States)

    Aldisi, Rana S; Elsidiq, Malaz S; Dargham, Soha R; Sahara, Afifah S; Al-Absi, Enas S; Nofal, Mariam Y; Mohammed, Layla I; Abu-Raddad, Laith J; Nasrallah, Gheyath K

    2018-03-22

    The number of diagnostic assays for the detection of herpes simplex virus type 1 (HSV-1) antibodies has increased over the years. However, their performance characteristics could vary among global populations. To investigate performance of two commercial ELISA kits, HerpeSelect ® 1 ELISA and Euroimmun Anti-HSV-1 (gC1) ELISA (IgG); and two commercial immunoblot (IB)/Western blot (WB) assays, HerpeSelect ® 1 and 2 Immunoblot IgG, and Euroimmun Anti-HSV-1/HSV-2 gG2 Euroline-WB (IgG/IgM); in detecting HSV-1 antibodies in a Middle East and North Africa (MENA) population. Blood specimens were collected from blood donors in Doha, Qatar, June 2013-2016. Twenty specimens were randomly selected from 10 MENA nationalities (Egypt, Iran, Jordan, Lebanon, Pakistan, Palestine, Qatar, Sudan, Syria, and Yemen; total = 200), and tested for HSV-1 antibodies. Across all six comparisons between assays, positive percent agreement ranged between 95.7% (95% CI: 91.4-98.3%) and 100.0% (95% CI: 97.8-100.0%). Negative percent agreement ranged between 86.2% (95% CI: 68.3-96.1%) and 96.2% (95% CI: 80.4-99.9%). Overall percent agreement ranged between 95.7% (95% CI: 91.7-97.8%) and 99.4% (95% CI: 96.7-99.9%). Cohen's kappa statistic ranged between 0.84 (95% CI: 0.73-0.95) and 0.98 (95% CI: 0.93-1.00). Compared against IB/WB, HerpeSelect ® and Euroimmun had sensitivities and specificities >96% and >86%, respectively. Positive and negative predictive values were >97% and >83%, respectively. The assays showed excellent concordance with one another, and with a high kappa statistic. The ELISA kits demonstrated robust diagnostic performance compared to the IB/WB assays. These findings support the assays' utility in clinical diagnosis and research in MENA populations. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Characterization and Validation of the LT-SYS Copper Assay on a Roche Cobas 8000 c502 Analyzer.

    Science.gov (United States)

    Kraus, F Bernhard; Mischereit, Marlies; Eller, Christoph; Ludwig-Kraus, Beatrice

    2017-02-01

    Validation of the LT-SYS quantitative in vitro copper assay on a Roche Cobas 8000 c502 analyzer and comparison with a BIOMED assay on a Roche Cobas Mira analyzer. Imprecision and bias were quantified at different concentration levels (serum and plasma) over a 20-day period. Linearity was assessed covering a range from 4.08 µmol/L to 33.8 µmol/L. Limit of blank (LoB) and limit of detection (LoD) were established based on a total of 120 blank and low-level samples. The method comparison was based on 58 plasma samples. Within-run imprecision ranged from 0.7% to 1.2% and within-laboratory imprecision from 1.4% to 3.3%. Relative bias for the 2 serum pools with known target values was less than 2.5%. The assay did not deviate from linearity over the tested measuring range. LoB and LoD were 0.12 µmol/L and 0.23 µmol/L, respectively. The method comparison revealed an average deviation of 11.5% (2.016 µmol/L), and the linear regression fit was y = 1.464 + 0.795x. The LT-SYS copper assay characterized in this study showed a fully acceptable performance with good degrees of imprecision and bias, no deviation from linearity in the relevant measuring rangem, and very low LoB and LoD. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies.

    Directory of Open Access Journals (Sweden)

    Emmanuel Bréard

    Full Text Available A newly developed Enzym Like Immuno Sorbant Assay (ELISA based on the recombinant nucleocapsid protein (N of Schmallenberg virus (SBV was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515 in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT or an indirect immuno-fluorescence assay (IFA. The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.

  5. Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark® for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

    Directory of Open Access Journals (Sweden)

    Jeffrey S. Larson

    2010-01-01

    Full Text Available We report here the results of the analytical validation of assays that measure HER2 total protein (H2T and HER2 homodimer (H2D expression in Formalin Fixed Paraffin Embedded (FFPE breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC (HercepTest. The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC or on indirect assessments of gene amplification (FISH.

  6. Development and validation of a 36-gene sequencing assay for hereditary cancer risk assessment

    Directory of Open Access Journals (Sweden)

    Valentina S. Vysotskaia

    2017-02-01

    Full Text Available The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs, short insertions and deletions (indels, and copy number variants (CNVs in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA. We also demonstrated the screen’s high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

  7. Enrofloxacin assay validation and pharmacokinetics following a single oral dose in chickens.

    Science.gov (United States)

    da Silva, R G; Reyes, F G R; Sartori, J R; Rath, S

    2006-10-01

    The pharmacokinetics of enrofloxacin (ENRO), a fluoroquinolone antimicrobial agent, was studied in male broiler chickens (Cobb) after single oral administration of 10 mg of ENRO/kg b.w. A high-performance liquid chromatography-photodiode array detector (DAD) (HPLC-DAD) method was developed and validated and used for quantitation of ENRO and its major metabolite ciprofloxacin in plasma. The HPLC analyses were carried out using a cationic-octadecyl mixed column and 0.05 mol/L phosphate buffer (pH 2.5)/acetonitrile as mobile phase. The sample preparation of plasma consisted of the precipitation of proteins followed by solid phase extraction on cationic-octadecyl mixed cartridges. The method was validated considering linear range, linearity, selectivity, sensitivity, limit of detection (LOD), limit of quantitation (LOQ), intra- and inter-day precisions and accuracy. The LOD and LOQ for both fluoroquinolones were 60 and 200 ng/mL for plasma. The plasma concentration vs. time graph was characteristic of a two-compartment open model. The maximal plasma concentration of 1.5 +/- 0.2 mg/mL was achieved at 9 +/- 2 h. The elimination half-life and the mean residence time of ENRO were 1.5 +/- 0.2 and 15.64 h, respectively. The area under the concentration-time curve was calculated as 35 +/- 4 mgxh/mL.

  8. Development of a simple, low cost chronoamperometric assay for fructose based on a commercial graphite-nanoparticle modified screen-printed carbon electrode.

    Science.gov (United States)

    Nicholas, Phil; Pittson, Robin; Hart, John P

    2018-02-15

    This paper describes the development of a simple, low cost chronoamperometric assay, for the measurement of fructose, using a graphite-nanoparticle modified screen-printed electrode (SPCE-G-COOH). Cyclic voltammetry showed that the response of the SPCE-G-COOH enhanced the sensitivity and precision, towards the enzymatically generated ferrocyanide species, over a plain SPCE; therefore the former was employed in subsequent studies. Calibration studies were carried out using chronoamperometry with a 40µl mixture containing fructose, mediator and FDH, deposited onto the SPCE-G-COOH. The response was linear from 0.1mM to 1.0mM. A commercial fruit juice sample was analysed using the developed assay and the fructose concentration was calculated to be 477mM with a precision of 3.03% (n=5). Following fortification (477mM fructose) the mean recovery was found to be 97.12% with a coefficient of variation of 6.42% (n=5); consequently, the method holds promise for the analysis of commercial fruit juices. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Polymerase chain reaction assay for verifying the labeling of meat and commercial meat products from game birds targeting specific sequences from the mitochondrial D-loop region.

    Science.gov (United States)

    Rojas, M; González, I; Pavón, M A; Pegels, N; Hernández, P E; García, T; Martín, R

    2010-05-01

    A PCR assay was developed for the identification of meats and commercial meat products from quail (Coturnix coturnix), pheasant (Phasianus colchicus), partridge (Alectoris spp.), guinea fowl (Numida meleagris), pigeon (Columba spp.), Eurasian woodcock (Scolopax rusticola), and song thrush (Turdus philomelos) based on oligonucleotide primers targeting specific sequences from the mitochondrial D-loop region. The primers designed generated specific fragments of 96, 100, 104, 106, 147, 127, and 154 bp in length for quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock, and song thrush tissues, respectively. The specificity of each primer pair was tested against DNA from various game and domestic species. In this work, satisfactory amplification was accomplished in the analysis of experimentally pasteurized (72 degrees C for 30 min) and sterilized (121 degrees C for 20 min) meats, as well as in commercial meat products from the target species. The technique was also applied to raw and sterilized muscular binary mixtures, with a detection limit of 0.1% (wt/wt) for each of the targeted species. The proposed PCR assay represents a rapid and straightforward method for the detection of possible mislabeling in game bird meat products.

  10. JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

    Science.gov (United States)

    Uno, Yoshifumi; Kojima, Hajime; Omori, Takashi; Corvi, Raffaella; Honma, Masamistu; Schechtman, Leonard M; Tice, Raymond R; Burlinson, Brian; Escobar, Patricia A; Kraynak, Andrew R; Nakagawa, Yuzuki; Nakajima, Madoka; Pant, Kamala; Asano, Norihide; Lovell, David; Morita, Takeshi; Ohno, Yasuo; Hayashi, Makoto

    2015-07-01

    The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. 11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

    Science.gov (United States)

    Islam, Rafiq; Briscoe, Chad; Bower, Joseph; Cape, Stephanie; Arnold, Mark; Hayes, Roger; Warren, Mark; Karnik, Shane; Stouffer, Bruce; Xiao, Yi Qun; van der Strate, Barry; Sikkema, Daniel; Fang, Xinping; Tudoroniu, Ariana; Tayyem, Rabab; Brant, Ashley; Spriggs, Franklin; Barry, Colin; Khan, Masood; Keyhani, Anahita; Zimmer, Jennifer; Caturla, Maria Cruz; Couerbe, Philippe; Khadang, Ardeshir; Bourdage, James; Datin, Jim; Zemo, Jennifer; Hughes, Nicola; Fatmi, Saadya; Sheldon, Curtis; Fountain, Scott; Satterwhite, Christina; Colletti, Kelly; Vija, Jenifer; Yu, Mathilde; Stamatopoulos, John; Lin, Jenny; Wilfahrt, Jim; Dinan, Andrew; Ohorodnik, Susan; Hulse, James; Patel, Vimal; Garofolo, Wei; Savoie, Natasha; Brown, Michael; Papac, Damon; Buonarati, Mike; Hristopoulos, George; Beaver, Chris; Boudreau, Nadine; Williard, Clark; Liu, Yansheng; Ray, Gene; Warrino, Dominic; Xu, Allan; Green, Rachel; Hayward-Sewell, Joanne; Marcelletti, John; Sanchez, Christina; Kennedy, Michael; Charles, Jessica St; Bouhajib, Mohammed; Nehls, Corey; Tabler, Edward; Tu, Jing; Joyce, Philip; Iordachescu, Adriana; DuBey, Ira; Lindsay, John; Yamashita, Jim; Wells, Edward

    2018-04-01

    The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

  12. Validating potential toxicity assays to assess petroleum hydrocarbon toxicity in polar soil.

    Science.gov (United States)

    Harvey, Alexis Nadine; Snape, Ian; Siciliano, Steven Douglas

    2012-02-01

    Potential microbial activities are commonly used to assess soil toxicity of petroleum hydrocarbons (PHC) and are assumed to be a surrogate for microbial activity within the soil ecosystem. However, this assumption needs to be evaluated for frozen soil, in which microbial activity is limited by liquid water (θ(liquid)). Influence of θ(liquid) on in situ toxicity was evaluated and compared to the toxicity endpoints of potential microbial activities using soil from an aged diesel fuel spill at Casey Station, East Antarctica. To determine in situ toxicity, gross mineralization and nitrification rates were determined by the stable isotope dilution technique. Petroleum hydrocarbon-contaminated soil (0-8,000 mg kg(-1)), packed at bulk densities of 1.4, 1.7, and 2.0 g cm(-3) to manipulate liquid water content, was incubated at -5°C for one, two, and three months. Although θ(liquid) did not have a significant effect on gross mineralization or nitrification, gross nitrification was sensitive to PHC contamination, with toxicity decreasing over time. In contrast, gross mineralization was not sensitive to PHC contamination. Toxic response of gross nitrification was comparable to potential nitrification activity (PNA) with similar EC25 (effective concentration causing a 25% effect in the test population) values determined by both measurement endpoints (400 mg kg(-1) for gross nitrification compared to 200 mg kg(-1) for PNA), indicating that potential microbial activity assays are good surrogates for in situ toxicity of PHC contamination in polar regions. Copyright © 2011 SETAC.

  13. Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies

    Science.gov (United States)

    Koestel, Carole; Simonin, Céline; Belcher, Sandrine

    2016-01-01

    Abstract Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices. PMID:27356183

  14. INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.

    Science.gov (United States)

    Franklin, Ashley D; Crosier, Adrienne E; Vansandt, Lindsey M; Mattson, Elliot; Xiao, Zhengguo

    2015-06-01

    Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of cheetahs (Acinonyx jubatus ; n=3) and stimulated with lipopolysaccharides (LPS) to induce the production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 for establishment of cross-reactivity between these cheetah cytokines and feline-specific cytokine antibodies provided in commercially available Feline DuoSet® ELISA kits (R&D Systems, Inc., Minneapolis, Minnesota 55413, USA). This study found that feline-specific cytokine antibodies bind specifically to cheetah proinflammatory cytokines TNF-α, IL-1β, and IL-6 from cell culture supernatants. The assays also revealed that cheetah PBMCs produce a measurable, cell concentration-dependent increase in proinflammatory cytokine production after LPS stimulation. To enable the use of these kits, which are designed for cell culture supernatants for analyzing cytokine concentrations in cheetah serum, percent recovery and parallelism of feline cytokine standards in cheetah serum were also evaluated. Cytokine concentrations in cheetah serum were approximated based on the use of domestic cat standards in the absence of cheetah standard material. In all cases (for cytokines TNF-α, IL-1β, and IL-6), percent recovery increased as the serum sample dilution increased, though percent recovery varied between cytokines at a given dilution factor. A 1:2 dilution of serum resulted in approximately 45, 82, and 7% recovery of TNF-α, IL-1β, and IL-6 standards, respectively. Adequate parallelism was observed across a large range of cytokine concentrations for TNF-α and IL-1β; however, a significant departure from parallelism was observed between the IL-6 standard and the serum samples (P=0.004). Therefore, based on our results, the Feline DuoSet ELISA (R&D Systems, Inc.) kits are valid assays for the measurement of TNF-α and IL-1β in cheetah serum but should not be used for accurate measurement of IL-6.

  15. Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays

    DEFF Research Database (Denmark)

    Møller, H J; Møller-Pedersen, T; Damsgaard, T E

    1995-01-01

    The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA...... cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate...... correlated well (r = 0.90) with the concentrations obtained with a traditional KS-ELISA that uses purified aggrecan as standard and coating antigen, and KS in both serum and synovial fluid could be measured with sufficient linearity....

  16. Commercial enzyme-linked immunosorbent assay versuspolymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    Pedro Emmanuel Alvarenga Americano do Brasil

    2016-01-01

    Full Text Available Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA and polymerase chain reaction (PCR diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects.

  17. Validation Study of Rapid Assays of Bioburden, Endotoxins and Other Contamination.

    Science.gov (United States)

    Shintani, Hideharu

    2016-01-01

    Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility.  For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent.  The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic

  18. Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay

    Science.gov (United States)

    Jackson, Petra

    2013-01-01

    The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor. PMID:24136994

  19. Validated spectophotometric methods for the assay of cinitapride hydrogen tartrate in pharmaceuticals

    Directory of Open Access Journals (Sweden)

    Satyanarayana K.V.V.

    2013-01-01

    Full Text Available Three simple, selective and rapid spectrophotometric methods have been established for the determination of cinitapride hydrogen tartrate (CHT in pharmaceutical tablets. The proposed methods are based on the diazotization of CHT with sodium nitrite and hydrochloric acid, followed by coupling with resorcinol, 1-benzoylacetone and 8-hydroxyquinoline in alkaline medium for methods A, B and C respectively. The formed azo dyes are measured at 442, 465 and 552 nm for methods A, B and C respectively. The parameters that affect the reaction were carefully optimized. Under optimum conditions, Beer’s law is obeyed over the ranges 2.0-32.0, 1.0-24.0 and 1.0-20.0 μg. mL-1 for methods A, B, and C, respectively. The calculated molar absorptivity values are 1.2853 x104, 1.9624 x104 and 3.92 x104 L.mol-1.cm-1 for methods A, B and C, respectively. The results of the proposed procedures were validated statistically according to ICH guidelines. The proposed methods were successfully applied to the determination of CHT in Cintapro tablets without interference from common excipients encountered.

  20. Validity of a Commercial Linear Encoder to Estimate Bench Press 1 RM from the Force-Velocity Relationship

    OpenAIRE

    Bosquet, Laurent; Porta-Benache, Jeremy; Blais, Jérôme

    2010-01-01

    The aim of this study was to assess the validity and accuracy of a commercial linear encoder (Musclelab, Ergotest, Norway) to estimate Bench press 1 repetition maximum (1RM) from the force - velocity relationship. Twenty seven physical education students and teachers (5 women and 22 men) with a heterogeneous history of strength training participated in this study. They performed a 1 RM test and a force - velocity test using a Bench press lifting task in a random order. Mean 1 RM was 61.8 ± 15...

  1. Validation of a commercial dry-form broth microdilution device (Sensititre) for testing tedizolid, a new oxazolidinone.

    Science.gov (United States)

    Jones, Ronald N; Holliday, Nicole M; Rhomberg, Paul R

    2015-02-01

    Tedizolid, a novel oxazolidinone antibacterial with potent activity against a wide range of Gram-positive pathogens, was recently approved by regulatory authorities for the treatment of acute bacterial skin and skin structure infections. A commercial broth microdilution device (Sensititre; Thermo Fisher Scientific) was validated using 285 selected Gram-positive isolates, and the device was documented to have 100.0% essential and categorical agreement with reference MIC results and excellent MIC endpoint reproducibility. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  3. Characterization of blood lipoproteins and validation of cholesterol and triacylglycerol assays for free-ranging polar bears (Ursus maritimus).

    Science.gov (United States)

    Whiteman, John P; Frank, Nicholas; Greller, Katie A; Harlow, Henry J; Ben-David, Merav

    2013-05-01

    Blood triacylglycerol (TG) and lipoproteins are important variables for evaluating nutritional status of wildlife, but measurements are often expensive and difficult. Performance of a small, portable blood analyzer intended for human medical diagnostics was evaluated in measuring these variables in plasma and serum from free-ranging polar bears (Ursus maritimus), which are experiencing nutritional stress related to sea ice loss. The analyzer accurately tracked changes in concentration of total cholesterol (Ctotal), cholesterol associated with high-density lipoprotein (CHDL), and TG during a validation protocol of diluting samples and spiking them with exogenous cholesterol and glycerol. Values of Ctotal and TG agreed well with values obtained by other methods (ultracentrifugation followed by colorimetric assays); agreement was variable for values of cholesterol associated with specific lipoproteins. Similar to a study of captive polar bears, ultracentrifugation methods revealed greater TG in very low-density lipoproteins than in low-density lipoprotein, which is unusual and merits additional study.

  4. Design and Validation Testing of TruckScan to Assay Large Sacks of Fukushima Radioactive Debris on a Truck

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Atsuo [Canberra-Japan (Japan); Bronson, Frazier [Canberra Industries Inc. (United States)

    2015-07-01

    As a result of the March 2011 earthquake and resulting tsunami in Japan, there was a serious accident at the Fukushima Dai-ichi Nuclear Power Plant. This accident has contaminated soil and vegetation in a wide area around the plant. Decontamination projects over the last 4 years have resulted in large numbers of 1 cubic meter canvas bags of debris, commonly called Super Sacks [SS]. These are currently stored nearby where they were generated, but starting in 2015, they will be moved to various Interim Storage Facilities [ISF]. Trucks will typically carry 8-20 of these SSs. When the trucks arrive at the ISF they need to be rapidly sorted into groups according to radioactivity level, for efficient subsequent processing. Canberra Industries, Inc. [CI] has designed a new truck monitoring system 'TruckScan' for use at these ISFs. The TruckScan system must measure the entire truck loaded with multiple closely packed SSs, and generate a nuclide specific assay report showing the radioactivity in each individual SS. The Canberra-Japan office, along with Obayashi Corporation have performed validation testing to demonstrate to the regulatory authorities that the proposed technique was sufficiently accurate. These validation tests were conducted at a temporary storage area in Fukushima prefecture. Decontaminated waste of various representative types and of various levels of radioactivity was gathered and mixed to create homogeneous volumes. These volumes were sampled multiple times and assayed with laboratory HPGe detectors to determine the reference concentration of each pile. Multiple SSs were loaded from each pile. Some of the SSs were filled 50% full, others 75% full, and others 100% full, to represent the typical loading configuration of the existing SSs in the field. The content of the SSs are either sand, soil, or vegetation with densities ranging from 0.3 g/cc - 1.6 g/cc. These SSs with known concentrations of Cs-134 and Cs-137 were then loaded onto trucks in

  5. Design and Validation Testing of TruckScan to Assay Large Sacks of Fukushima Radioactive Debris on a Truck

    International Nuclear Information System (INIS)

    Suzuki, Atsuo; Bronson, Frazier

    2015-01-01

    As a result of the March 2011 earthquake and resulting tsunami in Japan, there was a serious accident at the Fukushima Dai-ichi Nuclear Power Plant. This accident has contaminated soil and vegetation in a wide area around the plant. Decontamination projects over the last 4 years have resulted in large numbers of 1 cubic meter canvas bags of debris, commonly called Super Sacks [SS]. These are currently stored nearby where they were generated, but starting in 2015, they will be moved to various Interim Storage Facilities [ISF]. Trucks will typically carry 8-20 of these SSs. When the trucks arrive at the ISF they need to be rapidly sorted into groups according to radioactivity level, for efficient subsequent processing. Canberra Industries, Inc. [CI] has designed a new truck monitoring system 'TruckScan' for use at these ISFs. The TruckScan system must measure the entire truck loaded with multiple closely packed SSs, and generate a nuclide specific assay report showing the radioactivity in each individual SS. The Canberra-Japan office, along with Obayashi Corporation have performed validation testing to demonstrate to the regulatory authorities that the proposed technique was sufficiently accurate. These validation tests were conducted at a temporary storage area in Fukushima prefecture. Decontaminated waste of various representative types and of various levels of radioactivity was gathered and mixed to create homogeneous volumes. These volumes were sampled multiple times and assayed with laboratory HPGe detectors to determine the reference concentration of each pile. Multiple SSs were loaded from each pile. Some of the SSs were filled 50% full, others 75% full, and others 100% full, to represent the typical loading configuration of the existing SSs in the field. The content of the SSs are either sand, soil, or vegetation with densities ranging from 0.3 g/cc - 1.6 g/cc. These SSs with known concentrations of Cs-134 and Cs-137 were then loaded onto trucks in

  6. Preliminary validation of assays to measure parameters of calcium metabolism in captive Asian and African elephants in western Europe.

    Science.gov (United States)

    van Sonsbeek, Gerda R; van der Kolk, Johannes H; van Leeuwen, Johannes P T M; Schaftenaar, Willem

    2011-05-01

    Hypocalcemia is a well known cause of dystocia in animals, including elephants in captivity. In order to study calcium metabolism in elephants, it is of utmost importance to use properly validated assays, as these might be prone to specific matrix effects in elephant blood. The aim of the current study was to conduct preliminary work for validation of various parameters involved in calcium metabolism in both blood and urine of captive elephants. Basal values of these parameters were compared between Asian elephants (Elephas maximus) and African elephants (Loxodonta africana). Preliminary testing of total calcium, inorganic phosphorus, and creatinine appeared valid for use in plasma and creatinine in urine in both species. Furthermore, measurements of bone alkaline phosphatase and N-terminal telopeptide of type I collagen appeared valid for use in Asian elephants. Mean heparinized plasma ionized calcium concentration and pH were not significantly affected by 3 cycles of freezing and thawing. Storage at 4 °C, room temperature, and 37 °C for 6, 12, and 24 hr did not alter the heparinized plasma ionized calcium concentration in Asian elephants. The following linear regression equation using pH (range: 6.858-7.887) and ionized calcium concentration in heparinized plasma was utilized: iCa(7.4) (mmol/l) = -2.1075 + 0.3130·pH(actual) + 0.8296·iCa(actual) (mmol/l). Mean basal values for pH and plasma in Asian elephant whole blood were 7.40 ± 0.048 and 7.49 ± 0.077, respectively. The urinary specific gravity and creatinine concentrations in both Asian and African elephants were significantly correlated and both were significantly lower in Asian elephants. © 2011 The Author(s)

  7. An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round.

    Science.gov (United States)

    Ehling, G; Hecht, M; Heusener, A; Huesler, J; Gamer, A O; van Loveren, H; Maurer, Th; Riecke, K; Ullmann, L; Ulrich, P; Vandebriel, R; Vohr, H-W

    2005-08-15

    The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern. Similar to the EC(3) values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme.

  8. Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

    Science.gov (United States)

    Ahn, Ilyoung; Kim, Tae-Sung; Jung, Eun-Sun; Yi, Jung-Sun; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Jung, Mi-Sook; Jeon, Eun-Young; Yeo, Kyeong-Uk; Jo, Ji-Hoon; Park, Jung-Eun; Kim, Chang-Yul; Park, Yeong-Chul; Seong, Won-Keun; Lee, Ai-Young; Chun, Young Jin; Jeong, Tae Cheon; Jeung, Eui Bae; Lim, Kyung-Min; Bae, SeungJin; Sohn, Soojung; Heo, Yong

    2016-10-01

    Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

    Science.gov (United States)

    Galanti, L M; Cornu, C; Masson, P L; Robert, A R; Becheanu, D; Lamy, M E; Cambiaso, C L

    1991-05-01

    An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

  10. Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

    Science.gov (United States)

    Lobanov, Vladislav A; Peckle, Maristela; Massard, Carlos L; Brad Scandrett, W; Gajadhar, Alvin A

    2018-03-02

    Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing. We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84

  11. Validity of a commercial kit for detection of antibodies in bovine serum in an endemic area for fasciolosis

    Directory of Open Access Journals (Sweden)

    Aline Nunes Simões

    Full Text Available Abstract Fasciolosis is caused by Fasciola hepatica that affects the bile ducts and liver parenchyma of ruminants, which can result in economic loss. This study aimed to carry out the validity of the commercial kit ELISA® indirect front of the simple fecal sedimentation test used as the standard. 143 samples were collected blood and feces of cattle from Jerome, south of the Espírito Santo. Serum samples were left at -80 °C and used to perform the ELISA kit IDEXX®. All animals to stool examinations were also positive to the ELISA (22 and negative samples to test stool (121, 52 animals reacted positively against the antibody research. The frequency of fasciolosis was 15.4% in the stool examinations and 51.8% by ELISA. The validity was calculated by sensitivity (100%, specificity (57%, positive predictive value (29% and negative predictive value (100%, and the correlation between the tests calculated using the kappa index of 0.35. The better sensitivity of the ELISA commercial kit should not be a separately evaluated, since the cost benefit and the technical facility must be considered.

  12. High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format.

    Science.gov (United States)

    Mercurio, Meagan D; Dambergs, Robert G; Herderich, Markus J; Smith, Paul A

    2007-06-13

    The methyl cellulose precipitable (MCP) tannin assay and a modified version of the Somers and Evans color assay were adapted to high-throughput (HTP) analysis. To improve efficiency of the MCP tannin assay, a miniaturized 1 mL format and a HTP format using 96 well plates were developed. The Somers color assay was modified to allow the standardization of pH and ethanol concentrations of wine samples in a simple one-step dilution with a buffer solution, thus removing inconsistencies between wine matrices prior to analysis and allowing for its adaptation to a HTP format. Validation studies showed that all new formats were efficient, and results were reproducible and analogous to the original formats.

  13. Validation of a Pan-Orthopox Real-Time PCR Assay for the Detection and Quantification of Viral Genomes from Nonhuman Primate Blood

    Science.gov (United States)

    2017-06-19

    Validation of a pan-orthopox real-time PCR assay for the detection and quantification of viral genomes from nonhuman primate blood. Eric...medical countermeasures by the U.S. FDA, the assay was designed to quantitate poxvirus genomic DNA in a nonhuman primate (cynomolgus macaque) blood...monkeypox virus into nonhuman primate blood, we chose to use the HA standard after considering the potential biological safety and logistical issues with

  14. Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

    Science.gov (United States)

    Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

    2007-11-01

    The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

  15. Multi-institutional Validation Study of Commercially Available Deformable Image Registration Software for Thoracic Images

    International Nuclear Information System (INIS)

    Kadoya, Noriyuki; Nakajima, Yujiro; Saito, Masahide; Miyabe, Yuki; Kurooka, Masahiko; Kito, Satoshi; Fujita, Yukio; Sasaki, Motoharu; Arai, Kazuhiro; Tani, Kensuke; Yagi, Masashi; Wakita, Akihisa; Tohyama, Naoki; Jingu, Keiichi

    2016-01-01

    Purpose: To assess the accuracy of the commercially available deformable image registration (DIR) software for thoracic images at multiple institutions. Methods and Materials: Thoracic 4-dimensional (4D) CT images of 10 patients with esophageal or lung cancer were used. Datasets for these patients were provided by DIR-lab ( (dir-lab.com)) and included a coordinate list of anatomic landmarks (300 bronchial bifurcations) that had been manually identified. Deformable image registration was performed between the peak-inhale and -exhale images. Deformable image registration error was determined by calculating the difference at each landmark point between the displacement calculated by DIR software and that calculated by the landmark. Results: Eleven institutions participated in this study: 4 used RayStation (RaySearch Laboratories, Stockholm, Sweden), 5 used MIM Software (Cleveland, OH), and 3 used Velocity (Varian Medical Systems, Palo Alto, CA). The ranges of the average absolute registration errors over all cases were as follows: 0.48 to 1.51 mm (right-left), 0.53 to 2.86 mm (anterior-posterior), 0.85 to 4.46 mm (superior-inferior), and 1.26 to 6.20 mm (3-dimensional). For each DIR software package, the average 3-dimensional registration error (range) was as follows: RayStation, 3.28 mm (1.26-3.91 mm); MIM Software, 3.29 mm (2.17-3.61 mm); and Velocity, 5.01 mm (4.02-6.20 mm). These results demonstrate that there was moderate variation among institutions, although the DIR software was the same. Conclusions: We evaluated the commercially available DIR software using thoracic 4D-CT images from multiple centers. Our results demonstrated that DIR accuracy differed among institutions because it was dependent on both the DIR software and procedure. Our results could be helpful for establishing prospective clinical trials and for the widespread use of DIR software. In addition, for clinical care, we should try to find the optimal DIR procedure using thoracic 4D

  16. Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies.

    Science.gov (United States)

    Koestel, Carole; Simonin, Céline; Belcher, Sandrine; Rösti, Johannes

    2016-08-01

    Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices. © 2016 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists.

  17. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits.

    Science.gov (United States)

    Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin

    2014-10-01

    Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

  18. Inter-laboratory comparison to validate the dicentric assay as a cytogenetic triage tool for medical management of radiation accidents

    Energy Technology Data Exchange (ETDEWEB)

    Beinke, Christina, E-mail: christinabeinke@bundeswehr.org [Bundeswehr Institute of Radiobiology Affiliated to the University of Ulm, Neuherbergstrasse 11, 80937 Munich (Germany); Oestreicher, Ursula [Federal Office for Radiation Protection, Neuherberg (Germany); Riecke, Armin [Department for Internal Medicine, Federal Armed Forces Hospital, Ulm (Germany); Kulka, Ulrike [Federal Office for Radiation Protection, Neuherberg (Germany); Meineke, Viktor [Bundeswehr Institute of Radiobiology Affiliated to the University of Ulm, Neuherbergstrasse 11, 80937 Munich (Germany); Romm, Horst [Federal Office for Radiation Protection, Neuherberg (Germany)

    2011-09-15

    Radiation accidents with exposure of human beings can assume huge dimensions concerning occurring health impairments and essential medical resources such as personnel, patient care management and appropriate medical facilities. Particularly in mass-casualty events, a rapid sorting and allocation of victims to treatment is needed and their classification in medical treatment groups has to be conducted as fast as possible. For triage purposes several approaches can be considered. Clinical signs and symptoms are extremely helpful in estimating radiation effects on an organ-based level, whereas the assessment of radiation effects based on cytogenetic biodosimetry tools is the alternative approach. For both systems there are pros and cons with respect to the usefulness for specific applications, such as individual cases versus mass-casualty screening or whole- versus partial-body exposures. Among the biodosimetry tools the dicentric chromosome assay (DCA) is considered as the 'gold standard' for biodosimetry after an acute radiation exposure. Recently, steady progress in standardization and harmonization of the DCA has occurred, in order to enable the validated performance of the DCA in the frame of cooperative response of biodosimetry networks during a large scale radiological scenario. Using the DCA in triage mode which allows the stratification of radiation exposed victims into broad 1.0 Gy categories only 20-50 metaphase cells per subject are scored instead of the 500-1000 scored for routine analysis. Our data show that there are significant differences between the dicentric yields after 1.0 Gy and 3.0 Gy {gamma}-ray ex vivo exposure of blood suggesting this assay as suitable for the distinction between high and low dosed exposed individuals. These preliminary findings indicate the usefulness of the DCA also for therapeutic decision making.

  19. Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

    Directory of Open Access Journals (Sweden)

    Grazielle Cardoso da Graça

    2012-08-01

    Full Text Available In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70 regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL. A total of 70 Leishmania strains were analysed, including seven reference strains (RS and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP. This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region, internal transcribed spacer (ITS1 and glucose-6-phosphate dehydrogenase (G6pd. A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

  20. Full-Scale Structural and NDI Validation Tests of Bonded Composite Doublers for Commercial Aircraft Applications

    Energy Technology Data Exchange (ETDEWEB)

    Roach, D.; Walkington, P.

    1999-02-01

    Composite doublers, or repair patches, provide an innovative repair technique which can enhance the way aircraft are maintained. Instead of riveting multiple steel or aluminum plates to facilitate an aircraft repair, it is possible to bond a single Boron-Epoxy composite doubler to the damaged structure. Most of the concerns surrounding composite doubler technology pertain to long-term survivability, especially in the presence of non-optimum installations, and the validation of appropriate inspection procedures. This report focuses on a series of full-scale structural and nondestructive inspection (NDI) tests that were conducted to investigate the performance of Boron-Epoxy composite doublers. Full-scale tests were conducted on fuselage panels cut from retired aircraft. These full-scale tests studied stress reductions, crack mitigation, and load transfer capabilities of composite doublers using simulated flight conditions of cabin pressure and axial stress. Also, structures which modeled key aspects of aircraft structure repairs were subjected to extreme tension, shear and bending loads to examine the composite laminate's resistance to disbond and delamination flaws. Several of the structures were loaded to failure in order to determine doubler design margins. Nondestructive inspections were conducted throughout the test series in order to validate appropriate techniques on actual aircraft structure. The test results showed that a properly designed and installed composite doubler is able to enhance fatigue life, transfer load away from damaged structure, and avoid the introduction of new stress risers (i.e. eliminate global reduction in the fatigue life of the structure). Comparisons with test data obtained prior to the doubler installation revealed that stresses in the parent material can be reduced 30%--60% through the use of the composite doubler. Tests to failure demonstrated that the bondline is able to transfer plastic strains into the doubler and that

  1. Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Gornowicz-Porowska, Justyna; Seraszek-Jaros, Agnieszka; Bowszyc-Dmochowska, Monika; Kaczmarek, Elżbieta; Pietkiewicz, Paweł; Bartkiewicz, Paweł; Dmochowski, Marian

    2017-02-01

    Pemphigus and bullous pemphigoid (BP) are identified by autoantibodies (abs) against desmoglein 1, 3 (DSG1/3) and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF) using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection) and modified (IgG4 detection) mosaic for indirect immunofluorescence (IIFc - IIF commercial, IIFm - IIF modified) and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP) molecular diagnostics. To compare diagnostic accuracy of commercial (IgG detection) and modified (IgG4 detection) mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher's exact test. There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1)/desmoglein3 (DSG3)/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm.

  2. Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays

    Directory of Open Access Journals (Sweden)

    Justyna Gornowicz-Porowska

    2017-02-01

    Full Text Available Introduction : Pemphigus and bullous pemphigoid (BP are identified by autoantibodies (abs against desmoglein 1, 3 (DSG1/3 and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection and modified (IgG4 detection mosaic for indirect immunofluorescence (IIFc – IIF commercial, IIFm – IIF modified and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP molecular diagnostics. Aim : To compare diagnostic accuracy of commercial (IgG detection and modified (IgG4 detection mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Material and methods : Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher’s exact test. Results: There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1/desmoglein3 (DSG3/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. Conclusions : The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm.

  3. Performance and diagnostic usefulness of commercially available enzyme linked immunosorbent assay and rapid kits for detection of HIV, HBV and HCV in India

    Directory of Open Access Journals (Sweden)

    Maity Susmita

    2012-11-01

    Full Text Available Abstract Background HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. Results A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.; ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd. gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd., Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd. did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100% except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.. The kit efficiency didn’t vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p  Conclusions ELISA is a good screening assay for markers of HIV, HBV and HCV infections. Rapid tests are useful for

  4. Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.

    Science.gov (United States)

    Heilmann, Romy M; Cranford, Shannon M; Ambrus, Andy; Grützner, Niels; Schellenberg, Stefan; Ruaux, Craig G; Suchodolski, Jan S; Steiner, Jörg M

    2016-03-01

    Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers. The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces. Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated. Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations. The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs. © 2016 American Society for Veterinary Clinical Pathology.

  5. Validation of a second-generation multivariate index assay for malignancy risk of adnexal masses.

    Science.gov (United States)

    Coleman, Robert L; Herzog, Thomas J; Chan, Daniel W; Munroe, Donald G; Pappas, Todd C; Smith, Alan; Zhang, Zhen; Wolf, Judith

    2016-07-01

    Women with adnexal mass suspected of ovarian malignancy are likely to benefit from consultation with a gynecologic oncologist, but imaging and biomarker tools to ensure this referral show low sensitivity and may miss cancer at critical stages. The multivariate index assay (MIA) was designed to improve the detection of ovarian cancer among women undergoing surgery for a pelvic mass. To improve the prediction of benign masses, we undertook the redesign and validation of a second-generation MIA (MIA2G). MIA2G was developed using banked serum samples from a previously published prospective, multisite registry of patients who underwent surgery to remove an adnexal mass. Clinical validity was then established using banked serum samples from the OVA500 trial, a second prospective cohort of adnexal surgery patients. Based on the final pathology results of the OVA500 trial, this intended-use population for MIA2G testing was high risk, with an observed cancer prevalence of 18.7% (92/493). Coded samples were assayed for MIA2G biomarkers by an external clinical laboratory. Then MIA2G results were calculated and submitted to a clinical statistics contract organization for decoding and comparison to MIA results for each subject. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, among other measures, and stratified by menopausal status, stage, and histologic subtype. Three MIA markers (cancer antigen 125, transferrin, and apolipoprotein A-1) and 2 new biomarkers (follicle-stimulating hormone and human epididymis protein 4) were included in MIA2G. A single cut-off separated high and low risk of malignancy regardless of patient menopausal status, eliminating potential for confusion or error. MIA2G specificity (69%, 277/401 [n/N]; 95% confidence interval [CI], 64.4-73.4%) and PPV (40%, 84/208; 95% CI, 33.9-47.2%) were significantly improved over MIA (specificity, 54%, 215/401; 95% CI, 48.7-58.4%, and PPV, 31%, 85/271; 95

  6. High-throughput screening assay used in pharmacognosy: Selection, optimization and validation of methods of enzymatic inhibition by UV-visible spectrophotometry

    Directory of Open Access Journals (Sweden)

    Graciela Granados-Guzmán

    2014-02-01

    Full Text Available In research laboratories of both organic synthesis and extraction of natural products, every day a lot of products that can potentially introduce some biological activity are obtained. Therefore it is necessary to have in vitro assays, which provide reliable information for further evaluation in in vivo systems. From this point of view, in recent years has intensified the use of high-throughput screening assays. Such trials should be optimized and validated for accurate and precise results, i.e. reliable. The present review addresses the steps needed to develop and validate bioanalytical methods, emphasizing UV-Visible spectrophotometry as detection system. Particularly focuses on the selection of the method, the optimization to determine the best experimental conditions, validation, implementation of optimized and validated method to real samples, and finally maintenance and possible transfer it to a new laboratory.

  7. Validation and application of a high-performance liquid chromatography--tandem mass spectrometry assay for mosapride in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Chidambara, J; Varma, D P

    2005-09-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of mosapride (I), a novel and potent gastroprokinetic agent that enhances the upper gastrointestinal motility by stimulating 5-HT(4) receptor. The analyte and internal standard, tamsulosin (II), were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase Waters symmetry C(18) column with a mobile phase of 0.03% formic acid-acetonitrile (10:90, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 422.3 -->198.3 and m/z 409.1 -->228.1 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-100.0 ng/mL for mosapride in human plasma. The lower limit of quantitation was 500 pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. Copyright (c) 2005 John Wiley & Sons, Ltd.

  8. An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round.

    Science.gov (United States)

    Ehling, G; Hecht, M; Heusener, A; Huesler, J; Gamer, A O; van Loveren, H; Maurer, Th; Riecke, K; Ullmann, L; Ulrich, P; Vandebriel, R; Vohr, H-W

    2005-08-15

    The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8--10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A--C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies.

  9. An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round

    International Nuclear Information System (INIS)

    Ehling, G.; Hecht, M.; Heusener, A.; Huesler, J.; Gamer, A.O.; Loveren, H. van; Maurer, Th.; Riecke, K.; Ullmann, L.; Ulrich, P.; Vandebriel, R.; Vohr, H.-W.

    2005-01-01

    The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8-10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A-C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies

  10. A validated specific stability-indicating RP-HPLC assay method for Ambrisentan and its related substances.

    Science.gov (United States)

    Narayana, M B V; Chandrasekhar, K B; Rao, B M

    2014-09-01

    A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability). © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.

    Science.gov (United States)

    Hermanrud, Christina; Ryner, Malin; Luft, Thomas; Jensen, Poul Erik; Ingenhoven, Kathleen; Rat, Dorothea; Deisenhammer, Florian; Sørensen, Per Soelberg; Pallardy, Marc; Sikkema, Dan; Bertotti, Elisa; Kramer, Daniel; Creeke, Paul; Fogdell-Hahn, Anna

    2016-03-01

    Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low

  12. Cross-species assay validation using the AOP “deiodinase inhibition leading to impaired posterior chamber inflation”

    Science.gov (United States)

    High throughput screening assays able to detect chemical interactions with specific biological targets are increasingly being used to identify chemicals that could be hazardous to humans or wildlife. Most of these assays examine interaction with mammalian proteins. The present wo...

  13. Development and validation of a HPLC method for standardization of herbal and commercial extracts of Myrcia uniflora

    Directory of Open Access Journals (Sweden)

    Andrea N. de L. Batista

    2011-06-01

    Full Text Available Myrcia uniflora Barb. Rodr., Myrtaceae, popularly known as "pedra-hume-caá" in Brazil, is sold as dry extracts in capsules or as tinctures for the treatment of diabetes mellitus. Previous phytochemical studies on this species described the occurrence of the flavonoids mearnsitrin and myricitrin. In the present study, the chromatographic profiles of M. uniflora leaves and commercial extracts were determined using HPLC-PAD. Myricitrin was used as an external standard in the development and validation of the HPLC method. The proposed method is simple, rapid and reliable and can be successfully applied in industry for standardization of herbs and phytomedicines commercialised in Brazil as "pedra-hume-caá".

  14. Development and validation of a HPLC method for standardization of herbal and commercial extracts of Myrcia uniflora

    Directory of Open Access Journals (Sweden)

    Andrea N. de L. Batista

    2011-04-01

    Full Text Available Myrcia uniflora Barb. Rodr., Myrtaceae, popularly known as "pedra-hume-caá" in Brazil, is sold as dry extracts in capsules or as tinctures for the treatment of diabetes mellitus. Previous phytochemical studies on this species described the occurrence of the flavonoids mearnsitrin and myricitrin. In the present study, the chromatographic profiles of M. uniflora leaves and commercial extracts were determined using HPLC-PAD. Myricitrin was used as an external standard in the development and validation of the HPLC method. The proposed method is simple, rapid and reliable and can be successfully applied in industry for standardization of herbs and phytomedicines commercialised in Brazil as "pedra-hume-caá".

  15. Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Frederiksen, Hanne; Andersson, Anna-Maria

    2018-01-01

    BACKGROUND: The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantificat......BACKGROUND: The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method...

  16. Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar

    Directory of Open Access Journals (Sweden)

    Gortázar Christian

    2008-11-01

    Full Text Available Abstract Background Bovine tuberculosis (bTB remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures. Results An enzyme-linked immunosorbent assay (ELISA to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off. Conclusion Although some negative group animals showed an ELISA positive reaction (

  17. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

    Directory of Open Access Journals (Sweden)

    Ondigo Bartholomew N

    2012-12-01

    Full Text Available Abstract Background Multiplex cytometric bead assay (CBA have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

  18. Validity of a Commercial Linear Encoder to Estimate Bench Press 1 RM from the Force-Velocity Relationship.

    Science.gov (United States)

    Bosquet, Laurent; Porta-Benache, Jeremy; Blais, Jérôme

    2010-01-01

    The aim of this study was to assess the validity and accuracy of a commercial linear encoder (Musclelab, Ergotest, Norway) to estimate Bench press 1 repetition maximum (1RM) from the force - velocity relationship. Twenty seven physical education students and teachers (5 women and 22 men) with a heterogeneous history of strength training participated in this study. They performed a 1 RM test and a force - velocity test using a Bench press lifting task in a random order. Mean 1 RM was 61.8 ± 15.3 kg (range: 34 to 100 kg), while 1 RM estimated by the Musclelab's software from the force-velocity relationship was 56.4 ± 14.0 kg (range: 33 to 91 kg). Actual and estimated 1 RM were very highly correlated (r = 0.93, pvelocity relationship was a good measure for monitoring training induced adaptations, but also that it was not accurate enough to prescribe training intensities. Additional studies are required to determine whether accuracy is affected by age, sex or initial level. Key pointsSome commercial devices allow to estimate 1 RM from the force-velocity relationship.These estimations are valid. However, their accuracy is not high enough to be of practical help for training intensity prescription.Day-to-day reliability of force and velocity measured by the linear encoder has been shown to be very high, but the specific reliability of 1 RM estimated from the force-velocity relationship has to be determined before concluding to the usefulness of this approach in the monitoring of training induced adaptations.

  19. Validity of a Commercial Linear Encoder to Estimate Bench Press 1 RM from the Force-Velocity Relationship

    Science.gov (United States)

    Bosquet, Laurent; Porta-Benache, Jeremy; Blais, Jérôme

    2010-01-01

    The aim of this study was to assess the validity and accuracy of a commercial linear encoder (Musclelab, Ergotest, Norway) to estimate Bench press 1 repetition maximum (1RM) from the force - velocity relationship. Twenty seven physical education students and teachers (5 women and 22 men) with a heterogeneous history of strength training participated in this study. They performed a 1 RM test and a force - velocity test using a Bench press lifting task in a random order. Mean 1 RM was 61.8 ± 15.3 kg (range: 34 to 100 kg), while 1 RM estimated by the Musclelab’s software from the force-velocity relationship was 56.4 ± 14.0 kg (range: 33 to 91 kg). Actual and estimated 1 RM were very highly correlated (r = 0.93, p<0.001) but largely different (Bias: 5.4 ± 5.7 kg, p < 0.001, ES = 1.37). The 95% limits of agreement were ±11.2 kg, which represented ±18% of actual 1 RM. It was concluded that 1 RM estimated from the force-velocity relationship was a good measure for monitoring training induced adaptations, but also that it was not accurate enough to prescribe training intensities. Additional studies are required to determine whether accuracy is affected by age, sex or initial level. Key points Some commercial devices allow to estimate 1 RM from the force-velocity relationship. These estimations are valid. However, their accuracy is not high enough to be of practical help for training intensity prescription. Day-to-day reliability of force and velocity measured by the linear encoder has been shown to be very high, but the specific reliability of 1 RM estimated from the force-velocity relationship has to be determined before concluding to the usefulness of this approach in the monitoring of training induced adaptations. PMID:24149641

  20. Rapid validated HPTLC method for estimation of piperine and piperlongumine in root of Piper longum extract and its commercial formulation

    Directory of Open Access Journals (Sweden)

    Anagha A. Rajopadhye

    2012-12-01

    Full Text Available Piperine and piperlongumine, alkaloids having diverse biological activities, commonly occur in roots of Piper longum L., Piperaceae, which have high commercial, economical and medicinal value. In present study, rapid, validated HPTLC method has been established for the determination of piperine and piperlongumine in methanolic root extract and its commercial formulation 'Mahasudarshan churna®' using ICH guidelines. The use of Accelerated Solvent Extraction (ASE as an alternative to conventional techniques has been explored. The methanol extracts of root, its formulation and both standard solutions were applied on silica gel F254 HPTLC plates. The plates were developed in Twin chamber using mobile phase toluene: ethyl acetate (6:4, v/v and scanned at 342 and 325 nm (λmax of piperine and piperlongumine, respectively using Camag TLC scanner 3 with CATS 4 software. A linear relationship was obtained between response (peak area and amount of piperine and piperlongumine in the range of 20-100 and 30-150 ng/spot, respectively; the correlation coefficient was 0.9957 and 0.9941 respectively. Sharp, symmetrical and well resolved peaks of piperine and piperlongumine spots resolved at Rf 0.51 and 0.74, respectively from other components of the sample extracts. The HPTLC method showed good linearity, recovery and high precision of both markers. Extraction of plant using ASE and rapid HPTLC method provides a new and powerful approach to estimate piperine and piperlongumine as phytomarkers in the extract as well as its commercial formulations for routine quality control.

  1. A validated HPLC-MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: application to a clinical pharmacokinetic study.

    Science.gov (United States)

    Furlong, Michael T; Savant, Ishani; Yuan, Moucun; Scott, Laura; Mylott, William; Mariannino, Thomas; Kadiyala, Pathanjali; Roongta, Vikram; Arnold, Mark E

    2013-05-01

    Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies.

    Science.gov (United States)

    De La Vega, Francisco M; Dailey, David; Ziegle, Janet; Williams, Julie; Madden, Dawn; Gilbert, Dennis A

    2002-06-01

    Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.

  3. Validation of the (Troponin-only) Manchester ACS decision aid with a contemporary cardiac troponin I assay.

    Science.gov (United States)

    Va Den Berg, Patricia; Burrows, Gillian; Lewis, Philip; Carley, Simon; Body, Richard

    2018-04-01

    The Manchester Acute Coronary Syndromes (MACS) decision aid can 'rules in' and 'rule out' acute coronary syndromes (ACS) by combining a patient's symptoms with the results of a single blood test taken at the time of arrival in the Emergency Department (ED). The original model (MACS) included two biomarkers: high sensitivity cardiac troponin T (hs-cTnT) and heart-type fatty acid binding protein (h-FABP). A refined model without h-FABP was found to have comparable sensitivity but greater specificity. We sought to validate MACS and T-MACS using the contemporary Siemens Advia Centaur cardiac troponin I assay to increase usability in practice. This is a secondary analysis from prospective diagnostic cohort study at Stepping Hill Hospital, United Kingdom. Patients presenting with chest pain of suspected cardiac nature warranting rule out for ACS were included. All patients underwent hs-cTnT testing at least 12h after peak symptoms. The primary outcome was a diagnosis of ACS, defined as either prevalent acute myocardial infarction (AMI) or incident major adverse cardiac events (death, AMI or coronary revascularization) within 30days. Of 405 included patients, 76 (18.8%) had ACS. MACS and T-MACS had similar C-statistics (0.94 for each, p=0.36) and sensitivity (difference 1.3%, 95% CI -1.3 to 3.9%, p=1.00) but T-MACS had significantly greater specificity (difference 16.7%, 95% CI 14.6-18.9%, p<0.0001). T-MACS and MACS would have allowed 36.3% and 22.5% patients to be immediately discharged respectively. Of patients classified as 'very low risk', none had ACS when MACS was used compared to one (0.7%) with T-MACS. Both MACS and T-MACS effectively ruled out ACS even with a contemporary troponin I assay and could be used to reduce unnecessary hospital admissions. Copyright © 2017. Published by Elsevier Inc.

  4. Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments.

    Science.gov (United States)

    das Neves, José; Sarmento, Bruno; Amiji, Mansoor; Bahia, Maria Fernanda

    2012-12-12

    Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50μL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02-1.5μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02μg/mL), limit of detection (0.006μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti

  5. Performance and diagnostic usefulness of commercially available enzyme linked immunosorbent assay and rapid kits for detection of HIV, HBV and HCV in India.

    Science.gov (United States)

    Maity, Susmita; Nandi, Srijita; Biswas, Subrata; Sadhukhan, Salil Kumar; Saha, Malay Kumar

    2012-11-26

    HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.); ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd.) gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd.), Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd.) did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100%) except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.). The kit efficiency didn't vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p bank. For availability of quality commercial diagnostic assays, evaluation of kit may be helpful.

  6. Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta

    DEFF Research Database (Denmark)

    Hermanrud, Christina; Ryner, Malin; Luft, Thomas

    2016-01-01

    a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays......Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach...... to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines...

  7. Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

    Science.gov (United States)

    Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

    2010-08-01

    Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as

  8. Development and validation of a gas chromatography-mass spectrometry assay for opiates and cocaine in human teeth.

    Science.gov (United States)

    Pellegrini, Manuela; Casá, Adriana; Marchei, Emilia; Pacifici, Roberta; Mayné, Ruth; Barbero, Vanessa; Garcia-Algar, Oscar; Pichini, Simona

    2006-02-24

    A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for determination of opiates (6-monoacetylmorphine, morphine and codeine) and cocaine and metabolites (cocaine, benzoylecgonine and cocaethylene) in human teeth. After addition of nalorphine as internal standard, pulverized samples were incubated in HCl at 37 degrees C for 18 h. Then, after pH adjustment to 6, and the analytes were extracted with two volumes of 3 ml of chloroform/isopropanol (9:1). Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 7.5 (6.0 in case of codeine) to 500 ng/g with mean absolute recoveries ranged between 74.1 and 92.1% for the different analytes and precision and accuracy always better than 15%. The method was applied to the analysis of teeth from drug-addicts to assess past chronic consumption and verify self-reported declarations. In case of opiates, concentration range was 36.5-570.0 ng/g for 6-monoacetylmorphine, 8.7-154.8 ng/g for morphine and 7.9-127.9 ng/g for codeine. Cocaine concentration ranged between 5.6 and 57.2 ng/g with its principal metabolite benzoylecgonine varying from 12.6 to 81.7 ng/g and cocaethylene present in only one sample at 10 ng/g value. Teeth can be a promising non-invasive biological matrix in biomedical analysis for both clinical and forensic purposes.

  9. Development and validation of spectrophotometric method for assay determination and in vitro dissolution studies of sofosbuvir tablets

    International Nuclear Information System (INIS)

    Zaman, B.; Hassan, W.; Noreen, H.

    2017-01-01

    In vitro dissolution of sofosbuvir 400 mg tablets dosage form was performed, using USP dissolution apparatus type-II (paddle type), at 75rpm ± 4 %, and 900mL ± 1%, 0.05 M phosphate buffer pH 6.8 ± 0.05 equilibrated at 37.0 ± 0.5ºC as dissolution medium. Percentage of dissolved sofosbuvir as a function of time was determined using the straight line equation and linear regression using zero order and first order ANOVA based kinetics model. Comparative dissolution studies on two different generic brands A and B was performed comparing the drug release profile with innovator brand Sovaldi 400 mg tablets. The comparison of dissolution profiles was evaluated using model independent approach. The values of similarity factor f2 were (4 and 3) and the difference factor f1 were (64 and 50) for both generic products A and B respectively. A simple and precise spectrophotometric method was developed for estimation of sofosbuvir in dissolution medium based on spectrophotometric detection at wavelength 262 nm. The specific absorbance (A = 1%) of sofosbuvir was 178.5 ± 4% and Beer’s law was obeyed in the concentration ranges 4µg mL−1 to 48µg mL−1. The method was validated appropriately for accuracy, precision, linearity, and specificity, according the guidelines of United State Pharmacopoeia and International Conference on Harmonization. The calibration curve was linear with correlation coefficient (r > 0.9999) and there was no spectral interference from excipients present in the tablets dosage form. This method is precise, rapid and specific for determination of sofosbuvir in tablets dosage form and successfully applied for assay determination and in vitro dissolution studies. (author)

  10. Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2'-deoxyuridine incorporation.

    Science.gov (United States)

    Kojima, Hajime; Takeyoshi, Masahiro; Sozu, Takashi; Awogi, Takumi; Arima, Kazunori; Idehara, Kenji; Ikarashi, Yoshiaki; Kanazawa, Yukiko; Maki, Eiji; Omori, Takashi; Yuasa, Atsuko; Yoshimura, Isao

    2011-01-01

    The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5-bromo-2'-deoxyuridine (BrdU) incorporation (LLNA:BrdU-ELISA). The LLNA:BrdU-ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU-ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut-off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU-ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd.

  11. Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples.

    Science.gov (United States)

    Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Oldroyd, Nicola J; Hennessy, Lori K

    2011-07-01

    The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping. © 2011 American Academy of Forensic Sciences.

  12. Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1.

    Science.gov (United States)

    Decaro, Nicola; Amorisco, Francesca; Desario, Costantina; Lorusso, Eleonora; Camero, Michele; Bellacicco, Anna Lucia; Sciarretta, Rossana; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio

    2010-10-01

    A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  13. Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae

    DEFF Research Database (Denmark)

    Rashid, Ridwan Bin; Ferdous, Jannataul; Tulsiani, Suhella

    2017-01-01

    is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24–1.32) and remarkable reproducibility (CV%: 1.08–3.7). Amplification efficiencies in the 89–100% range were observed. The assay is more economical than Taqman-based multiplex...

  14. Evaluation and validation of a real-time PCR assay for detection and quantitation of human adenovirus 14 from clinical samples.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available In 2007, the Centers for Disease Control and Prevention (CDC reported that Human adenovirus type 14 (HAdV-14 infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10(2 to 10(7 copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.

  15. VALIDITY OF A COMMERCIAL LINEAR ENCODER TO ESTIMATE BENCH PRESS 1 RM FROM THE FORCE-VELOCITY RELATIONSHIP

    Directory of Open Access Journals (Sweden)

    Laurent Bosquet

    2010-09-01

    Full Text Available The aim of this study was to assess the validity and accuracy of a commercial linear encoder (Musclelab, Ergotest, Norway to estimate Bench press 1 repetition maximum (1RM from the force - velocity relationship. Twenty seven physical education students and teachers (5 women and 22 men with a heterogeneous history of strength training participated in this study. They performed a 1 RM test and a force - velocity test using a Bench press lifting task in a random order. Mean 1 RM was 61.8 ± 15.3 kg (range: 34 to 100 kg, while 1 RM estimated by the Musclelab's software from the force-velocity relationship was 56.4 ± 14.0 kg (range: 33 to 91 kg. Actual and estimated 1 RM were very highly correlated (r = 0.93, p<0.001 but largely different (Bias: 5.4 ± 5.7 kg, p < 0.001, ES = 1.37. The 95% limits of agreement were ±11.2 kg, which represented ±18% of actual 1 RM. It was concluded that 1 RM estimated from the force-velocity relationship was a good measure for monitoring training induced adaptations, but also that it was not accurate enough to prescribe training intensities. Additional studies are required to determine whether accuracy is affected by age, sex or initial level.

  16. Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

    Science.gov (United States)

    Ondigo, Bartholomew N; Park, Gregory S; Gose, Severin O; Ho, Benjamin M; Ochola, Lyticia A; Ayodo, George O; Ofulla, Ayub V; John, Chandy C

    2012-12-21

    Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in

  17. Validation of the Thermo Scientific SureTect Escherichia coli O157:H7 Real-Time PCR Assay for Raw Beef and Produce Matrixes.

    Science.gov (United States)

    Cloke, Jonathan; Crowley, Erin; Bird, Patrick; Bastin, Ben; Flannery, Jonathan; Agin, James; Goins, David; Clark, Dorn; Radcliff, Roy; Wickstrand, Nina; Kauppinen, Mikko

    2015-01-01

    The Thermo Scientific™ SureTect™ Escherichia coli O157:H7 Assay is a new real-time PCR assay which has been validated through the AOAC Research Institute (RI) Performance Tested Methods(SM) program for raw beef and produce matrixes. This validation study specifically validated the assay with 375 g 1:4 and 1:5 ratios of raw ground beef and raw beef trim in comparison to the U.S. Department of Agriculture, Food Safety Inspection Service, Microbiology Laboratory Guidebook (USDS-FSIS/MLG) reference method and 25 g bagged spinach and fresh apple juice at a ratio of 1:10, in comparison to the reference method detailed in the International Organization for Standardization 16654:2001 reference method. For raw beef matrixes, the validation of both 1:4 and 1:5 allows user flexibility with the enrichment protocol, although which of these two ratios chosen by the laboratory should be based on specific test requirements. All matrixes were analyzed by Thermo Fisher Scientific, Microbiology Division, Vantaa, Finland, and Q Laboratories Inc, Cincinnati, Ohio, in the method developer study. Two of the matrixes (raw ground beef at both 1:4 and 1:5 ratios) and bagged spinach were additionally analyzed in the AOAC-RI controlled independent laboratory study, which was conducted by Marshfield Food Safety, Marshfield, Wisconsin. Using probability of detection statistical analysis, no significant difference was demonstrated by the SureTect kit in comparison to the USDA FSIS reference method for raw beef matrixes, or with the ISO reference method for matrixes of bagged spinach and apple juice. Inclusivity and exclusivity testing was conducted with 58 E. coli O157:H7 and 54 non-E. coli O157:H7 isolates, respectively, which demonstrated that the SureTect assay was able to detect all isolates of E. coli O157:H7 analyzed. In addition, all but one of the nontarget isolates were correctly interpreted as negative by the SureTect Software. The single isolate giving a positive result was an E

  18. Validation, optimisation, and application data in support of the development of a targeted selected ion monitoring assay for degraded cardiac troponin T

    Directory of Open Access Journals (Sweden)

    Alexander S. Streng

    2016-06-01

    Full Text Available Cardiac troponin T (cTnT fragmentation in human serum was investigated using a newly developed targeted selected ion monitoring assay, as described in the accompanying article: “Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T” [1]. This article presents data describing aspects of the validation and optimisation of this assay. The data consists of several figures, an excel file containing the results of a sequence identity search, and a description of the raw mass spectrometry (MS data files, deposited in the ProteomeXchange repository with id PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003187.

  19. Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard

    DEFF Research Database (Denmark)

    Børsting, Claus; Rockenbauer, Eszter; Morling, Niels

    2009-01-01

    cases and 33 twin cases were typed at least twice for the 49 SNPs. All electropherograms were analysed independently by two expert analysts prior to approval. Based on these results, detailed guidelines for analysis of the SBE products were developed. With these guidelines, the peak height ratio...... of a heterozygous allele call or the signal to noise ratio of a homozygous allele call is compared with previously obtained ratios. A laboratory protocol for analysis of SBE products was developed where allele calls with unusual ratios were highlighted to facilitate the analysis of difficult allele calls......A multiplex assay with 49 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was validated for forensic genetic casework and accredited according to the ISO 17025 standard. The multiplex assay was based on the SNPforID 52plex SNP assay [J.J. Sanchez, C. Phillips, C...

  20. Evaluation of a commercial rubella IgM assay for use on oral fluid samples for diagnosis and surveillance of congenital rubella syndrome and postnatal rubella.

    Science.gov (United States)

    Vijaylakshmi, P; Muthukkaruppan, V R; Rajasundari, A; Korukluoglu, G; Nigatu, W; L A Warrener; Samuel, D; Brown, D W G

    2006-12-01

    Clinical diagnosis (surveillance) of rubella is unreliable and laboratory confirmation is essential. Detection of virus specific IgM in serum is the most commonly used method. However, the use of serum necessitates the drawing of blood, either through venipuncture or finger/heel prick, which can be difficult in young babies. Oral fluid samples have proved useful as an alternative, less invasive sample for virus specific IgM detection however until recently no commercial rubella IgM tests were available, restricting the usefulness of this approach. To evaluate the performance of the Microimmune Rubella IgM capture EIA using oral fluid samples from outbreaks as well as in cases of suspected congenital rubella syndrome (CRS). Paired serum and oral fluids were collected from cases during a rubella outbreak in three provinces in Turkey. Matched serum and oral fluid samples were collected from children with suspected CRS in an active surveillance programme at the Aravind Eye Hospital in South India. Serum samples were collected as part of the measles surveillance programme in Ethiopia. On serum samples the sensitivity and specificity of the Microimmune Rubella IgM capture EIA compared to Behring Enzygnost rubella IgM test was 96.9% (62/64; 95% CI 94.2-100%) and 100% (53/53; 95% CI 93.2-100%). On oral fluids compared to matched Behring results on serum the sensitivity was 95.5% (42/44; 95% CI 84.5-99.4%). The sensitivity and specificity of Microimmune Rubella IgM capture EIA on oral fluids from suspected CRS cases compared to serum results using Behring Enzygnost IgM assay was 100% (95% CI 84.5-100%) and 100% (95% CI 95.8-100.0%) respectively. Microimmune Rubella IgM capture EIA has adequate performance for diagnosis and surveillance of rubella in outbreak using either serum or oral fluid specimens.

  1. Combination of Pharmacophore Matching, 2D Similarity Search, and In Vitro Biological Assays in the Selection of Potential 5-HT6 Antagonists from Large Commercial Repositories.

    Science.gov (United States)

    Dobi, Krisztina; Flachner, Beáta; Pukáncsik, Mária; Máthé, Enikő; Bognár, Melinda; Szaszkó, Mária; Magyar, Csaba; Hajdú, István; Lőrincz, Zsolt; Simon, István; Fülöp, Ferenc; Cseh, Sándor; Dormán, György

    2015-10-01

    Rapid in silico selection of target-focused libraries from commercial repositories is an attractive and cost-effective approach. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compound databases, but the generated library requires further focusing. We report here a combination of the 2D approach with pharmacophore matching which was used for selecting 5-HT6 antagonists. In the first screening round, 12 compounds showed >85% antagonist efficacy of the 91 screened. For the second-round (hit validation) screening phase, pharmacophore models were built, applied, and compared with the routine 2D similarity search. Three pharmacophore models were created based on the structure of the reference compounds and the first-round hit compounds. The pharmacophore search resulted in a high hit rate (40%) and led to novel chemotypes, while 2D similarity search had slightly better hit rate (51%), but lacking the novelty. To demonstrate the power of the virtual screening cascade, ligand efficiency indices were also calculated and their steady improvement was confirmed. © 2015 John Wiley & Sons A/S.

  2. Validation of clinical testing for warfarin sensitivity: comparison of CYP2C9-VKORC1 genotyping assays and warfarin-dosing algorithms.

    Science.gov (United States)

    Langley, Michael R; Booker, Jessica K; Evans, James P; McLeod, Howard L; Weck, Karen E

    2009-05-01

    Responses to warfarin (Coumadin) anticoagulation therapy are affected by genetic variability in both the CYP2C9 and VKORC1 genes. Validation of pharmacogenetic testing for warfarin responses includes demonstration of analytical validity of testing platforms and of the clinical validity of testing. We compared four platforms for determining the relevant single nucleotide polymorphisms (SNPs) in both CYP2C9 and VKORC1 that are associated with warfarin sensitivity (Third Wave Invader Plus, ParagonDx/Cepheid Smart Cycler, Idaho Technology LightCycler, and AutoGenomics Infiniti). Each method was examined for accuracy, cost, and turnaround time. All genotyping methods demonstrated greater than 95% accuracy for identifying the relevant SNPs (CYP2C9 *2 and *3; VKORC1 -1639 or 1173). The ParagonDx and Idaho Technology assays had the shortest turnaround and hands-on times. The Third Wave assay was readily scalable to higher test volumes but had the longest hands-on time. The AutoGenomics assay interrogated the largest number of SNPs but had the longest turnaround time. Four published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy for predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. The predicted doses from both the Washington University and UCSF algorithms demonstrated the best correlation with actual warfarin doses.

  3. Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

    Directory of Open Access Journals (Sweden)

    Elisa Fanunza

    2018-02-01

    Full Text Available The interferon (IFN system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24 is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE. Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

  4. Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Heidi A. Neubauer

    2017-03-01

    Full Text Available Sphingosine kinase 2 (SK2 is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs. Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout (Sphk2-/- MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

  5. SU-E-J-52: Validation of 3D Structure Projection Onto 2D DRR in Commercial Treatment Planning Systems.

    Science.gov (United States)

    Zhang, L; Court, L; Balter, P; Dong, L

    2012-06-01

    The use of structure overlay on setup DRRs can aid the image alignment procedure for daily image-guided setup procedures. However, the accuracy of a 3D region-of-interest (ROI) projected on a 2D digitally reconstructed radiograph (DRR) has rarely been evaluated quantitatively. The goal of this study is to test the accuracy of two commercial treatment planning systems (TPS) in producing overlay structures on setup DRRs. We designed a novel method to identify landmarks which were on the boundary of the projected ROI on a DRR. The 3D ROIvolume is composed of a stack of 2D curves. We first mathematically project each 2D curve onto a beams-eye-view (BEV) plane. Next, we detectthe boundary points of the projected curves. Those boundary points serve aslandmarks. Finally, we project the binary mask of the 3D ROI volume using ray tracing method onto the BEV plane. This projected binary mask is used to exclude the false landmarks. Once those landmarks are detected, wecompute the distance between the landmarks and ROI outlines from the TPS. We applied our validation method to 13 ROIs from a lung patient and 4 simulated ROIs on 2 BEV DRRs for two different TPS (Eclipse and Pinnacle). Average distance between the landmarks and ROIoutlines was 0.5mm for both Eclipse and Pinnacle approaches, which is close to the pixel resolution of the DRR. The maximum distance andaverage maximum distance was 2mm and 1 mm, respectively, for both TPS.The maximum distance occurred at points where the ROI curve has a sharpchange between slices. The accuracy of Eclipse and Pinnacle ROI projection method seems to be acceptable to within 1mm althoughprojection error can be as large as 2mm when structure shape has a sharp variation from one slice to the next. © 2012 American Association of Physicists in Medicine.

  6. Evaluation of Commercial Diagnostic Assays for the Specific Detection of Avian Influenza A (H7N9) Virus RNA Using a Quality-Control Panel and Clinical Specimens in China

    Science.gov (United States)

    Chen, Suhong; Wang, Dayan; Li, Changgui; Wu, Xing; Li, Lili; Bai, Dongting; Zhang, Chuntao; Wang, Junzhi

    2015-01-01

    A novel avian influenza A H7N9-subtype virus emerged in China in 2013 and threatened global public health. Commercial kits that specifically detect avian influenza A (H7N9) virus RNA are urgently required to prepare for the emergence and potential pandemic of this novel influenza virus. The safety and effectiveness of three commercial molecular diagnostic assays were evaluated using a quality-control panel and clinical specimens collected from over 90 patients with confirmed avian influenza A (H7N9) virus infections. The analytical performance evaluation showed that diverse influenza H7N9 viruses can be detected with high within- and between-lot reproducibility and without cross-reactivity to other influenza viruses (H1N1 pdm09, seasonal H1N1, H3N2, H5N1 and influenza B). The detection limit of all the commercial assays was 2.83 Log10 copies/μl [0.7 Log10TCID50/mL of avian influenza A (H7N9) virus strain A/Zhejiang/DTID-ZJU01/2013], which is comparable to the method recommended by the World Health Organization (WHO). In addition, using a WHO-Chinese National Influenza Center (CNIC) method as a reference for clinical evaluation, positive agreement of more than 98% was determined for all of the commercial kits, while negative agreement of more than 99% was observed. In conclusion, our findings provide comprehensive evidence for the high performance of three commercial diagnostic assays and suggest the application of these assays as rapid and effective diagnostic tools for avian influenza A (H7N9) virus in the routine clinical practice of medical laboratories. PMID:26361351

  7. A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

    Science.gov (United States)

    Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

    2015-12-01

    Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

  8. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

    Science.gov (United States)

    Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

    2013-01-01

    The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the

  9. A practical approach for the validation and clinical implementation of a high-sensitivity cardiac troponin I assay across a North American city

    Directory of Open Access Journals (Sweden)

    Peter A. Kavsak

    2015-04-01

    Full Text Available Objectives: Despite several publications on the analytical performance of high-sensitivity cardiac troponin (hs-cTn assays, there has been little information on how laboratories should validate and implement these assays into clinical service. Our study provides a practical approach for the validation and implementation of a hs-cTn assay across a large North American City. Design and methods: Validation for the Abbott ARCHITECT hs-cTnI assay (across 5 analyzers consisted of verification of limit of blank (LoB, precision (i.e., coefficient of variation; CV testing at the reported limit of detection (LoD and within and outside the 99th percentile, linearity testing, cTnI versus hs-cTnI patient comparison within and between analyzers (Passing and Bablok and non-parametric analyses. Education, clinical communications, and memorandums were issued in advance to inform all staff across the city as well as a selected reminder the day before live-date to important users. All hospitals switched to the hs-cTnI assay concurrently (the contemporary cTnI assay removed with laboratory staff instructed to repeat samples previously measured with the contemporary cTnI assay with the hs-cTnI assay only by physician request. Results: Across the 5 analyzers and 6 reagent packs the overall LoB was 0.6 ng/L (n=60 with a CV of 33% at an overall mean of 1.2 ng/L (n=60; reported LoD=1.0 ng/L, with linearity demonstrated from 45,005 ng/L to 1.1 ng/L. Precision testing with a normal patient-pool QC material (mean range across 5 analyzers was 3.9–4.4 ng/L yielded a range of CVs from 7% to 10% (within-run and CVs from 7% to 18% (between-run with the high patient-pool QC material (mean range across 5 analyzers was 29.6–36.3 ng/L yielding a range of CVs from 2% to 5% (within-run and CVs from 4% to 8% (between-run. There was agreement between hs-cTnI versus cTnI with the patient samples (slope ranges: 0.89–1.03; intercept ranges: 1.9–3

  10. Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

    DEFF Research Database (Denmark)

    Kruse, N.; Persson, S.; Alcolea, D.

    2015-01-01

    (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when...... the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings...... assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program -Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits...

  11. An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis.

    Directory of Open Access Journals (Sweden)

    Emma R Travis

    Full Text Available Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100% and high sensitivity (97% at levels of 10(5 cells g(-1 and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level b

  12. Validation and pharmacological characterisation of MK-801-induced locomotor hyperactivity in BALB/C mice as an assay for detection of novel antipsychotics.

    Science.gov (United States)

    Bradford, Andrea M; Savage, Kevin M; Jones, Declan N C; Kalinichev, Mikhail

    2010-10-01

    We evaluated locomotor hyperactivity induced in BALB/C mice by an N-methyl-D-aspartate receptor antagonist MK-801 as an assay for the detection of antipsychotic drugs. We assessed the effects of antipsychotic drugs to validate the assay (study 1), selective dopamine and serotonin ligands for pharmacological characterisation of the model (study 2) and a number of compounds with efficacy in models of schizophrenia to understand the predictive validity of the model (study 3). Adult males (n  = 9/group) were pretreated with a test compound, habituated to locomotor activity cages before receiving MK-801 (0.32 mg/kg) and activity recorded for a further 75 or 120 min. In study 1, we tested haloperidol, clozapine, olanzapine, risperidone, ziprasidone, aripiprazole, sertindole and quetiapine. In study 2, we tested SCH23390 (D(1) antagonist), sulpiride (D(2)/D(3) antagonist), raclopride (D(2)/D(3) antagonist), SB-277011 (D(3) antagonist), L-745,870 (D(4) antagonist), WAY100635 (5-HT(1A) antagonist), 8-OH-DPAT (5-HT(1A) agonist), ketanserin (5-HT(2A)/5-HT(2C) antagonist) and SB-242084 (5-HT(2C) antagonist). In study 3, we tested xanomeline (M(1)/M(4) receptor agonist), LY379268 (mGluR2/3 receptor agonist), diazepam (GABA(A) modulator) and thioperamide (H(3) receptor antagonist). All antipsychotics suppressed MK-801-induced hyperactivity in a dose-dependent and specific manner. The effects of antipsychotics appear to be mediated via dopamine D(1), D(2) and 5-HT(2) receptors. Xanomeline, LY379268 and diazepam were active in this assay while thioperamide was not. MK-801-induced hyperactivity in BALB/C mice model of positive symptoms has shown predictive validity with novel compounds acing at M(1)/M(4), mGluR2/3 and GABA(A) receptors and can be used as a screening assay for detection of novel pharmacotherapies targeting those receptors.

  13. Validation and Application of a Dried Blood Spot Assay for Biofilm-Active Antibiotics Commonly Used for Treatment of Prosthetic Implant Infections

    Science.gov (United States)

    Knippenberg, Ben; Page-Sharp, Madhu; Clark, Ben; Dyer, John; Batty, Kevin T.; Davis, Timothy M. E.

    2016-01-01

    Dried blood spot (DBS) antibiotic assays can facilitate pharmacokinetic (PK)/pharmacodynamic (PD) studies in situations where venous blood sampling is logistically difficult. We sought to develop, validate, and apply a DBS assay for rifampin (RIF), fusidic acid (FUS), and ciprofloxacin (CIP). These antibiotics are considered active against organisms in biofilms and are therefore commonly used for the treatment of infections associated with prosthetic implants. A liquid chromatography-mass spectroscopy DBS assay was developed and validated, including red cell partitioning and thermal stability for each drug and the rifampin metabolite desacetyl rifampin (Des-RIF). Plasma and DBS concentrations in 10 healthy adults were compared, and the concentration-time profiles were incorporated into population PK models. The limits of quantification for RIF, Des-RIF, CIP, and FUS in DBS were 15 μg/liter, 14 μg/liter, 25 μg/liter, and 153 μg/liter, respectively. Adjusting for hematocrit, red cell partitioning, and relative recovery, DBS-predicted plasma concentrations were comparable to measured plasma concentrations for each antibiotic (r > 0.95; P < 0.0001), and Bland-Altman plots showed no significant bias. The final population PK estimates of clearance, volume of distribution, and time above threshold MICs for measured and DBS-predicted plasma concentrations were comparable. These drugs were stable in DBSs for at least 10 days at room temperature and 1 month at 4°C. The present DBS antibiotic assays are robust and can be used as surrogates for plasma concentrations to provide valid PK and PK/PD data in a variety of clinical situations, including therapeutic drug monitoring or studies of implant infections. PMID:27270283

  14. Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

    Science.gov (United States)

    Plappert-Helbig, Ulla; Junker-Walker, Ursula; Martus, Hans-Joerg

    2015-07-01

    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. A validated HPLC assay to monitor riluzole plasma or serum concentrations in patients with amyotrophic lateral sclerosis

    NARCIS (Netherlands)

    van Kan, H. J. M.; Spieksma, M.; Groeneveld, G. J.; Toraño, J. Sastre; van den Berg, L. H.; Guchelaar, H. J.

    2004-01-01

    A specific, accurate and precise high-performance liquid chromatographic assay was developed for the determination of riluzole, a drug used to treat patients with amyotrophic lateral sclerosis. Samples were treated by extraction with dichloromethane followed by reversed-phase chromatography with

  16. Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

    NARCIS (Netherlands)

    Damen, Carola W. N.; de Groot, Els R.; Heij, Marianne; Boss, David S.; Schellens, Jan H. M.; Rosing, Hilde; Beijnen, Jos H.; Aarden, Lucien A.

    2009-01-01

    Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to

  17. The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA.

    Science.gov (United States)

    Koel-Simmelink, Marleen J A; Vennegoor, Anke; Killestein, Joep; Blankenstein, Marinus A; Norgren, Niklas; Korth, Carsten; Teunissen, Charlotte E

    2014-01-15

    Neurofilament (Nf) proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases. The aim of this study was to evaluate the effects of pre-analytical variables on the concentration of neurofilament heavy (NfH) and neurofilament light (NfL) proteins. For NfH an in-house newly-developed and validated SinglePlex Luminex assay was used; ELISA was used to analyze NfL. For the NfL ELISA assay, the intra- and inter-assay variation was respectively, 1.5% and 16.7%. Analytical performance of the NfH SinglePlex Luminex assay in terms of sensitivity (6.6pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6-1250pg/mL), and inter- and intra-assay variation (<8%) were good. Concentrations of both NfL and NfH appeared not negatively affected by blood contamination, repeated freeze-thaw cycles (up to 4), delayed processing (up to 24hours) and during long-term storage at -20°C, 4°C, and room temperature. A decrease in concentration was observed during storage of both neurofilament proteins up to 21days at 37°C, which was significant by day 5. The newly developed NfH SinglePlex Luminex assay has a good sensitivity and is robust. Moreover, both NfH and NfL are stable under the most prevalent pre-analytical variations. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Calibration and validation of the {sup 14}C-labelled polyethylene glycol-binding assay for tannins in tropical browse

    Energy Technology Data Exchange (ETDEWEB)

    Mlambo, V. [Animal Production Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf (Austria)]. E-mail: vmlambo@agric.uniswa.sz; Makkar, H.P.S. [Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Agriculture and Food, International Atomic Energy Agency, Vienna (Austria)

    2005-08-19

    This study evaluates the radiolabelled polyethylene glycol (PEG)-binding procedure [Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Polyethylene glycol-binding to plant samples as an assay for the biological effects of tannins: predicting the negative effects of tannins in Mediterranean browse on rumen degradation. J. Agric. Food Chem. 44, 3230-3234] for tannin analysis, using 27 tropical browse plants. In this method, the amount of PEG bound to a plant sample is assumed to be a reflection of its tannin content. The method was modified to exclude the use of non-tanniniferous substrate for estimating non-specific binding (NSB) in tannin-containing substrates. Non-specific binding values varied widely (0.4-2.8 mg PEG/100 mg DM tannin-free substrate) when the tannin-free substrate was changed from wheat straw to either rye grass or maize shoots. We therefore propose a modified radiolabelled PEG-binding method to estimate the level of PEG-binding (PEGb) to tannin-containing foliage without using tannin-free substrate to correct for non-specific binding. In this approach, incremental levels of each tanniniferous substrate were used to generate PEGb values. The resultant linear response was analysed and tannin activity was expressed as the slope of the response curve (PEGbSlope) observed for each substrate. The slope takes into account the non-specific binding in each substrate, thus PEGbSlope does not require correction for NSB using tannin-free samples. This approach improved the correlation between PEGb and the {sup 125}I-labelled bovine serum albumin precipitation assay. Relationships between the modified PEG-binding assay and radiolabelled bovine serum albumin assay, in vitro tannin bioassay and colorimetric assays are presented. (author)

  19. Calibration and validation of the 14C-labelled polyethylene glycol-binding assay for tannins in tropical browse

    International Nuclear Information System (INIS)

    Mlambo, V.; Makkar, H.P.S.

    2005-01-01

    This study evaluates the radiolabelled polyethylene glycol (PEG)-binding procedure [Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Polyethylene glycol-binding to plant samples as an assay for the biological effects of tannins: predicting the negative effects of tannins in Mediterranean browse on rumen degradation. J. Agric. Food Chem. 44, 3230-3234] for tannin analysis, using 27 tropical browse plants. In this method, the amount of PEG bound to a plant sample is assumed to be a reflection of its tannin content. The method was modified to exclude the use of non-tanniniferous substrate for estimating non-specific binding (NSB) in tannin-containing substrates. Non-specific binding values varied widely (0.4-2.8 mg PEG/100 mg DM tannin-free substrate) when the tannin-free substrate was changed from wheat straw to either rye grass or maize shoots. We therefore propose a modified radiolabelled PEG-binding method to estimate the level of PEG-binding (PEGb) to tannin-containing foliage without using tannin-free substrate to correct for non-specific binding. In this approach, incremental levels of each tanniniferous substrate were used to generate PEGb values. The resultant linear response was analysed and tannin activity was expressed as the slope of the response curve (PEGbSlope) observed for each substrate. The slope takes into account the non-specific binding in each substrate, thus PEGbSlope does not require correction for NSB using tannin-free samples. This approach improved the correlation between PEGb and the 125 I-labelled bovine serum albumin precipitation assay. Relationships between the modified PEG-binding assay and radiolabelled bovine serum albumin assay, in vitro tannin bioassay and colorimetric assays are presented. (author)

  20. Development and Validation of a Rapid Turbidimetric Assay to Determine the Potency of Cefuroxime Sodium in Powder for Dissolution for Injection

    Directory of Open Access Journals (Sweden)

    Daniela C. M. Vieira

    2014-07-01

    Full Text Available The cefuroxime sodium is a second generation cephalosporin indicated for infections caused by Gram-positive and Gram-negative microorganisms. Although this drug is highly studied and researched regarding the antimicrobial activity, pharmacokinetics and pharmacodynamics, there are few studies regarding the development of analytical methodology for this cephalosporin. Thus, research involving analytical methods is essential and highly relevant to optimize its analysis in the pharmaceutical industry and guarantee the quality of the product already sold. This study describes the development and validation of a microbiological assay applying the turbidimetric method for the determination of cefuroxime, using Micrococcus luteus ATCC 9341 as micro-organism test and 3x3 parallel line assay design, with nine tubes for each assay, as recommended by the Brazilian Pharmacopoeia. The developed and validated method showed excellent results of linearity, seletivity, precision and robustness, in the concentration range from 30.0 to 120.0 mg/mL, with 100.21% accuracy and content 99.97% to cefuroxime sodium in injectable pharmaceutical form.

  1. Development and Validation of a Novel LC-MS/MS Opioid Confirmation Assay: Evaluation of β-glucuronidase Enzymes and Sample Cleanup Methods.

    Science.gov (United States)

    Yang, He S; Wu, Alan H B; Lynch, Kara L

    2016-06-01

    With the rise in the use and misuse of prescription opioids, there is an increasing need for the confirmed identification of opioid analgesics in toxicology laboratories. The goals of this study were to (i) systematically evaluate the hydrolysis efficiency of four β-glucuronidase enzymes under optimized condition; (ii) evaluate compound recovery, matrix effects and precision of three protein precipitation plates and (iii) develop and validate a qualitative liquid-chromatography mass spectrometry (LC-MS/MS) assay to identify 13 opioids in urine. A recombinant β-glucuronidase exhibited the best overall hydrolysis efficiency for seven opioid glucuronide conjugates compared with β-glucuronidase from red abalone, Escherichia coli and Patella vulgata One of the protein precipitation plates tested exhibited overall better recovery of the opioids and lower ion suppression compared with the other two plates. An ESI positive mode LC-MS/MS assay for qualitative opioid analysis was developed and validated. Linearity, LOD, precision, matrix effect, recovery, carryover and interference of the method were evaluated. Sixty-two patient samples were analyzed by both a legacy GC-MS opioid method and the LC-MS/MS method, and 22 samples were analyzed by the LC-MS/MS and an LC-MS/MS reference method. The results of the comparisons showed good concordance. Overall, we described an efficient sample preparation procedure for a sensitive qualitative opioid confirmation assay in urine. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds.

    Science.gov (United States)

    Carneiro, Greice Amaral; Matić, Slavica; Ortu, Giuseppe; Garibaldi, Angelo; Spadaro, Davide; Gullino, Maria Lodovica

    2017-07-01

    Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

  3. Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

    Directory of Open Access Journals (Sweden)

    Francielle Gibson da Silva-Zacarias

    2009-11-01

    Full Text Available Chlamydophila abortus (C. abortus is associated with reproductive problems in cattle, sheep, and goats. Diagnosis of C. abortus using embryonated chicken eggs or immortalized cell lines has a very low sensitivity. Polymerase chain reaction (PCR assays have been used to detect C. abortus infection in clinical specimens and organ fragments, such as placenta, fetal organs, vaginal secretions, and semen. The aim of this study was to develop a PCR assay for the amplification of an 856-bp fragment of the rRNA gene of the Chlamydiaceae family. The PCR assay was evaluated using organs from 15 mice experimentally infected with the S26/3 reference strain of C. abortus. The results of the rRNA PCR were compared to the results from another PCR system (Omp2 PCR that has been previously described for the Omp2 (outer major protein gene from the Chlamydiaceae family. From the 15 C. abortus-inoculated mice, 13 (K=0.84, standard error =0.20 tested positive using the rRNA PCR assay and 9 (K=0.55, standard error=0.18 tested positive using the Omp2 PCR assay. The detection limit, measured using inclusion-forming units (IFU, for C. abortus with the rRNA PCR (1.05 IFU was 100-fold lower than for the Omp2 PCR (105 IFU. The higher sensitivity of the rRNA PCR, as compared to the previously described PCR assay, and the specificity of the assay, demonstrated using different pathogenic microorganisms of the bovine reproductive system, suggest that the new PCR assay developed in this study can be used for the molecular diagnosis of C. abortus in abortion and other reproductive failures in bovines, caprines, and ovines.Chlamydophila abortus (C. abortus é frequentemente associada a distúrbios reprodutivos em bovinos, ovinos e caprinos. Para o diagnóstico, os métodos de cultivo em ovo embrionado de galinha e em células de linhagem contínua apresentam baixa sensibilidade. A reação em cadeia da polimerase (PCR tem sido utilizada em placenta, órgãos fetais, secre

  4. Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence

    Directory of Open Access Journals (Sweden)

    Yicheng Xie

    2018-04-01

    Full Text Available Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6% of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

  5. Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

    Science.gov (United States)

    Xie, Yicheng; Wahab, Laith; Gill, Jason J

    2018-04-12

    Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

  6. Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

    Science.gov (United States)

    Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

    2015-07-01

    Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus.

    Science.gov (United States)

    Faye, Martin; Dacheux, Laurent; Weidmann, Manfred; Diop, Sylvie Audrey; Loucoubar, Cheikh; Bourhy, Hervé; Sall, Amadou Alpha; Faye, Ousmane

    2017-05-01

    Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The Manchester Acute Coronary Syndromes (MACS) decision rule: validation with a new automated assay for heart-type fatty acid binding protein.

    Science.gov (United States)

    Body, Richard; Burrows, Gillian; Carley, Simon; Lewis, Philip S

    2015-10-01

    The Manchester Acute Coronary Syndromes (MACS) decision rule may enable acute coronary syndromes to be immediately 'ruled in' or 'ruled out' in the emergency department. The rule incorporates heart-type fatty acid binding protein (h-FABP) and high sensitivity troponin T levels. The rule was previously validated using a semiautomated h-FABP assay that was not practical for clinical implementation. We aimed to validate the rule with an automated h-FABP assay that could be used clinically. In this prospective diagnostic cohort study we included patients presenting to the emergency department with suspected cardiac chest pain. Serum drawn on arrival was tested for h-FABP using an automated immunoturbidimetric assay (Randox) and high sensitivity troponin T (Roche). The primary outcome, a diagnosis of acute myocardial infarction (AMI), was adjudicated based on 12 h troponin testing. A secondary outcome, major adverse cardiac events (MACE; death, AMI, revascularisation or new coronary stenosis), was determined at 30 days. Of the 456 patients included, 78 (17.1%) had AMI and 97 (21.3%) developed MACE. Using the automated h-FABP assay, the MACS rule had the same C-statistic for MACE as the original rule (0.91; 95% CI 0.88 to 0.92). 18.9% of patients were identified as 'very low risk' and thus eligible for immediate discharge with no missed AMIs and a 2.3% incidence of MACE (n=2, both coronary stenoses). 11.1% of patients were classed as 'high-risk' and had a 92.0% incidence of MACE. Our findings validate the performance of a refined MACS rule incorporating an automated h-FABP assay, facilitating use in clinical settings. The effectiveness of this refined rule should be verified in an interventional trial prior to implementation. UK CRN 8376. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  9. Development and validation of a microRNA based diagnostic assay for primary tumor site classification of liver core biopsies

    DEFF Research Database (Denmark)

    Perell, Katharina; Vincent, Martin; Vainer, Ben

    2015-01-01

    for normal liver tissue contamination. Performance was estimated by cross-validation, followed by independent validation on 55 liver core biopsies with a tumor content as low as 10%. A microRNA classifier developed, using the statistical contamination model, showed an overall classification accuracy of 74...... on classification. MicroRNA profiling was performed using quantitative Real-Time PCR on formalin-fixed paraffin-embedded samples. 278 primary tumors and liver metastases, representing nine primary tumor classes, as well as normal liver samples were used as a training set. A statistical model was applied to adjust.......5% upon independent validation. Two-thirds of the samples were classified with high-confidence, with an accuracy of 92% on high-confidence predictions. A classifier trained without adjusting for liver tissue contamination, showed a classification accuracy of 38.2%. Our results indicate that surrounding...

  10. Molecular Methods for Typing of Streptococcus agalactiae with Special Emphasis on the Development and Validation of a Multi-Locus Variable Number of Tandem Repeats Assay (MLVA)

    OpenAIRE

    Radtke, Andreas

    2012-01-01

    Molekylære metoder for typing av Streptococcus agalactiae med særlig vektlegging av utvikling og validering av et multi-locus variable number of tandem repeats assay (MLVA) Sammendraget: Streptococcus agalactiae eller gruppe B streptokokker (GBS) forårsaker livsfarlige infeksjoner hos nyfødte, gravide eller voksne med kroniske sykdommer. Den forårsaker også jurbetennelse i storfe. Typing av GBS gir innblikk i bakteriens epidemiologi og dens fylogenetiske slektskap. Ulike deler av bakterie...

  11. Prospective validation of an automated chemiluminescence-based assay of renin and aldosterone for the work-up of arterial hypertension.

    Science.gov (United States)

    Rossi, Gian Paolo; Ceolotto, Giulio; Rossitto, Giacomo; Seccia, Teresa Maria; Maiolino, Giuseppe; Berton, Chiara; Basso, Daniela; Plebani, Mario

    2016-09-01

    The availability of simple and accurate assays of plasma active renin (DRC) and aldosterone concentration (PAC) can improve the detection of secondary forms of arterial hypertension. Thus, we investigated the performance of an automated chemiluminescent assay for DRC and PAC in referred hypertensive patients. We prospectively recruited 260 consecutive hypertensive patients referred to an ESH Center for Hypertension. After exclusion of six protocol violations, 254 patients were analyzed: 67.3% had primary hypertension, 17.3% an aldosterone producing adenoma (APA), 11.4% idiopathic hyperaldosteronism (IHA), 2.4% renovascular hypertension (RVH), 0.8% familial hyperaldosteronism type 1 (FH-1), 0.4% apparent mineralocorticoid excess (AME), 0.4% a renin-producing tumor, and 3.9% were adrenalectomized APA patients. Bland-Altman plots and Deming regression were used to analyze results. The diagnostic accuracy (area under the curve, AUC of the ROC) of the DRC-based aldosterone-renin ratio (ARRCL) was compared with that of the PRA-based ARR (ARRRIA) using as reference the conclusive diagnosis of APA. At Bland-Altman plot, the DRC and PAC assay showed no bias as compared to the PRA and PAC assay. A tight relation was found between the DRC and the PRA values (concordance correlation coefficient=0.92, pAPA identification the AUC of the ARRCL was higher than that of the ARRRIA [0.974 (95% CI 0.940-0.991) vs. 0.894 (95% CI 0.841-0.933), p=0.02]. This rapid automated chemiluminescent DRC/PAC assay performed better than validated PRA/PAC radioimmunoassays for the identification of APA in referred hypertensive patients.

  12. Variation in the measurement of DNA damage by comet assay measured by the ECVAG dagger inter-laboratory validation trial

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Johansson, Clara; Loft, Steffen

    2010-01-01

    the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation...... by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P test). A large fraction...

  13. Field Validation of a Transcriptional Assay for the Prediction of Age of Uncaged Aedes aegypti Mosquitoes in Northern Australia

    Science.gov (United States)

    Hugo, Leon E.; Cook, Peter E.; Johnson, Petrina H.; Rapley, Luke P.; Kay, Brian H.; Ryan, Peter A.; Ritchie, Scott A.; O'Neill, Scott L.

    2010-01-01

    Background New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. Methodology/Principal Findings We produced “free-range” test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R2 value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. Conclusions/Significance The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito

  14. Field validation of a transcriptional assay for the prediction of age of uncaged Aedes aegypti mosquitoes in Northern Australia.

    Directory of Open Access Journals (Sweden)

    Leon E Hugo

    Full Text Available BACKGROUND: New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. METHODOLOGY/PRINCIPAL FINDINGS: We produced "free-range" test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R(2 value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. CONCLUSIONS/SIGNIFICANCE: The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where

  15. Validating DNA Polymorphisms Using KASP Assay in Prairie Cordgrass (Spartina pectinata Link Populations in the U.S.

    Directory of Open Access Journals (Sweden)

    Hannah eGraves

    2016-01-01

    Full Text Available Single nucleotide polymorphisms (SNPs are one of the most abundant DNA variants found in plant genomes and are highly efficient when comparing genome and transcriptome sequences. SNP marker analysis can be used to analyze genetic diversity, create genetic maps, and utilize marker-assisted selection breeding in many crop species. In order to utilize these technologies, one must first identify and validate putative SNPs. In this study, 121 putative SNPs, developed from a nuclear transcriptome of prairie cordgrass (Spartina pectinata Link, were analyzed using KASP technology in order to validate the SNPs. Fifty-nine SNPs were validated using a core collection of 38 natural populations and a phylogenetic tree was created with one main clade. Samples from the same population tended to cluster in the same location on the tree. Polymorphisms were identified within 52.6% of the populations, split evenly between the tetraploid and octoploid cytotypes. Twelve selected SNP markers were used to assess the fidelity of tetraploid crosses of prairie cordgrass and their resulting F2 population. These markers were able to distinguish true crosses and selfs. This study provides insight into the genomic structure of prairie cordgrass, but further analysis must be done on other cytotypes to fully understand the structure of this species. This study validates putative SNPs and confirms the potential usefulness of SNP marker technology in future breeding programs of this species.

  16. Comparison of the Abbott RealTime CT new formulation assay with two other commercial assays for detection of wild-type and new variant strains of Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth; Pedersen, Lisbeth Nørum; Persson, Kenneth

    2010-01-01

    In an analytical methods comparison study on clinical samples, the Abbott RealTime CT new formulation assay (m2000 real-time PCR) consisting of a duplex PCR targeting different parts of the cryptic plasmid in Chlamydia trachomatis was compared with version 2 of the Roche COBAS(R) TaqMan(R) CT ass...

  17. Evaluation and validation of a single-dilution potency assay based upon serology of vaccines containing diphtheria toxoid: analysis for consistency in production and testing at the laboratory of the Control of Biological Products of the RIVM

    NARCIS (Netherlands)

    Akkermans AM; Hendriksen CFM; Marsman FR; de Jong WH; van de Donk HJM

    1993-01-01

    A single-dilution assay can be a valid procedure to demonstrate that a product exceeds the minimal requirement given for potency provided that consistency in production and testing has been proven. Information is presented justifying the use of a single dilution assay based upon quantitative

  18. Quantification of imatinib in human serum: validation of a high-performance liquid chromatography-mass spectrometry method for therapeutic drug monitoring and pharmacokinetic assays.

    Science.gov (United States)

    Rezende, Vinicius Marcondes; Rivellis, Ariane; Novaes, Mafalda Megumi Yoshinaga; de Alencar Fisher Chamone, Dalton; Bendit, Israel

    2013-01-01

    Imatinib mesylate has been a breakthrough treatment for chronic myeloid leukemia. It has become the ideal tyrosine kinase inhibitor and the standard treatment for chronic-phase leukemia. Striking results have recently been reported, but intolerance to imatinib and noncompliance with treatment remain to be solved. Molecular monitoring by quantitative real-time polymerase chain reaction is the gold standard for monitoring patients, and imatinib blood levels have also become an important tool for monitoring. A fast and cheap method was developed and validated using high-performance liquid chromatography-mass spectrometry for quantification of imatinib in human serum and tamsulosin as the internal standard. Remarkable advantages of the method includes use of serum instead of plasma, less time spent on processing and analysis, simpler procedures, and requiring reduced amounts of biological material, solvents, and reagents. Stability of the analyte was also studied. This research also intended to drive the validation scheme in clinical centers. The method was validated according to the requirements of the US Food and Drug Administration and Brazilian National Health Surveillance Agency within the range of 0.500-10.0 μg/mL with a limit of detection of 0.155 μg/mL. Stability data for the analyte are also presented. Given that the validated method has proved to be linear, accurate, precise, and robust, it is suitable for pharmacokinetic assays, such as bioavailability and bioequivalence, and is being successfully applied in routine therapeutic drug monitoring in the hospital service.

  19. Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

    2015-06-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

  20. Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

    2015-01-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

  1. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics.

    Science.gov (United States)

    Gupta, Shalini; Devanarayan, Viswanath; Finco, Deborah; Gunn, George R; Kirshner, Susan; Richards, Susan; Rup, Bonita; Song, An; Subramanyam, Meena

    2011-07-15

    The administration of biological therapeutics may result in the development of anti-drug antibodies (ADAs) in treated subjects. In some cases, ADA responses may result in the loss of therapeutic efficacy due to the formation of neutralizing ADAs (NAbs). An important characteristic of anti-drug NAbs is their direct inhibitory effect on the pharmacological activity of the therapeutic. Neutralizing antibody responses are of particular concern for biologic products with an endogenous homolog whose activity can be potentially dampened or completely inhibited by the NAbs leading to an autoimmune-type deficiency syndrome. Therefore, it is important that ADAs are detected and characterized appropriately using sensitive and reliable methods. The design, development and optimization of cell-based assays used for detection of NAbs have been published previously by Gupta et al. 2007 [1]. This paper provides recommendations on best practices for the validation of cell-based NAb assay and suggested validation parameters based on the experience of the authors. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Investigation of sodium arsenite, thioacetamide, and diethanolamine in the alkaline comet assay: Part of the JaCVAM comet validation exercise.

    Science.gov (United States)

    Beevers, Carol; Henderson, Debbie; Lillford, Lucinda

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined sodium arsenite, thioacetamide, and diethanolamine. Using the JaCVAM approved study protocol version 14.2, each chemical was tested in male rats up to maximum tolerated dose levels and DNA damage in the liver and stomach was assessed approximately 3h after the final administration by gavage. Histopathology assessments of liver and stomach sections from the same animals were also examined for evidence of cytotoxicity or necrosis. No evidence of DNA damage was observed in the stomach of animals treated with sodium arsenite at 7.5, 15, or 30 mg/kg/day. However, equivocal findings were found in the liver, where increases in DNA migration were observed in two independent experiments, but not in all treated animals and not at the same dose levels. Thioacetamide caused an increase in DNA migration in the stomach of rats treated at 19, 38, and 75 mg/kg/day, but not in the liver, despite evidence of marked hepatotoxicity following histopathology assessments. No evidence of DNA damage was observed in the stomach or liver of animals treated with diethanolamine at 175, 350, or 700 mg/kg/day. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry

    Science.gov (United States)

    Jimmerson, Leah C.; Zheng, Jia-Hua; Bushman, Lane R.; MacBrayne, Christine E.; Anderson, Peter L.; Kiser, Jennifer J.

    2014-01-01

    Efficient, inexpensive and sensitive assays for the measurement of drugs are of interest for pharmacokinetic and pharmacodynamics (PK-PD) analysis. Dried blood spots (DBS) are a unique bioanaltyical matrix with the potential to fulfill this interest for the measurement of numerous analytes. Here we describe the development and validation of a reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of ribavirin (RBV) in DBS. A 3mm punch from spotted and dried whole blood was extracted in methanol utilizing isotopically labeled internal standard for LC-MS/MS analysis. Validation was performed over a range of 0.05 μg/mL to 10.0 μg/mL and the method was shown to be precise (coefficient of variation ≤ 15%) and accurate (within ±15% of control). These acceptance criteria were met for hematocrit ranges of 20-54%, for center versus edge punches and for spot volumes from 10-60 μL. RBV was stable for up to 140 days at room temperature and −20°C as well as for three freeze/thaw cycles. Correlation of RBV in DBS versus in plasma yielded r2 ≥ 0.98 demonstrating that DBS can be used as an alternative to plasma for PK-PD studies in human subjects. PMID:24291608

  4. Optimization and Validation of Real Time PCR Assays for Absolute Quantification of toxigenic Vibrio cholerae and Escherichia coli

    DEFF Research Database (Denmark)

    Ferdous, J.; Hossain, Z. Z.; Tulsiani, S.

    2016-01-01

    and quantify DNA by real-time PCR for two pathogenic species, Escherichia coli (E. coli) and Vibrio cholerae (V.cholerae). In order to generate a standard curve, total bacterial DNA was diluted in a 10-fold series and each sample was adjusted to an estimated cell count. The starting bacterial DNA concentration......Quantitative real-time PCR (qPCR) is a dynamic and cogent assay for the detection and quantification of specified nucleic acid sequences and is more accurate compared to both traditional culture based techniques and ‘end point’ conventional PCR. Serial dilution of bacterial cell culture provides...... significant, low F ratios indicated that there was some variation in CT values when genomic DNA dilution was compared to dilution of cell suspension in media. Different water samples spiked with pure cultures of E. coli and V. cholerae were used as unknown samples. The standard curve constructed by the serial...

  5. Development and validation of an assay for the simultaneous determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Kromdijk, W; Pereira, S A; Rosing, H; Mulder, J W; Beijnen, J H; Huitema, A D R

    2013-03-01

    This paper describes the development and validation of an assay for the simultaneous quantification of the antiviral and antiretroviral drugs zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 0.1% (v/v) formic acid in methanol, evaporation and reconstitution. Chromatographic separation was performed on a Synergy Polar reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 μm) using a stepwise gradient with 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol at a flow rate of 300 μL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for drug detection and quantification. Isotopically labeled zidovudine, lamivudine, tenofovir and ribavirin were used as internal standards. The method was validated over a clinical range of 20-2500 ng/mL for zidovudine, lamivudine and tenofovir, 4-500 ng/mL for abacavir and emtricitabine and 160-20,000 ng/mL for ribavirin. The inter and intra-assay accuracies and precisions were between -8.47% and 14.2% for zidovudine, emtricitabine and ribavirin. For abacavir, lamivudine and tenofovir, the inter and intra-assay accuracies and precisions at the lower limit of quantification were between -11.0% and 18.3%, whereas at all other levels these accuracies and precisions were between -11.7% and 12.0%. The described method is suitable for the determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma in clinical practice to monitor plasma concentrations in selected cases to optimize therapy. Copyright © 2013. Published by Elsevier B.V.

  6. Development and application of a PCR assay to detect chicken and turkey parvoviruses in commercial poultry flocks in the United States.

    Science.gov (United States)

    Comparative sequence analysis of six independent chicken and turkey parvovirus nonstructural (NS) genes revealed specific genomic regions with 100% nucleotide sequence identity. A PCR assay with primers targeting these conserved genome sequences proved to be highly specific and sensitive to detect p...

  7. Evaluation of a commercial competitive enzyme-linked immunosorbent assay for detection of avian influenza virus subtype H5 antibodies in zoo birds

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Andersen, Jannie Holmegaard; Hjulsager, Charlotte Kristiane

    2017-01-01

    The hemagglutination inhibition (HI) test is the current gold standard for detecting antibodies to avian influenza virus (AIV). Enzyme-linked immunosorbent assays (ELISAs) have been explored for use in poultry and certain wild bird species because of high efficiency and lower cost. This study com...

  8. Stability indicating method development and validation of assay method for the estimation of rizatriptan benzoate in tablet

    Directory of Open Access Journals (Sweden)

    Chandrashekhar K. Gadewar

    2017-05-01

    Full Text Available A simple, sensitive, precise and specific high performance liquid chromatography method was developed and validated for the determination of rizatriptan in rizatriptan benzoate tablet. The separation was carried out by using a mobile phase consisting of acetonitrile: pH 3.4 phosphate buffer in ratio of 20:80. The column used was Zorbax SB CN 250 mm × 4.6 mm, 5 μ with a flow rate of 1 ml/min using UV detection at 225 nm. The retention time of rizatriptan and benzoic acid was found to be 4.751 and 8.348 min respectively. A forced degradation study of rizatriptan benzoate in its tablet form was conducted under the condition of hydrolysis, oxidation, thermal and photolysis. Rizatriptan was found to be stable in basic buffer while in acidic buffer was found to be degraded (water bath at 60 °C for 15 min. The detector response of rizatriptan is directly proportional to concentration ranging from 30% to 160% of test concentration i.e. 15.032 to 80.172 mcg/ml. Results of analysis were validated statistically and by recovery studies (mean recovery = 99.44. The result of the study showed that the proposed method is simple, rapid, precise and accurate, which is useful for the routine determination of rizatriptan in pharmaceutical dosage forms.

  9. Evaluation of three commercial enzyme-linked imuunosorbent assays for the detection of antibodies against Salmonella spp. in meat juice from finishing pigs in Spain

    NARCIS (Netherlands)

    Vico, J.P.; Engel, B.; Buist, W.G.; Mainar-Jaime, R.C.

    2010-01-01

    The control of animal salmonellosis is considered as a major objective in Europe and indirect ELISAs will be important tools for the implementation of control programs for this infection in pigs. We analyse the results yielded by three commercial ELISAs (Herdcheck Swine Salmonella, SALMOTYPE Pig

  10. Quantification of imatinib in human serum: validation of a high-performance liquid chromatography-mass spectrometry method for therapeutic drug monitoring and pharmacokinetic assays

    Directory of Open Access Journals (Sweden)

    Rezende VM

    2013-08-01

    Full Text Available Vinicius Marcondes Rezende,1 Ariane Rivellis,1 Mafalda Megumi Yoshinaga Novaes,1 Dalton de Alencar Fisher Chamone,2 Israel Bendit1,21Laboratory of Tumor Biology, 2Department of Hematology, School of Medicine, University of São Paulo, São Paulo, BrazilBackground: Imatinib mesylate has been a breakthrough treatment for chronic myeloid leukemia. It has become the ideal tyrosine kinase inhibitor and the standard treatment for chronic-phase leukemia. Striking results have recently been reported, but intolerance to imatinib and noncompliance with treatment remain to be solved. Molecular monitoring by quantitative real-time polymerase chain reaction is the gold standard for monitoring patients, and imatinib blood levels have also become an important tool for monitoring.Methods: A fast and cheap method was developed and validated using high-performance liquid chromatography-mass spectrometry for quantification of imatinib in human serum and tamsulosin as the internal standard. Remarkable advantages of the method includes use of serum instead of plasma, less time spent on processing and analysis, simpler procedures, and requiring reduced amounts of biological material, solvents, and reagents. Stability of the analyte was also studied. This research also intended to drive the validation scheme in clinical centers. The method was validated according to the requirements of the US Food and Drug Administration and Brazilian National Health Surveillance Agency within the range of 0.500–10.0 µg/mL with a limit of detection of 0.155 µg/mL. Stability data for the analyte are also presented.Conclusion: Given that the validated method has proved to be linear, accurate, precise, and robust, it is suitable for pharmacokinetic assays, such as bioavailability and bioequivalence, and is being successfully applied in routine therapeutic drug monitoring in the hospital service.Keywords: imatinib, high-performance liquid chromatography-mass spectrometry, therapeutic

  11. Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

    Science.gov (United States)

    Karjalainen, Katja; Pasqualini, Renata; Cortes, Jorge E.; Kornblau, Steven M.; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M.; Sidman, Richard L.; Arap, Wadih; Koivunen, Erkki

    2015-01-01

    Background We introduce an ex vivo methodology to perform drug library screening against human leukemia. Method Our strategy relies on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. We demonstrate several advantages: (I) partial recapitulation of the leukemia microenvironment, (ii) use of native hemoglobin oxygenation as real-time sensor/reporter, (iii) cost-effectiveness, (iv) species-specificity, and (v) format that enables high-throughput screening. Results As a proof-of-concept, we screened a chemical library (size ∼20,000) against human leukemia cells. We identified 70 compounds (“hit” rate=0.35%; Z-factor=0.71) with activity; we examined 20 to find 18 true-positives (90%). Finally, we show that carbonohydraxonic diamide group-containing compounds are potent anti-leukemia agents that induce cell death in leukemia cells and patient-derived samples. Conclusions This unique functional assay can identify novel drug candidates as well as find future applications in personalized drug selection for leukemia patients. PMID:24496871

  12. Differences in serum thyroglobulin measurements by 3 commercial immunoradiometric assay kits and laboratory standardization using Certified Reference Material 457 (CRM-457).

    Science.gov (United States)

    Lee, Ji In; Kim, Ji Young; Choi, Joon Young; Kim, Hee Kyung; Jang, Hye Won; Hur, Kyu Yeon; Kim, Jae Hyeon; Kim, Kwang-Won; Chung, Jae Hoon; Kim, Sun Wook

    2010-09-01

    Serum thyroglobulin (Tg) is essential in the follow-up of patients with differentiated thyroid carcinoma (DTC). However, interchangeability and standardization between Tg assays have not yet been achieved, even with the development of an international Tg standard (Certified Reference Material 457 [CRM-457]). Serum Tg from 30 DTC patients and serially diluted CRM-457 were measured using 3 different immunoradiometric assays (IRMA-1, IRMA-2, IRMA-3). The intraclass correlation coefficient (ICC) method was used to describe the concordance of each IRMA to CRM-457. The serum Tg measured by 3 different IRMAs correlated well (r > .85, p CRM-457, showed the best ICC (p(1) = .98) for the CRM-457. Hospitals caring for patients with DTC should either set their own cutoffs for IRMAs for Tg based on their patient pools, or adopt IRMAs standardized to CRM-457 and calibrate their laboratory using CRM-457.

  13. Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

    Science.gov (United States)

    Nguyen, Duc Doan; Busetti, Francesco; Johnson, Stuart Keith; Solah, Vicky Ann

    2018-03-01

    This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

  14. Clinical and preclinical validation of the serum free light chain assay: identification of the critical difference for optimized clinical use.

    Science.gov (United States)

    Hansen, Charlotte T; Münster, Anna-Marie; Nielsen, Lars; Pedersen, Per; Abildgaard, Niels

    2012-12-01

    The use of the assay for the measurements of free light chains in serum (sFLCs) is increasing. However, there are technical limitations that potentially affect the use in serial measurements. We need further knowledge on the standards of analytical precision, the utility of conventional population-based reference values and the critical difference (CD) between serial results required for significance. To answer these questions, the biological variation must be known. We determined the biological variation in healthy individuals and patients with plasma cell dyscrasia (PCD). We assessed the imprecision of the analysis in use from FreeLite™. We determined the reference interval (RI) in 170 healthy individuals. The biological variation is identical for healthy individuals and patients with PCD. The imprecision of the sFLC analysis cannot fulfil the desirable performance standards for a laboratory test, but are within the manufacturer's ±20% variation for quality control samples. RI showed a significant increase for κ FLC and κ/λ ratio with age, but not for λ. Critical difference was calculated to be 24% and 23% for κ and λ, respectively. We suggest the use of an age-dependent RI. When monitoring patients with PCD, their own former results are the best reference, and knowledge on CD is a valuable tool, which we describe for the first time. Also, it challenges the recently proposed International Myeloma Working Group 'paraprotein relapse criteria', recommending an increase of more than 25% in the involved FLC to indicate the need for initiation of retreatment. We recommend revision of this criterion. © 2012 John Wiley & Sons A/S.

  15. Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

    Science.gov (United States)

    Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

    2012-10-01

    Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.

    Science.gov (United States)

    Thålin, Charlotte; Daleskog, Maud; Göransson, Sophie Paues; Schatzberg, Daphne; Lasselin, Julie; Laska, Ann-Charlotte; Kallner, Anders; Helleday, Thomas; Wallén, Håkan; Demers, Mélanie

    2017-06-01

    There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

  17. Validation of low-volume enrichment protocols for detection of Escherichia coli O157 in raw ground beef components, using commercial kits.

    Science.gov (United States)

    Ahmed, Imtiaz; Hughes, Denise; Jenson, Ian; Karalis, Tass

    2009-03-01

    Testing of beef destined for use in ground beef products for the presence of Escherichia coli O157:H7 has become an important cornerstone of control and verification activities within many meat supply chains. Validation of the ability of methods to detect low levels of E. coli O157:H7 is critical to confidence in test systems. Many rapid methods have been validated against standard cultural methods for 25-g samples. In this study, a number of previously validated enrichment broths and commercially available test kits were validated for the detection of low numbers of E. coli O157:H7 in 375-g samples of raw ground beef component matrices using 1 liter of enrichment broth (large-sample:low-volume enrichment protocol). Standard AOAC International methods for 25-g samples in 225 ml of enrichment broth, using the same media, incubation conditions, and test kits, were used as reference methods. No significant differences were detected in the ability of any of the tests to detect low levels of E. coli O157:H7 in samples of raw ground beef components when enriched according to standard or large-sample:low-volume enrichment protocols. The use of large-sample:low-volume enrichment protocols provides cost savings for media and logistical benefits when handling and incubating large numbers of samples.

  18. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

    Directory of Open Access Journals (Sweden)

    Vinci Maria

    2012-03-01

    Full Text Available Abstract Background There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K-mammalian target of rapamycin (mTOR inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG (heat shock protein 90 (HSP90 inhibitor and CCT130234 (in-house phospholipase C (PLCγ inhibitor. Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration

  19. The Abbott RealTime High Risk HPV test is a clinically validated human papillomavirus assay for triage in the referral population and use in primary cervical cancer screening in women 30 years and older: a review of validation studies.

    Science.gov (United States)

    Poljak, Mario; Oštrbenk, Anja

    2013-01-01

    Human papillomavirus (HPV) testing has become an essential part of current clinical practice in the management of cervical cancer and precancerous lesions. We reviewed the most important validation studies of a next-generation real-time polymerase chain reaction-based assay, the RealTime High Risk HPV test (RealTime)(Abbott Molecular, Des Plaines, IL, USA), for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older published in peer-reviewed journals from 2009 to 2013. RealTime is designed to detect 14 high-risk HPV genotypes with concurrent distinction of HPV-16 and HPV-18 from 12 other HPV genotypes. The test was launched on the European market in January 2009 and is currently used in many laboratories worldwide for routine detection of HPV. We concisely reviewed validation studies of a next-generation real-time polymerase chain reaction (PCR)-based assay: the Abbott RealTime High Risk HPV test. Eight validation studies of RealTime in referral settings showed its consistently high absolute clinical sensitivity for both CIN2+ (range 88.3-100%) and CIN3+ (range 93.0-100%), as well as comparative clinical sensitivity relative to the currently most widely used HPV test: the Qiagen/Digene Hybrid Capture 2 HPV DNA Test (HC2). Due to the significantly different composition of the referral populations, RealTime absolute clinical specificity for CIN2+ and CIN3+ varied greatly across studies, but was comparable relative to HC2. Four validation studies of RealTime performance in cervical cancer screening settings showed its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, as well as comparative clinical sensitivity and specificity relative to HC2 and GP5+/6+ PCR. RealTime has been extensively evaluated in the last 4 years. RealTime can be considered clinically validated for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older.

  20. Qualification testing of fiber-based laser transmitters and on-orbit validation of a commercial laser system

    Science.gov (United States)

    Wright, M. W.; Wilkerson, M. W.; Tang, R. R.

    2017-11-01

    Qualification testing of fiber based laser transmitters is required for NASA's Deep Space Optical Communications program to mature the technology for space applications. In the absence of fully space qualified systems, commercial systems have been investigated in order to demonstrate the robustness of the technology. To this end, a 2.5 W fiber based laser source was developed as the transmitter for an optical communications experiment flown aboard the ISS as a part of a technology demonstration mission. The low cost system leveraged Mil Standard design principles and Telcordia certified components to the extent possible and was operated in a pressure vessel with active cooling. The laser was capable of high rate modulation but was limited by the mission requirements to 50 Mbps for downlinking stored video from the OPALS payload, externally mounted on the ISS. Environmental testing and space qualification of this unit will be discussed along with plans for a fully space qualified laser transmitter.

  1. Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

    Science.gov (United States)

    Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

    2015-08-26

    Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

  2. Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

    Science.gov (United States)

    Welti, Jonathan; Rodrigues, Daniel Nava; Sharp, Adam; Sun, Shihua; Lorente, David; Riisnaes, Ruth; Figueiredo, Ines; Zafeiriou, Zafeiris; Rescigno, Pasquale; de Bono, Johann S; Plymate, Stephen R

    2016-10-01

    The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; pprostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  3. Development and validation of LC-MS/MS assay for the determination of Butoconazole in human plasma: Evaluation of systemic absorption following topical application in healthy volunteers

    Directory of Open Access Journals (Sweden)

    Eman G. Nouman

    2017-12-01

    Full Text Available Butoconazole is an imidazole antifungal that is more effective than miconazole and clotrimazole for treatment of vaginal candidiasis. A highly sensitive tandem mass spectrometric assay was developed and validated to evaluate systemic absorption of Butoconazole following intravaginal administration. Chromatographic separation was achieved using Waters Xterra C18 column (3 µm, 3.0 × 50.0 mm. Liquid-liquid extraction using tert-butyl methyl ether was used for preparation of plasma samples. The mobile phase was solvent A: 0.1% formic acid in water and solvent B: acetonitrile: methanol (30:70, v/v, using gradient elution mode at 0.5 mL/min. Detection at positive electrospray ionization in the MRM mode was then employed. Analysis was carried out within 5.5 min over a linear concentration range of 0.10–30.00 ng/mL. Validation was carried out according to US FDA guidelines for bioanalytical method validation. Matrix effect, recovery efficiency and process efficiency have been investigated for the analyte and internal standard in neat solvent, post-extraction matrix and plasma. The mean percentage recoveries were higher than 80%, the accuracy was 93.51–106.85% and the RSD was below 10% throughout the studied concentration range. Results indicated sufficient stability of the target analyte in plasma at the employed experimental conditions. Results of incurred sample re-analysis and incurred sample stability revealed less than 5% variability. The applicability of the assay for monitoring of the systemic absorption of Butoconazole following intra vaginal application to healthy volunteers was demonstrated. Results confirmed that Butoconazole was detected shortly after intra vaginal administration with Cmax and tmax of 30 ng/mL and 6 h, respectively. Keywords: Butoconazole, LC-MS/MS, Matrix effect, Process efficiency, Recovery efficiency

  4. 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.

  5. A Molecular Host Response Assay to Discriminate Between Sepsis and Infection-Negative Systemic Inflammation in Critically Ill Patients: Discovery and Validation in Independent Cohorts.

    Directory of Open Access Journals (Sweden)

    Leo McHugh

    2015-12-01

    group of patients (validation cohorts 2-5; 249 patients after excluding 37 patients with an infection likelihood of "possible" gave an AUC of 0.89 (95% CI 0.85-0.93. Disease severity, as measured by Sequential Organ Failure Assessment (SOFA score or Acute Physiology and Chronic Health Evaluation (APACHE IV score, was not a significant confounding variable. The diagnostic utility of SeptiCyte Lab was evaluated by comparison to various clinical and laboratory parameters available to a clinician within 24 h of ICU admission. SeptiCyte Lab was significantly better at differentiating cases from controls than all tested parameters, both singly and in various logistic combinations, and more than halved the diagnostic error rate compared to procalcitonin in all tested cohorts and cohort combinations. Limitations of this study relate to (1 cohort compositions that do not perfectly reflect the composition of the intended use population, (2 potential biases that could be introduced as a result of the current lack of a gold standard for diagnosing sepsis, and (3 lack of a complete, unbiased comparison to C-reactive protein.SeptiCyte Lab is a rapid molecular assay that may be clinically useful in managing ICU patients with systemic inflammation. Further study in population-based cohorts is needed to validate this assay for clinical use.

  6. Dengue Virus NS1 Protein as a Diagnostic Marker: Commercially Available ELISA and Comparison to qRT-PCR and Serological Diagnostic Assays Currently Used by the State of Florida

    Directory of Open Access Journals (Sweden)

    Jason H. Ambrose

    2017-01-01

    Full Text Available Background. The proper management of patients infected with dengue virus requires early detection. Here, real-time molecular assays have proven useful but have limitations, whereas ELISAs that detect antibodies are still favored but results are obtained too late to be of clinical value. The production of DENV NS1 peaks early during infection and its detection can combine the advantages of both diagnostic approaches. Methods. This study compared assays currently used for detecting DENV infection at the Florida Department of Health including anti-DENV IgM and IgG ELISAs as well as qRT-PCR, against a commercially available DENV NS1 ELISA. These comparisons were made among a group of 21 human sera. Results. Nine of 14 (64.3% DENV qRT-PCR+ samples were also DENV NS1+. Interestingly, the 5 NS1− samples that were qRT-PCR+ were additionally IgM− and IgG+ suggesting a nonprimary infection. Compared to qRT-PCR, the NS1 assay had a sensitivity of 64.3%, specificity 100%, PPV of 100%, and NPV of 58.3%. Conclusions. The NS1 ELISA performed as expected in known DENV qRT-PCR+ samples; however negative NS1 results for qRT-PCR+ and IgG+ sera seemingly reduced the usefulness of the NS1 ELISA for nonprimary cases. We therefore conclude that diagnosis obtained via DENV NS1 ELISA deserves further investigation.

  7. Commissioning and Validation of the First Monte Carlo Based Dose Calculation Algorithm Commercial Treatment Planning System in Mexico

    International Nuclear Information System (INIS)

    Larraga-Gutierrez, J. M.; Garcia-Garduno, O. A.; Hernandez-Bojorquez, M.; Galvan de la Cruz, O. O.; Ballesteros-Zebadua, P.

    2010-01-01

    This work presents the beam data commissioning and dose calculation validation of the first Monte Carlo (MC) based treatment planning system (TPS) installed in Mexico. According to the manufacturer specifications, the beam data commissioning needed for this model includes: several in-air and water profiles, depth dose curves, head-scatter factors and output factors (6x6, 12x12, 18x18, 24x24, 42x42, 60x60, 80x80 and 100x100 mm 2 ). Radiographic and radiochromic films, diode and ionization chambers were used for data acquisition. MC dose calculations in a water phantom were used to validate the MC simulations using comparisons with measured data. Gamma index criteria 2%/2 mm were used to evaluate the accuracy of MC calculations. MC calculated data show an excellent agreement for field sizes from 18x18 to 100x100 mm 2 . Gamma analysis shows that in average, 95% and 100% of the data passes the gamma index criteria for these fields, respectively. For smaller fields (12x12 and 6x6 mm 2 ) only 92% of the data meet the criteria. Total scatter factors show a good agreement ( 2 ) that show a error of 4.7%. MC dose calculations are accurate and precise for clinical treatment planning up to a field size of 18x18 mm 2 . Special care must be taken for smaller fields.

  8. Development and validation of a rapid ultra-high performance liquid chromatography method for the assay of benzalkonium chloride using a quality-by-design approach.

    Science.gov (United States)

    Mallik, Rangan; Raman, Srividya; Liang, Xiaoli; Grobin, Adam W; Choudhury, Dilip

    2015-09-25

    A rapid robust reversed-phase UHPLC method has been developed for the analysis of total benzalkonium chloride in preserved drug formulation. A systematic Quality-by-Design (QbD) method development approach using commercial, off the shelf software (Fusion AE(®)) has been used to optimize the column, mobile phases, gradient time, and other HPLC conditions. Total benzalkonium chloride analysis involves simple sample preparation. The method uses gradient elution from an ACE Excel 2 C18-AR column (50mm×2.1mm, 2.0μm particle size), ammonium phosphate buffer (pH 3.3; 10mM) as aqueous mobile phase and methanol/acetonitrile (85/15, v/v) as the organic mobile phase with UV detection at 214nm. Using these conditions, major homologs of the benzalkonium chloride (C12 and C14) have been separated in less than 2.0min. The validation results confirmed that the method is precise, accurate and linear at concentrations ranging from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The recoveries ranged from 99% to 103% at concentrations from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The validation results also confirmed the robustness of the method as predicted by Fusion AE(®). Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

    Directory of Open Access Journals (Sweden)

    Tri Joko Raharjo

    2017-07-01

    Full Text Available Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR, based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3' was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.

  10. Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

    Science.gov (United States)

    Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Interlaboratory validation of 1% pluronic l92 surfactant as a suitable, aqueous vehicle for testing pesticide formulations using the murine local lymph node assay.

    Science.gov (United States)

    Boverhof, Darrell R; Wiescinski, Connie M; Botham, Phil; Lees, David; Debruyne, Eric; Repetto-Larsay, Marina; Ladics, Gregory; Hoban, Denise; Gamer, Armin; Remmele, Marina; Wang-Fan, Weizheng; Ullmann, Ludwig G; Mehta, Jyotigna; Billington, Richard; Woolhiser, Michael R

    2008-09-01

    The mouse local lymph node assay (LLNA) has become the preferred test for evaluating the dermal sensitization potential of chemicals and requirements are now emerging for its use in the evaluation of their formulated products, especially in the European Union. However, despite its widespread use and extensive validation, the use of this assay for directly testing mixtures and formulated products has been questioned, which could lead to repeat testing using multiple animal models. As pesticide formulations are typically a specific complex blend of chemicals for use as aqueous-based dilutions, traditional vehicles prescribed for the LLNA may change the properties of these formulations leading to inaccurate test results and hazard identification. The objective of this study was to evaluate the effectiveness of an aqueous solution of Pluronic L92 block copolymer surfactant (L92) as a vehicle in the mouse LLNA across five laboratories. Three chemicals with known sensitization potential and four pesticide formulations for which the sensitization potential in guinea pigs and/or humans had previously been assessed were used. Identical LLNA protocols and test materials were used in the evaluation. Assessment of the positive control chemicals, hexylcinnamaldehyde, formaldehyde, and potassium dichromate revealed positive results when using 1% aqueous L92 as the vehicle. Furthermore, results for these chemicals were reproducible among the five laboratories and demonstrated consistent relative potency determinations. The four pesticide formulations diluted in 1% aqueous L92 also demonstrated reproducible results in the LLNA among the five laboratories. Results for these test materials were also consistent with those generated previously using guinea pigs or from human experience. These data support testing aqueous compatible chemicals or pesticide formulations using the mouse LLNA, and provide additional support for the use of 1% aqueous L92 as a suitable, aqueous-based vehicle.

  12. Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

    Science.gov (United States)

    Liu, Ang; Kozhich, Alexander; Passmore, David; Gu, Huidong; Wong, Richard; Zambito, Frank; Rangan, Vangipuram S; Myler, Heather; Aubry, Anne-Françoise; Arnold, Mark E; Wang, Jian

    2015-10-01

    Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds.

    Science.gov (United States)

    Fu, Qiang; Shou, Minshan; Chien, Dwight; Markovich, Robert; Rustum, Abu M

    2010-02-05

    Betamethasone (9alpha-fluoro-16beta-methylprednisolone) is one of the members of the corticosteriod family of active pharmaceutical ingredient (API), which is widely used as an anti-inflammatory agent and also as a starting material to manufacture various esters of betamethasone. A stability-indicating reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate low levels of 26 betamethasone related compounds. The stability-indicating capability of the method was demonstrated through adequate separation of all potential betamethasone related compounds from betamethasone and also from each other that are present in aged and stress degraded betamethasone stability samples. Chromatographic separation of betamethasone and its related compounds was achieved by using a gradient elution at a flow rate of 1.0mL/min on a ACE 3 C18 column (150mmx4.6mm, 3microm particle size, 100A pore size) at 40 degrees C. Mobile phase A of the gradient was 0.1% methanesulfonic acid in aqueous solution and mobile phase B was a mixture of tert-butanol and 1,4-dioxane (7:93, v/v). UV detection at 254nm was employed to monitor the analytes. For betamethasone 21-aldehyde, the QL and DL were 0.02% and 0.01% respectively. For betamethasone and the rest of the betamethasone related compounds, the QL and DL were 0.05% and 0.02%. The precision of betamethasone assay is 0.6% and the accuracy of betamethasone assay ranged from 98.1% to 99.9%.

  14. An immunohistochemical and fluorescence in situ hybridization-based comparison between the Oracle HER2 Bond Immunohistochemical System, Dako HercepTest, and Vysis PathVysion HER2 FISH using both commercially validated and modified ASCO/CAP and United Kingdom HER2 IHC scoring guidelines.

    LENUS (Irish Health Repository)

    O'Grady, Anthony

    2010-12-01

    Immunohistochemistry (IHC) is used as the frontline assay to determine HER2 status in invasive breast cancer patients. The aim of the study was to compare the performance of the Leica Oracle HER2 Bond IHC System (Oracle) with the current most readily accepted Dako HercepTest (HercepTest), using both commercially validated and modified ASCO\\/CAP and UK HER2 IHC scoring guidelines. A total of 445 breast cancer samples from 3 international clinical HER2 referral centers were stained with the 2 test systems and scored in a blinded fashion by experienced pathologists. The overall agreement between the 2 tests in a 3×3 (negative, equivocal and positive) analysis shows a concordance of 86.7% and 86.3%, respectively when analyzed using commercially validated and modified ASCO\\/CAP and UK HER2 IHC scoring guidelines. There is a good concordance between the Oracle and the HercepTest. The advantages of a complete fully automated test such as the Oracle include standardization of key analytical factors and improved turn around time. The implementation of the modified ASCO\\/CAP and UK HER2 IHC scoring guidelines has minimal effect on either assay interpretation, showing that Oracle can be used as a methodology for accurately determining HER2 IHC status in formalin fixed, paraffin-embedded breast cancer tissue.

  15. An immunohistochemical and fluorescence in situ hybridization-based comparison between the Oracle HER2 Bond Immunohistochemical System, Dako HercepTest, and Vysis PathVysion HER2 FISH using both commercially validated and modified ASCO/CAP and United Kingdom HER2 IHC scoring guidelines.

    Science.gov (United States)

    O'Grady, Anthony; Allen, David; Happerfield, Lisa; Johnson, Nicola; Provenzano, Elena; Pinder, Sarah E; Tee, Lilian; Gu, Mai; Kay, Elaine W

    2010-12-01

    Immunohistochemistry (IHC) is used as the frontline assay to determine HER2 status in invasive breast cancer patients. The aim of the study was to compare the performance of the Leica Oracle HER2 Bond IHC System (Oracle) with the current most readily accepted Dako HercepTest (HercepTest), using both commercially validated and modified ASCO/CAP and UK HER2 IHC scoring guidelines. A total of 445 breast cancer samples from 3 international clinical HER2 referral centers were stained with the 2 test systems and scored in a blinded fashion by experienced pathologists. The overall agreement between the 2 tests in a 3×3 (negative, equivocal and positive) analysis shows a concordance of 86.7% and 86.3%, respectively when analyzed using commercially validated and modified ASCO/CAP and UK HER2 IHC scoring guidelines. There is a good concordance between the Oracle and the HercepTest. The advantages of a complete fully automated test such as the Oracle include standardization of key analytical factors and improved turn around time. The implementation of the modified ASCO/CAP and UK HER2 IHC scoring guidelines has minimal effect on either assay interpretation, showing that Oracle can be used as a methodology for accurately determining HER2 IHC status in formalin fixed, paraffin-embedded breast cancer tissue.

  16. Validation and utilization of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinoma and alveolar soft part sarcoma.

    Science.gov (United States)

    Pradhan, Dinesh; Roy, Somak; Quiroga-Garza, Gabriela; Cieply, Kathleen; Mahaffey, Alyssa L; Bastacky, Sheldon; Dhir, Rajiv; Parwani, Anil V

    2015-09-29

    Xp11.2 or TFE3 translocation renal cell carcinomas (RCC) and alveolar soft part sarcoma (ASPS) are characterized by chromosome translocations involving the Xp11.2 breakpoint resulting in transcription factor TFE3 gene fusions. The most common translocations documented in TFE3 RCCs are t(X;1) (p11.2;q21) and t(X;17) (p11.2;q25) which leads to fusion of TFE3 gene on Xp11.2 with PRCC or ASPL respectively. TFE3 immunohistochemistry (IHC) has been inconsistent over time due to background staining problems in part related to fixation issues. Karyotyping to detect TFE3 gene rearrangement requires typically unavailable fresh tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) is generally very challenging due to degradation of RNA in archival material. The study objective was to develop and validate a TFE3 break-apart fluorescence in situ hybridization (FISH) assay to confirm Xp11 translocation RCCs and ASPS. Representative sections of formalin-fixed paraffin-embedded tissue blocks were selected in 40 possible cases. Approximately 60 tumor cells were analyzed in the targeted region. The validation of TFE3 FISH was done with 11 negative and two positive cases. Cut off for a positive result was validated as >7.15 % positive nuclei with any pattern of break-apart signals. FISH evaluation was done blinded of the immunohistochemical or karyotype data. Three out of forty cases were positive for the TFE3 break-apart signals by FISH. The negative cases were reported as clear cell RCC with papillary features (10), clear cell RCC with sarcomatoid areas (2), Papillary RCC with clear cell areas (9), Chromophobe RCC (2), RCC, unclassified type (3) and renal medullary carcinoma (1). 3 of the negative cases were consultation cases for renal tumor with unknown histology. Seven negative cases were soft tissue tumor suspicious for ASPS. Our study validates the utility of TFE3 break-apart FISH on formalin-fixed paraffin-embedded tissue sections for diagnosis and confirmation of

  17. A New Enzyme-Linked Immunosorbent Assay for a Total Anti-T Lymphocyte Globulin Determination: Development, Analytical Validation, and Clinical Applications.

    Science.gov (United States)

    Montagna, Michela; La Nasa, Giorgio; Bernardo, Maria E; Piras, Eugenia; Avanzini, Maria A; Regazzi, Mario; Locatelli, Franco

    2017-06-01

    Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological posttransplant complications, in particular rejection and graft-versus-host disease, after allogeneic hematopoietic stem cell transplantation (HSCT). We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with β-Thalassemia, receiving allogeneic HSCT. Diluted serum samples were incubated with Goat-anti-Rabbit IgG antibody coated on a microtiter plate and then, with Goat-anti-Human IgG labeled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492 nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200-0.095 ng/mL), prepared by diluting a known amount of ATLG in phosphate-buffered saline (PBS). Low, medium, and high-quality control concentrations were 1.56, 6.25, and 25 ng/mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095 ng/mL. We analyzed serum samples from 14 children with β-Thalassemia who received ATLG (Grafalon) at a dose of 10 mg/kg administered as intravenous (IV) infusion on days -5, -4, and -3 before HSCT (day 0). Blood sampling for PK evaluation was performed on days -5, -4, and -3 before and after drug infusion; and then from day -2 to +56. The median total ATLG levels pre-IVand post-IV were 0 and 118 mcg/mL on day -5; 85.9 and 199.2 mcg/mL on day -4; 153 and 270.9 mcg/mL on day -3, respectively. The median PK values of CL was 0.0029 (range: 0.0028-0.0057) L·kg·d, Vd was 0.088 (range: 0.025-0.448) L/kg and t1/2 was 20.2 (range: 5.8-50.2) days. These data suggest that given the marked interindividual variability of total ATLG disposition, the development of

  18. Development and validation of a chiral high-performance liquid chromatography assay for rogletimide and rogletimide-N-oxide isomers in plasma.

    Science.gov (United States)

    Etienne, M C; Oster, W; Milano, G

    1996-01-01

    The purpose of the present study was to develop and validate a stereo-specific high-performance liquid chromatography (HPLC) assay for rogletimide (Rog) and rogletimide-N-oxide (Nox) isomers in plasma. The assay was performed with a chiral cellulose-[4-methylbenzoate]ester column (Chiracel OJ). Optimal separation was achieved isocratically with a mobile phase consisting of n-hexane/anhydrous ethanol (65/35, v/v) at a flow rate of 0.9 ml/min, with the column being thermostated at +35 degrees C (UV detection at 257 nm). Under these conditions, retention times were approximately 17, 28, 31 and 76 min for R-Rog, S-Rog, R-Nox and S-Nox, respectively. S-aminoglutethimide (S-Ag) served as the internal standard (retention time 70 min). An extraction procedure from plasma samples was developed on Bond Elut RP8 500-mg cartridges; conditioning was performed with 5 ml methanol and 5 ml water, after which 1 ml plasma that had previously been spiked with 5 microM S-Ag was applied. Washing was done with 6 ml water and elution, with 4 ml methanol. After evaporation to dryness, residues were dissolved in 400 microliters anhydrous ethanol and 12-48 microliters was injected onto the HPLC system. Blank plasma from healthy donors showed the random presence of a small interference eluting at the retention time of R-Rog, precluding the accurate quantification of R-Rog concentrations below 2.5 microM. Reproducibility assays demonstrated the need to use an internal standard. Taking into account the internal standard, at 2.5 microM the intra- and inter-assay coefficients of variation were 10.5% and 21.0% for R-Rog 5.5% and 8.7% for S-Rog, 7.6% and 20.8% for R-Nox and 11.7% and 6.4% for S-Nox, respectively. The detection limit was 2.5 microM for R-Rog, 0.5 microM for S-Rog, 0.25 microM for R-Nox and 0.5 microM for S-Nox. Linearity was satisfactory at concentrations ranging from 2.5 to 10 microM for R-Rog, from 0.5 to 10 microM for S-Rog, from 0.25 to 2.5 microM for R-Nox and from 0.50 to 2

  19. Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's wort preparations.

    Science.gov (United States)

    de los Reyes, G C; Koda, R T

    2001-12-01

    A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol-water (80:20, v/v) containing 5% HP-beta-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 degrees C, were injected into a C18 column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5-2.5 microg/ml (hypericin), 0.35-1.6 microg/ml (pseudohypericin) and 5-50 microg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were < or = 4.4%, < or = 5.4%, and < or = 2.8%, respectively; inter-day CVs were < or = 5.8%, < or = 4.9%, and < or = 2.5%, respectively. This method may be applied for the routine standardization of St. John's wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.

  20. Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay.

    Science.gov (United States)

    De Boeck, Marlies; van der Leede, Bas-jan; De Vlieger, Kathleen; Geys, Helena; Vynckier, An; Van Gompel, Jacky

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Lucile Courtial

    2017-04-01

    Full Text Available The extracellular signal-regulated protein kinase (ERK signalling pathway controls key cellular processes, such as cell cycle regulation, cell fate determination and the response to external stressors. Although ERK functions are well studied in a variety of living organisms ranging from yeast to mammals, its functions in corals are still poorly known. The present work aims to give practical tools to study the expression level of ERK protein and the activity of the ERK signalling pathway in corals. The antibody characterisation experiment was performed five times and identical results were obtained. The present study validated the immune-reactivity of commercially available antibodies directed against ERK and its phosphorylated/activated forms on protein extracts of the reef-building coral Stylophora pistillata.

  2. Adaptation and validation of a commercial head phantom for cranial radiosurgery dosimetry end-to-end audit.

    Science.gov (United States)

    Dimitriadis, Alexis; Palmer, Antony L; Thomas, Russell A S; Nisbet, Andrew; Clark, Catharine H

    2017-06-01

    To adapt and validate an anthropomorphic head phantom for use in a cranial radiosurgery audit. Two bespoke inserts were produced for the phantom: one for providing the target and organ at risk for delineation and the other for performing dose measurements. The inserts were tested to assess their positional accuracy. A basic treatment plan dose verification with an ionization chamber was performed to establish a baseline accuracy for the phantom and beam model. The phantom and inserts were then used to perform dose verification measurements of a radiosurgery plan. The dose was measured with alanine pellets, EBT extended dose film and a plastic scintillation detector (PSD). Both inserts showed reproducible positioning (±0.5 mm) and good positional agreement between them (±0.6 mm). The basic treatment plan measurements showed agreement to the treatment planning system (TPS) within 0.5%. Repeated film measurements showed consistent gamma passing rates with good agreement to the TPS. For 2%-2 mm global gamma, the mean passing rate was 96.7% and the variation in passing rates did not exceed 2.1%. The alanine pellets and PSD showed good agreement with the TPS (-0.1% and 0.3% dose difference in the target) and good agreement with each other (within 1%). The adaptations to the phantom showed acceptable accuracies. The presence of alanine and PSD do not affect film measurements significantly, enabling simultaneous measurements by all three detectors. Advances in knowledge: A novel method for thorough end-to-end test of radiosurgery, with capability to incorporate all steps of the clinical pathway in a time-efficient and reproducible manner, suitable for a national audit.

  3. Determination of bioactive marker glycyrrhizin in Glycyrrhiza glabra root and commercial formulation by validated HPTLC-densitometric method

    Directory of Open Access Journals (Sweden)

    Perwez Alam

    2014-11-01

    Full Text Available Objective: To develop a simple sensitive high performance thin layer chromatography (HPTLC- densitometric method for quantification of glycyrrhizin in the Glycyrrhiza glabra root methanol extract and licorice root capsule methanol extract. Methods: Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the green solvents ethyl acetate: glacial acetic acid: methanol: water in proportion of 6:2:1:0.5, v/v/ v/v as mobile phase. Scanning and quantification of developed plate was done densitometrically at 254 nm. The method was validated for detection and quantification limits, precision, recovery and robustness according to International Conference on Harmonization guidelines. Results: The system gave compact spot for glycyrrhizin (Rf =0.280±0.001. The regression curve of standard was found to be Y=4.213x+22.078. The limit of detection (15.7 ng per band, limit of quantification (47.1 ng per band, recovery (99.4%-99.8% and precision (≤1.62% and ≤1.84%; intraday and interday were satisfactory for glycyrrhizin. Linearity range for glycyrrhizin was 20- 200 ng (r 2 =0.996. The content of glycyrrhizin was estimated as 5.9% and 11.2% w/w in glycyrrhizin in the Glycyrrhiza glabra root methanol extract and licorice root capsule methanol extract, respectively. Conclusions: This estimation technique is very much useful for the estimation of glycyrrhizin present in various formulations as well as for quality control of crude drugs containing glycyrrhizin.

  4. Validation study of a quantitative multigene reverse transcriptase-polymerase chain reaction assay for assessment of recurrence risk in patients with stage II colon cancer.

    Science.gov (United States)

    Gray, Richard G; Quirke, Philip; Handley, Kelly; Lopatin, Margarita; Magill, Laura; Baehner, Frederick L; Beaumont, Claire; Clark-Langone, Kim M; Yoshizawa, Carl N; Lee, Mark; Watson, Drew; Shak, Steven; Kerr, David J

    2011-12-10

    We developed quantitative gene expression assays to assess recurrence risk and benefits from chemotherapy in patients with stage II colon cancer. We sought validation by using RNA extracted from fixed paraffin-embedded primary colon tumor blocks from 1,436 patients with stage II colon cancer in the QUASAR (Quick and Simple and Reliable) study of adjuvant fluoropyrimidine chemotherapy versus surgery alone. A recurrence score (RS) and a treatment score (TS) were calculated from gene expression levels of 13 cancer-related genes (n = 7 recurrence genes and n = 6 treatment benefit genes) and from five reference genes with prespecified algorithms. Cox proportional hazards regression models and log-rank methods were used to analyze the relationship between the RS and risk of recurrence in patients treated with surgery alone and between TS and benefits of chemotherapy. Risk of recurrence was significantly associated with RS (hazard ratio [HR] per interquartile range, 1.38; 95% CI, 1.11 to 1.74; P = .004). Recurrence risks at 3 years were 12%, 18%, and 22% for predefined low, intermediate, and high recurrence risk groups, respectively. T stage (HR, 1.94; P < .001) and mismatch repair (MMR) status (HR, 0.31; P < .001) were the strongest histopathologic prognostic factors. The continuous RS was associated with risk of recurrence (P = .006) beyond these and other covariates. There was no trend for increased benefit from chemotherapy at higher TS (P = .95). The continuous 12-gene RS has been validated in a prospective study for assessment of recurrence risk in patients with stage II colon cancer after surgery and provides prognostic value that complements T stage and MMR. The TS was not predictive of chemotherapy benefit.

  5. Validated RP-HPLC Method for the Assay of Etoricoxib (A Non-Steroidal Anti-Inflammatory Drug in Pharmaceutical Dosage Forms

    Directory of Open Access Journals (Sweden)

    Srinivasu Topalli

    2012-01-01

    Full Text Available A simple, accurate, sensitive and reproducible reverse phase high performance liquid chromatographic method has been developed for the quantitative determination of Etoricoxib in pharmaceutical dosage forms. The assay was performed on Hypersil ODS C-18 (250 x 4.6 mm., 5µm particle size column using acetonitrile and potassium dihydrogen phosphate buffer (pH 4.2 (46:54 % v/v as mobile phase with UV detection at 280 nm (flow rate 1.2 ml/min. Bromhexine was used as an internal standard. Quantization was achieved by measurement of the peak area ratio of the drug to the internal standard. The limit of detection (LOD and the limit of quantification (LOQ were 0.0704 µg ml-1 and 0.2134 µg ml-1 respectively. Each analysis required no longer than 10 minutes. The calibration curve was linear over the concentration range from 0.5-85.0 µg ml-1. The retention times of Etoricoxib and Bromhexine were found to be 3.083 and 7.631 minutes respectively. The proposed method was validated according to the ICH guidelines and can be used successfully to analyse marketed formulations.

  6. Validation of the World Health Organization Enzyme-Linked Immunosorbent Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti-Pneumococcal Antibodies in Human Serum.

    Science.gov (United States)

    Lee, Hyunju; Lim, Soo Young; Kim, Kyung Hyo

    2017-10-01

    The World Health Organization (WHO) enzyme-linked immunosorbent assay (ELISA) guideline is currently accepted as the gold standard for the evaluation of immunoglobulin G (IgG) antibodies specific to pneumococcal capsular polysaccharide. We conducted validation of the WHO ELISA for 7 pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by evaluating its specificity, precision (reproducibility and intermediate precision), accuracy, spiking recovery test, lower limit of quantification (LLOQ), and stability at the Ewha Center for Vaccine Evaluation and Study, Seoul, Korea. We found that the specificity, reproducibility, and intermediate precision were within acceptance ranges (reproducibility, coefficient of variability [CV] ≤ 15%; intermediate precision, CV ≤ 20%) for all serotypes. Comparisons between the provisional assignments of calibration sera and the results from this laboratory showed a high correlation > 94% for all 7 serotypes, supporting the accuracy of the ELISA. The spiking recovery test also fell within an acceptable range. The quantification limit, calculated using the LLOQ, for each of the serotypes was 0.05-0.093 μg/mL. The freeze-thaw stability and the short-term temperature stability were also within an acceptable range. In conclusion, we showed good performance using the standardized WHO ELISA for the evaluation of serotype-specific anti-pneumococcal IgG antibodies; the WHO ELISA can evaluate the immune response against pneumococcal vaccines with consistency and accuracy. © 2017 The Korean Academy of Medical Sciences.

  7. Validation and development of an immunonephelometric assay for the determination of alpha-1 antitrypsin levels in dried blood spots from patients with COPD

    Directory of Open Access Journals (Sweden)

    Laura Russo Zillmer

    2013-09-01

    Full Text Available OBJECTIVE: To validate and develop an immunonephelometric assay for the determination of alpha-1 antitrypsin (AAT levels in dried blood spots from COPD patients in Brazil. METHODS: We determined AAT levels in serum samples and dried blood spots from 192 COPD patients. For the preparation of dried blood spots, a disk (diameter, 6 mm was placed into a tube, eluted with 200 µL of PBS, and stored overnight at 4ºC. All of the samples were analyzed by immunonephelometry in duplicate. We used the bootstrap resampling method in order to determine a cut-off point for AAT levels in dried blood spots. RESULTS: The correlation coefficient between the AAT levels in serum samples and those in dried blood spots was r = 0.45. For dried blood spots, the cut-off value was 2.02 mg/dL (97% CI: 1.45-2.64 mg/dL, with a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 95.7%, 27.2%, and 100%, respectively. CONCLUSIONS: This method for the determination of AAT levels in dried blood spots appears to be a reliable screening tool for patients with AAT deficiency.

  8. Specific and sensitive enzyme-linked immunosorbent assays for analysis of residual allergenic food proteins in commercial bottled wine fined with egg white, milk, and nongrape-derived tannins.

    Science.gov (United States)

    Rolland, Jennifer M; Apostolou, Effie; de Leon, Maria P; Stockley, Creina S; O'Hehir, Robyn E

    2008-01-23

    Regulations introduced by the Food Standards Australia New Zealand in December 2002 require all wine and wine product labels in Australia to identify the presence of a processing aid, additive or other ingredient, which is known to be a potential allergen. The objective of this study was to establish sensitive assays to detect and measure allergenic proteins from commonly used processing aids in final bottled wine. Sensitive and specific enzyme-linked immunosorbent assays (ELISA) were developed and established for the proteins casein, ovalbumin, and peanut. Lower limits of detection of these proteins were 8, 1, and 8 ng/mL, respectively. A panel of 153 commercially available bottled Australian wines were tested by these ELISA, and except for two red wines known to contain added whole eggs, residuals of these food allergens were not detected in any wine. These findings are consistent with a lack of residual potentially allergenic egg-, milk-, or nut-derived processing aids in final bottled wine produced in Australia according to good manufacturing practice at a concentration that could cause an adverse reaction in egg, milk, or peanut/tree-nut allergic adult consumers.

  9. Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA

    DEFF Research Database (Denmark)

    Bosteen, Markus Høybye; Dahlbäck, Björn; Nielsen, Lars Bo

    2015-01-01

    that specifically recognizes human apoM in plasma using commercially available reagents. Commercial apoM antibodies were screened for compatibility in a sandwich ELISA-based assay. One optimal pair of antibodies was chosen, and sample preparation, buffers, and incubation times were optimized to generate a simple...... and reproducible method. Validation and comparison to a previously described ELISA for apoM confirmed that the assay displays a high degree of sensitivity, specificity, and precision. Our results show that commercially available antibodies can be used to accurately measure human plasma apoM. This method can...

  10. DNA detection of Schistosoma japonicum: diagnostic validity of a LAMP assay for low-intensity infection and effects of chemotherapy in humans.

    Directory of Open Access Journals (Sweden)

    Jing Xu

    2015-04-01

    Full Text Available Schistosomiasis has decreased significantly in prevalence and intensity of infection in China, thus more accurate and sensitive methods are desperately needed for the further control of schistosomiasis. The present work aimed to assess the utility of the loop-mediated isothermal amplification (LAMP for detection of light intensity infection or false-negative patients and patients post-treatment, targeting the highly repetitive retrotransposon SjR2 of Schistosoma japonicum.LAMP was first assessed in rabbits with low intensity infection (EPG<10. Then 110 patient sera from Hunan Province, China, and 47 sera after treatment by praziquantel were used to evaluate the diagnostic validity of LAMP. Meanwhile, 42 sera from healthy individuals in a non-endemic area, and 60 sera from "healthy" residents who were identified as being negative for feces examination and immuno-methods in an endemic area were also examined. The results showed that LAMP could detect S. japonicum DNA in sera from rabbits at 3rd day post-infection. Following administration of praziquantel, the S. japonicum DNA in rabbit sera became negative at 10 weeks post-treatment. Of 110 sera from patients, LAMP showed 95.5% sensitivity, and even for 41 patients with less than 10 EPG, the sensitivity of LAMP still reached to 95.1%. For 47 patients after treatment, the negative conversion rate of S. japonicum DNA in patient sera increased from 23.4%, 61.7% to 83.0% at 3 months, 6 months and 9 months post-treatment, respectively. No false-positive result was obtained for 42 human sera from non-endemic area, while for the 60 "healthy" individuals from endemic area, 10 (16.7% individuals were positive by LAMP, which suggested that these individuals might be false-negative patients.The present study demonstrated that the LAMP assay is sensitive, specific, and affordable, which would help reduce schistosomiasis transmission through targeted treatment of individuals, particularly for those with

  11. Validation of an accelerated solvent extraction liquid chromatography-tandem mass spectrometry method for Pacific ciguatoxin-1 in fish flesh and comparison with the mouse neuroblastoma assay.

    Science.gov (United States)

    Wu, Jia Jun; Mak, Yim Ling; Murphy, Margaret B; Lam, James C W; Chan, Wing Hei; Wang, Mingfu; Chan, Leo L; Lam, Paul K S

    2011-07-01

    Ciguatera fish poisoning (CFP) is a global foodborne illness caused by consumption of seafood containing ciguatoxins (CTXs) originating from dinoflagellates such as Gambierdiscus toxicus. P-CTX-1 has been suggested to be the most toxic CTX, causing ciguatera at 0.1 μg/kg in the flesh of carnivorous fish. CTXs are structurally complex and difficult to quantify, but there is a need for analytical methods for CFP toxins in coral reef fishes to protect human health. In this paper, we describe a sensitive and rapid extraction method using accelerated solvent extraction combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the detection and quantification of P-CTX-1 in fish flesh. By the use of a more sensitive MS system (5500 QTRAP), the validated method has a limit of quantification (LOQ) of 0.01 μg/kg, linearity correlation coefficients above 0.99 for both solvent- and matrix-based standard solutions as well as matrix spike recoveries ranging from 49% to 85% in 17 coral reef fish species. Compared with previous methods, this method has better overall recovery, extraction efficiency and LOQ. Fish flesh from 12 blue-spotted groupers (Cephalopholis argus) was assessed for the presence of CTXs using HPLC-MS/MS analysis and the commonly used mouse neuroblastoma assay, and the results of the two methods were strongly correlated. This method is capable of detecting low concentrations of P-CTX-1 in fish at levels that are relevant to human health, making it suitable for monitoring of suspected ciguateric fish both in the environment and in the marketplace.

  12. SU-F-T-152: Experimental Validation and Calculation Benchmark for a Commercial Monte Carlo Pencil BeamScanning Proton Therapy Treatment Planning System in Heterogeneous Media

    Energy Technology Data Exchange (ETDEWEB)

    Lin, L; Huang, S; Kang, M; Ainsley, C; Simone, C; McDonough, J; Solberg, T [University of Pennsylvania, Philadelphia, PA (United States)

    2016-06-15

    Purpose: Eclipse AcurosPT 13.7, the first commercial Monte Carlo pencil beam scanning (PBS) proton therapy treatment planning system (TPS), was experimentally validated for an IBA dedicated PBS nozzle in the CIRS 002LFC thoracic phantom. Methods: A two-stage procedure involving the use of TOPAS 1.3 simulations was performed. First, Geant4-based TOPAS simulations in this phantom were experimentally validated for single and multi-spot profiles at several depths for 100, 115, 150, 180, 210 and 225 MeV proton beams, using the combination of a Lynx scintillation detector and a MatriXXPT ionization chamber array. Second, benchmark calculations were performed with both AcurosPT and TOPAS in a phantom identical to the CIRS 002LFC, with the exception that the CIRS bone/mediastinum/lung tissues were replaced with similar tissues that are predefined in AcurosPT (a limitation of this system which necessitates the two stage procedure). Results: Spot sigmas measured in tissue were in agreement within 0.2 mm of TOPAS simulation for all six energies, while AcurosPT was consistently found to have larger spot sigma (<0.7 mm) than TOPAS. Using absolute dose calibration by MatriXXPT, the agreements between profiles measurements and TOPAS simulation, and calculation benchmarks are over 97% except near the end of range using 2 mm/2% gamma criteria. Overdosing and underdosing were observed at the low and high density side of tissue interfaces, respectively, and these increased with increasing depth and decreasing energy. Near the mediastinum/lung interface, the magnitude can exceed 5 mm/10%. Furthermore, we observed >5% quenching effect in the conversion of Lynx measurements to dose. Conclusion: We recommend the use of an ionization chamber array in combination with the scintillation detector to measure absolute dose and relative PBS spot characteristics. We also recommend the use of an independent Monte Carlo calculation benchmark for the commissioning of a commercial TPS. Partially

  13. Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

    International Nuclear Information System (INIS)

    Nielsen, Torsten; Storhoff, James; Wallden, Brett; Schaper, Carl; Ferree, Sean; Liu, Shuzhen; Gao, Dongxia; Barry, Garrett; Dowidar, Naeem; Maysuria, Malini

    2014-01-01

    NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories. Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test. The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area). The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories

  14. AR2, a novel automatic muscle artifact reduction software method for ictal EEG interpretation: Validation and comparison of performance with commercially available software [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Shennan Aibel Weiss

    2017-04-01

    Full Text Available Objective: To develop a novel software method (AR2 for reducing muscle contamination of ictal scalp electroencephalogram (EEG, and validate this method on the basis of its performance in comparison to a commercially available software method (AR1 to accurately depict seizure-onset location. Methods: A blinded investigation used 23 EEG recordings of seizures from 8 patients. Each recording was uninterpretable with digital filtering because of muscle artifact and processed using AR1 and AR2 and reviewed by 26 EEG specialists. EEG readers assessed seizure-onset time, lateralization, and region, and specified confidence for each determination. The two methods were validated on the basis of the number of readers able to render assignments, confidence, the intra-class correlation (ICC, and agreement with other clinical findings. Results: Among the 23 seizures, two-thirds of the readers were able to delineate seizure-onset time in 10 of 23 using AR1, and 15 of 23 using AR2 (p<0.01. Fewer readers could lateralize seizure-onset (p<0.05. The confidence measures of the assignments were low (probable-unlikely, but increased using AR2 (p<0.05. The ICC for identifying the time of seizure-onset was 0.15 (95% confidence interval (CI, 0.11-0.18 using AR1 and 0.26 (95% CI 0.21-0.30 using AR2.  The EEG interpretations were often consistent with behavioral, neurophysiological, and neuro-radiological findings, with left sided assignments correct in 95.9% (CI 85.7-98.9%, n=4 of cases using AR2, and 91.9% (77.0-97.5% (n=4 of cases using AR1. Conclusions: EEG artifact reduction methods for localizing seizure-onset does not result in high rates of interpretability, reader confidence, and inter-reader agreement. However, the assignments by groups of readers are often congruent with other clinical data. Utilization of the AR2 software method may improve the validity of ictal EEG artifact reduction.

  15. Re-evaluation of Assay Data of Spent Nuclear Fuel obtained at Japan Atomic Energy Research Institute for validation of burnup calculation code systems

    Energy Technology Data Exchange (ETDEWEB)

    Suyama, Kenya, E-mail: suyama.kenya@jaea.go.jp [Office of International Relations, Nuclear Safety Division, Ministry of Education, Culture, Sports, Science and Technology - Japan, 3-2-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-8959 (Japan); Murazaki, Minoru; Ohkubo, Kiyoshi [Fuel Cycle Safety Research Group, Nuclear Safety Research Center, Japan Atomic Energy Agency, 2-4 Shirakata Shirane, Tokai-mura, Ibaraki 319-1195 (Japan); Nakahara, Yoshinori [Research Group for Analytical Science, Nuclear Science and Engineering Directorate, Japan Atomic Energy Agency, 2-4 Shirakata Shirane, Tokai-mura, Ibaraki 319-1195 (Japan); Uchiyama, Gunzo [Fuel Cycle Safety Research Group, Nuclear Safety Research Center, Japan Atomic Energy Agency, 2-4 Shirakata Shirane, Tokai-mura, Ibaraki 319-1195 (Japan)

    2011-05-15

    Highlights: > The specifications required for the analyses of the destructive assay data taken from irradiated fuel in Ohi-1 and Ohi-2 PWRs were documented in this paper. > These data were analyzed using the SWAT2.1 code, and the calculation results showed good agreement with experimental results. > These destructive assay data are suitable for the benchmarking of the burnup calculation code systems. - Abstract: The isotopic composition of spent nuclear fuels is vital data for studies on the nuclear fuel cycle and reactor physics. The Japan Atomic Energy Agency (JAEA) has been active in obtaining such data for pressurized water reactor (PWR) and boiling water reactor (BWR) fuels, and some data has already been published. These data have been registered with the international Spent Fuel Isotopic Composition Database (SFCOMPO) and widely used as international benchmarks for burnup calculation codes and libraries. In this paper, Assay Data of Spent Nuclear Fuel from two fuel assemblies irradiated in the Ohi-1 and Ohi-2 PWRs in Japan are shown. The destructive assay data from Ohi-2 have already been published. However, these data were not suitable for the benchmarking of calculation codes and libraries because several important specifications and data were not included. This paper summarizes the details of destructive assay data and specifications required for analyses of isotopic composition from Ohi-1 and Ohi-2. For precise burnup analyses, the burnup values of destructive assay samples were re-evaluated in this study. These destructive assay data were analyzed using the SWAT2.1 code, and the calculation results showed good agreement with experimental results. This indicates that the quality of destructive assay data from Ohi-1 and Ohi-2 PWRs is high, and that these destructive assay data are suitable for the benchmarking of burnup calculation code systems.

  16. Development and Validation of a Multiplex PCR-Based Assay for the Upper Respiratory Tract Bacterial Pathogens Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.

    Science.gov (United States)

    Post; White; Aul; Zavoral; Wadowsky; Zhang; Preston; Ehrlich

    1996-06-01

    Background: Conventional simplex polymerase chain reaction (PCR)-based assays are limited in that they only provide for the detection of a single infectious agent. Many clinical diseases, however, present in a nonspecific, or syndromic, fashion, thereby necessitating the simultaneous assessment of multiple pathogens. Panel-based molecular diagnostic testing can be accomplished by the development of multiplex PCR-based assays, which can detect, individually or severally, different pathogens that are associated with syndromic illness. As part of a larger program of panel development, an assay that can simultaneously detect Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was developed. These organisms were chosen as they are the most common bacterial pathogens associated with both the acute and chronic forms of otitis media; they are also responsible for a high percentage of sinus infections in both children and adults. In addition, H. influenzae and S. pneumoniae are commonly associated with septic meningitits. Methods and Results: Multiple individual PCR-based assays were developed for each of the three target organisms which were then evaluated for sensitivity and specificity. Utilizing the simplex assays that met our designated performance criteria, a matrix style approach was used to develop a duplex H. influenzae-S. pneumoniae assay. The duplex assay was then used as a single component in the development of a triplex assay, wherein the various M. catarrhalis primer-probe sets were tested for compatibility with the existing assay. A single-step PCR protocol, with species-specific primers for each of the three target organisms and a liquid hybridization-gel retardation amplimer detection system, was developed, which amplifies and then discriminates among each of the amplification products according to size. This assay is able to detect all three organisms in a specific manner, either individually or severally. Dilutional experiments

  17. Ensaio imunoenzimático comercial no diagnóstico sorológico das infecções por herpesvírus bovino 1 A commercial enzyme immune assay in serodiagnosis of bovine herpesvirus 1 infections

    Directory of Open Access Journals (Sweden)

    Kerlei Cristina Médici

    2000-04-01

    Full Text Available Avaliou-se o desempenho de um ensaio imunoenzimático, obtido de fonte comercial, na identificação de anticorpos contra herpesvírus bovino tipo 1 (BHV-1, induzidos tanto por infecção natural quanto por vacinação, em 1000 amostras de soros sangüíneos de bovinos. A análise comparativa dos resultados obtidos no sistema avaliado e na técnica padrão de soroneutralização mostrou uma concordância de 97,05% (K=0,94 entre as duas metodologias de diagnóstico sorológico.The performance of a commercial immune assay in the identification antibody of natural infection or vaccination against bovine herpesvirus type 1 (BHV-1 in 1000 samples of bovine serum was evaluated. The comparative analysis from the result of the evaluated system and standard serum neutralization technique showed a rate of agreement of 97.05% (K=0.94 between the two serologic diagnotic methods.

  18. Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

    Science.gov (United States)

    Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

    2015-02-18

    In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

  19. 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.

  20. Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

    Directory of Open Access Journals (Sweden)

    Sanchita Bhadra

    Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

  1. Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples.

    LENUS (Irish Health Repository)

    Jones, S

    2011-01-01

    Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche\\/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche\\/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6%, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.

  2. Characterization of the commercially-available fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a marker for chloroquine resistance and uptake in a 96-well plate assay.

    Directory of Open Access Journals (Sweden)

    Cheryl C Y Loh

    Full Text Available Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.

  3. Considerations on development, validation, application, and quality control of immuno(metric) biomarker assays in clinical cancer research: an EORTC-NCI working group report.

    NARCIS (Netherlands)

    Sweep, C.G.J.; Fritsche, H.A.; Gion, M.; Klee, G.G.; Schmitt, M.

    2003-01-01

    A major dilemma associated with immuno(metric) assays for biomarkers is that various kits employing antibodies with differing specificities and binding affinities may generate non-equivalent test results. Also, variation in sample processing and the use of different standards (reference material)

  4. Development and validation of an indirect Enzyme-linked Immunosorbent Assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants

    NARCIS (Netherlands)

    Heijden, van der H.M.J.F.; Bouwstra, R.J.; Mars, M.H.; Poel, van der W.H.M.; Wellenberg, G.J.; Maanen, van C.

    2013-01-01

    To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood

  5. Development and independent validation of a prognostic assay for stage II colon cancer using formalin-fixed paraffin-embedded tissue.

    LENUS (Irish Health Repository)

    Kennedy, Richard D

    2011-12-10

    Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples.

  6. Commercial lumber

    Science.gov (United States)

    Kent A. McDonald; David E. Kretschmann

    1999-01-01

    In a broad sense, commercial lumber is any lumber that is bought or sold in the normal channels of commerce. Commercial lumber may be found in a variety of forms, species, and types, and in various commercial establishments, both wholesale and retail. Most commercial lumber is graded by standardized rules that make purchasing more or less uniform throughout the country...

  7. Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

    Science.gov (United States)

    Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

    2018-04-06

    Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

  8. MMP Mediated Degradation of Type VI Collagen Is Highly Associated with Liver Fibrosis - Identification and Validation of a Novel Biochemical Marker Assay

    DEFF Research Database (Denmark)

    Veidal, Sanne Skovgard; Karsdal, Morten Asser; Vassiliadis, Efstathios

    2011-01-01

    Background and Aims: During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small...... fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis. Methods: Mass spectrometric...... analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon...

  9. Laboratory Validation of Four Black Carbon Measurement Methods for Determination of the Nonvolatile Particulate Matter (nvPM) Mass Emissions from Commercial Aircraft Engines

    Science.gov (United States)

    Four candidate black carbon (BC) measurement techniques have been identified by the SAE International E-31 Committee for possible use in determining nonvolatile particulate matter (nvPM) mass emissions during commercial aircraft engine certification. These techniques are carbon b...

  10. Validation of Performance of the Gen-Probe Human Immunodeficiency Virus Type 1 Viral Load Assay with Genital Swabs and Breast Milk Samples

    Science.gov (United States)

    DeVange Panteleeff, Dana; Emery, Sandra; Richardson, Barbra A.; Rousseau, Christine; Benki, Sarah; Bodrug, Sharon; Kreiss, Joan K.; Overbaugh, Julie

    2002-01-01

    Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in ≥77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load. PMID:12409354

  11. Comparing in vivo biodistribution with radiolabeling and Franz cell permeation assay to validate the efficacy of both methodologies in the evaluation of nanoemulsions: a safety approach

    International Nuclear Information System (INIS)

    Cerqueira-Coutinho, C S; De Campo, V E B; Mansur, C R E; Rossi, A L; Veiga, V F; Holandino, C; Freitas, Z M F; Ricci-Junior, E; Santos, E P; Santos-Oliveira, R

    2016-01-01

    The Franz cells permeation assay has been performed for over 25 years. However, the advent of nanotechnology created a whole new world, especially with regard to topical products. In this new global scenario an increasing number of nanostructure-based delivery systems (NDSs) have emerged and a global warning relating to the safety of these NDSs is arising. This work studied the efficacy of the Franz cells assay, comparing it with the radiolabeling biodistribution test. For this purpose a formulation of sunscreen based on an NDS was developed and characterized. The results demonstrated both that the NDS did not present in vitro cytotoxicity and that the radiolabeling biodistribution test is more precise for the evaluation of NDS cosmetics than the Franz cells assay, since it detected the permeation of the NDS at a picogram order. Due to this fact, and considering all the concerns related to NDSs and nanoparticles in general, more precise methods must be used in order to guarantee the safe use of these new classes of products. (paper)

  12. Commercially available avian and mammalian whole prey diet items targeted for consumption by managed exotic and domestic pet felines: true metabolizable energy and amino acid digestibility using the precision-fed cecectomized rooster assay.

    Science.gov (United States)

    Kerr, K R; Kappen, K L; Garner, L M; Utterback, P L; Parsons, C M; Swanson, K S

    2014-10-01

    Whole prey diets are commonly used in the zoo and home setting for captive exotic and domestic cats, respectively. Despite their increase in popularity, nutrient digestibility of such diets has been poorly studied. In this study, the precision-fed cecectomized rooster assay was used to determine the protein quality and nitrogen-corrected true ME (TMEn) of 17 whole prey samples (mice [1 to 2 , 10 to 13 , 21 to 25 , 30 to 40 , and 150 to 180 d old], rats [1 to 4, 10 to 13, 21 to 25, 32 to 42, and >60 d old], rabbits [stillborn, 30 to 45 d old, and >65 d old], chicken [1 to 3 d old], and quail [1 to 3, 21 to 40, and >60 d old]) and 2 ground poultry-based products (chicken and duck). Amino acid score (AAS) and protein digestibility corrected AAS (PDCAAS) were calculated using the nutrient profile recommendations for domestic cat food as a reference value (AAFCO, 2012). Average individual indispensable AA (IAA) and total IAA (TIAA) digestibility coefficients were variable anddepended on AA (84 to 94% TIAA, 85 to 95% Arg, 87 to 96% His, 82 to 92% Ile, 84 to 94% Leu, 85 to 93% Lys, 89 to 97% Met, 83 to 94% Phe, 80 to 95% Thr, 84 to 94% Trp, and 80 to 93% Val) and sample. For a majority of the whole prey items, AA concentrations were greater than the Association of American Feed Control Officials ( AAFCO: , 2012) domestic cat nutrient profile recommendations for growth and reproduction and adult maintenance; however, some whole prey had AA concentrations below the AAFCO (2012) recommendations: Met + Cys (1.10% DM) in ground duck (1.06% DM) and taurine (Tau; 0.20% DM) in 30-to-45- and >65-d-old rabbits (0.01 and 0.10% DM, respectively), 150-to-180-d-old mice (0.18% DM), and ground duck (0.15% DM). The TMEn (3.76 to 6.44 kcal/g DM) expressed as the percent of GE (i.e., TMEn/GE) ranged from 66 to 85%, demonstrating how variable the digestibility of these items may be and justifying more research in this area. Both Met and Tau are commonly added to commercial pet foods, so

  13. NCI-FDA Interagency Oncology Task Force Workshop Provides Guidance for Analytical Validation of Protein-based Multiplex Assays | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    An NCI-FDA Interagency Oncology Task Force (IOTF) Molecular Diagnostics Workshop was held on October 30, 2008 in Cambridge, MA, to discuss requirements for analytical validation of protein-based multiplex technologies in the context of its intended use. This workshop developed through NCI's Clinical Proteomic Technologies for Cancer initiative and the FDA focused on technology-specific analytical validation processes to be addressed prior to use in clinical settings. In making this workshop unique, a case study approach was used to discuss issues related to

  14. The OECD validation program of the H295R steroidogenesis assay for the identification of in vitro inhibitors and inducers of testosterone and estradiol production. Phase 2: Inter-laboratory pre-validation studies

    DEFF Research Database (Denmark)

    Hecker, Markus; Hollert, Henner; Cooper, Ralph

    2007-01-01

    plate was run in conjunction with the chemical exposure plate to account for inter-assay variation. Each chemical exposure was conducted two or three times. Results. All laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the different...

  15. The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

    Science.gov (United States)

    Townsend, Todd A; Parrish, Marcus C; Engelward, Bevin P; Manjanatha, Mugimane G

    2017-08-01

    DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, "EpiComet," which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Development of robotic plasma radiochemical assays for positron emission tomography

    International Nuclear Information System (INIS)

    Alexoff, D.L.; Shea, C.; Fowler, J.S.; Gatley, S.J.; Schlyer, D.J.

    1995-01-01

    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 [ 11 C]Benztropine, [ 11 C]cocaine, [ 11 C]clorgyline, [ 11 C]deprenyl, [ 11 C]methadone, [ 11 C]methylphenidate, [ 11 C]raclorpride, and [ 11 C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers

  17. Validation of the FDG (18F) radiochemical purity assay by thin layer chromatography; Validacao do ensaio de pureza radioquimica do FDG (18F) por cromatografia em camada delgada

    Energy Technology Data Exchange (ETDEWEB)

    Leao, R.L.C.; Oliveira, M.L.; Nascimento, J.E.; Nascimento, N.C.E.S., E-mail: renata.lleao@hotmail.com [Centro Regional de Ciencias Nucleares do Nordeste (CRCN-NE/CNEN-PE), Recife, PE (Brazil). Div. de Producao de Radiofarmacos

    2013-08-15

    All methodologies utilized in radiopharmaceutical industry should be validated in order to prove that they meet the requirements of analytical applications, ensuring the reliability of the results. At a radiopharmaceutical industry there is one challenge aspect: sometimes it is not possible use a stable standard to perform the validation analysis. In order to overcome this difficulty, the objective of this study was to suggest a validation protocol for these methodologies, based on the recommendations of RE n° 899/Agencia Nacional de Vigilancia Sanitaria (ANVISA), and prove its efficiency, performing the radiochemical purity validation test of FDG (18F), by TLC. To obtain the calibration curve, we suggested that the theoretical activity values should be determined using a dose calibrator, simultaneously of each analysis performed by TLC, for 5 hours. The method was linear (R{sup 2} of 0.996), precise (CV% <5%) and accurate (96.85% < accuracy < 102.56%). In relation to the robustness test, our experiments evaluated the influence of the distance travelled by mobile phase, variations at mobile phase concentration and type of chromatographic plate (silica gel on glass or aluminium plates). The detection and quantification limits were determined (321.9 and 1065.6 kBq, respectively). As expected, this methodology was nonspecific, showing a slight spot corresponding to the FDM. The proposed protocol was efficient and the methodology tested was effective to determine the radiochemical purity of FDG (18F), in accordance to the limits recommended by ANVISA. (author)

  18. Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

    Science.gov (United States)

    Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

    2016-04-19

    A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

  19. Development and validation of a hydrophilic interaction chromatography-mass spectrometry assay for taurine and methionine in matrices rich in carbohydrates.

    Science.gov (United States)

    de Person, Marine; Hazotte, Aurélie; Elfakir, Claire; Lafosse, Michel

    2005-07-22

    A new procedure based on hydrophilic interaction chromatography coupled to tandem mass spectrometry (ionisation process by pneumatically assisted electrospray in negative ion mode), is developed and validated for the simultaneous determination of underivatised taurine and methionine in beverages rich in carbohydrates such as energy drinks. No initial clean-up procedure and no sample derivatisation are required. Satisfactory analysis was obtained on an Astec apHera NH2 (150 mm x 4.6 mm; 5 microm) column with methanol-water (60/40) as mobile phase. The method was validated in terms of specificity, detection limits, linearity, accuracy, precision and stability, using threonine as internal standard. The potential effects of matrix and endogenous amino acid content were also examined. The limits of detection in the beverage varied from 20 microg L(-1) for taurine to 50 micro L(-1) for methionine.

  20. Analytical method (HPLC, validation used for identification and assay of the pharmaceutical active ingredient, Tylosin tartrate for veterinary use and its finite product Tilodem 50, hydrosoluble powder

    Directory of Open Access Journals (Sweden)

    Maria Neagu

    2010-12-01

    Full Text Available In SC DELOS IMPEX ’96 SRL the quality of the active pharmaceutical ingredient (API for the finite product Tilodem 50 - hydrosoluble powder was acomkplished in the respect of last European Pharmacopoeia.The method for analysis used in this purpose was the compendial method „Tylosin tartrate for veterinary use” in EurPh. in vigour edition and represent a variant developed and validation „in house”.The parameters which was included in the methodology validation for chromatographic method are the followings: Selectivity, Linearity, Linearity range, Detection and Quantification limits, Precision, Repeatability (intra day, Inter-Day Reproductibility, Accuracy, Robustness, Solutions’ stability and System suitability. According to the European Pharmacopoeia, the active pharmaceutical ingredient is consistent, in terms of quality, if it contains Tylosin A - minimum 80% and the amount of Tylosin A, B, C, D, at minimum 95%. Identification and determination of each component separately (Tylosin A, B, C, D is possible by chromatographic separation-HPLC. Validation of analytical methods is presented below.

  1. DNA detection of Schistosoma japonicum: diagnostic validity of a LAMP assay for low-intensity infection and effects of chemotherapy in humans.

    Science.gov (United States)

    Xu, Jing; Guan, Zhi-Xun; Zhao, Bo; Wang, Yan-Yan; Cao, Yun; Zhang, Hui-Qin; Zhu, Xing-Quan; He, Yong-Kang; Xia, Chao-Ming

    2015-04-01

    Schistosomiasis has decreased significantly in prevalence and intensity of infection in China, thus more accurate and sensitive methods are desperately needed for the further control of schistosomiasis. The present work aimed to assess the utility of the loop-mediated isothermal amplification (LAMP) for detection of light intensity infection or false-negative patients and patients post-treatment, targeting the highly repetitive retrotransposon SjR2 of Schistosoma japonicum. LAMP was first assessed in rabbits with low intensity infection (EPGLAMP. Meanwhile, 42 sera from healthy individuals in a non-endemic area, and 60 sera from "healthy" residents who were identified as being negative for feces examination and immuno-methods in an endemic area were also examined. The results showed that LAMP could detect S. japonicum DNA in sera from rabbits at 3rd day post-infection. Following administration of praziquantel, the S. japonicum DNA in rabbit sera became negative at 10 weeks post-treatment. Of 110 sera from patients, LAMP showed 95.5% sensitivity, and even for 41 patients with less than 10 EPG, the sensitivity of LAMP still reached to 95.1%. For 47 patients after treatment, the negative conversion rate of S. japonicum DNA in patient sera increased from 23.4%, 61.7% to 83.0% at 3 months, 6 months and 9 months post-treatment, respectively. No false-positive result was obtained for 42 human sera from non-endemic area, while for the 60 "healthy" individuals from endemic area, 10 (16.7%) individuals were positive by LAMP, which suggested that these individuals might be false-negative patients. The present study demonstrated that the LAMP assay is sensitive, specific, and affordable, which would help reduce schistosomiasis transmission through targeted treatment of individuals, particularly for those with negative stool examinations who may yet remain infected. The LAMP assay may provide a potential tool to support schistosomiasis control and elimination strategies.

  2. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  3. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  4. Validation of a commercial insulated isothermal PCR-based POCKIT test for rapid and easy detection of white spot syndrome virus infection in Litopenaeus vannamei.

    Directory of Open Access Journals (Sweden)

    Yun-Long Tsai

    Full Text Available Timely pond-side detection of white spot syndrome virus (WSSV plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR, and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction. The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV, monodon baculovirus (MBV, and hepatopancreatic parvovirus (HPV positive samples. Accuracy analysis using 700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61-95.56% and specificity 97% (95% CI: 94.31-98.50%. Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei.

  5. Validation of the Monte Carlo criticality program KENO IV and the Hansen-Roach sixteen-energy-group-cross sections for high-assay uranium systems

    International Nuclear Information System (INIS)

    Handley, G.R.; Masters, L.C.; Stachowiak, R.V.

    1981-01-01

    Validation of the Monte Carlo criticality code, KENO IV, and the Hansen-Roach sixteen-energy-group cross sections was accomplished by calculating the effective neutron multiplication constant, k/sub eff/, of 29 experimentally critical assemblies which had uranium enrichments of 92.6% or higher in the uranium-235 isotope. The experiments were chosen so that a large variety of geometries and of neutron energy spectra were covered. Problems, calculating the k/sub eff/ of systems with high-uranium-concentration uranyl nitrate solution that were minimally reflected or unreflected, resulted in the separate examination of five cases

  6. A Validated Stability-indicating Reverse Phase HPLC Assay Method for the Determination of Memantine Hydrochloride Drug Substance with UV-Detection Using Precolumn Derivatization Technique

    Directory of Open Access Journals (Sweden)

    Bhavil Narola

    2010-01-01

    Full Text Available This present paper deals with the development and validation of a stability indicating high performance liquid chromatographic method for the quantitative determination of Memantine hydrochloride. Memantine hydrochloride was derivatized with 0.015 M 9-fluorenylmethyl chloroformate (FMOC and 0.5 M borate buffer solution by keeping it at room temperature for about 20 minutes and the chromatographic separation achieved by injecting 10 μL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a kromasil C18 column (150 × 4.6 mm, 5 μ, The mobile phase consisting of 80% acetonitrile and 20% phosphate buffer solution and a flow rate of 2 milliliter/minute. The Memantine was eluted at approximately 7.5 minutes. The volume of FMOC used in derivatization, concentration of FMOC and derivatization time was optimized and used. Forced degradation studies were performed on bulk sample of Memantine hydrochloride using acid (5.0 Normal (N hydrochloric acid, base (1.0 N sodium hydroxide, oxidation (30% hydrogen peroxide, thermal (105 ° C, photolytic and humidity conditions. The developed LC method was validated with respect to specificity, precision (% RSD about 0.70%, linearity (linearity of range about 70-130 μg/mL, ruggedness (Overall % RSD about 0.35%, stability in analytical solution (Cumulative % RSD about 0.11% after 1450 min. and robustness.

  7. A Validated Stability-indicating Reverse Phase HPLC Assay Method for the Determination of Memantine Hydrochloride Drug Substance with UV-Detection Using Precolumn Derivatization Technique

    Directory of Open Access Journals (Sweden)

    Bhavil Narola

    2010-07-01

    Full Text Available This present paper deals with the development and validation of a stability indicating high performance liquid chromatographic method for the quantitative determination of Memantine hydrochloride. Memantine hydrochloride was derivatized with 0.015 M 9-fluorenylmethyl chloroformate (FMOC and 0.5 M borate buffer solution by keeping it at room temperature for about 20 minutes and the chromatographic separation achieved by injecting 10 µL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a kromasil C18 column (150 × 4.6 mm, 5 µ. The mobile phase consisting of 80% acetonitrile and 20% phosphate buffer solution and a flow rate of 2 milliliter/minute. The Memantine was eluted at approximately 7.5 minutes. The volume of FMOC used in derivatization, concentration of FMOC and derivatization time was optimized and used. Forced degradation studies were performed on bulk sample of Memantine hydrochloride using acid (5.0 Normal (N hydrochloric acid, base (1.0 N sodium hydroxide, oxidation (30% hydrogen peroxide, thermal (105°C, photolytic and humidity conditions. The developed LC method was validated with respect to specificity, precision (% RSD about 0.70%, linearity (linearity of range about 70–130 µg/mL, ruggedness (Overall % RSD about 0.35%, stability in analytical solution (Cumulative % RSD about 0.11% after 1450 min. and robustness.

  8. Detection of genetically modified rice: Collaborative validation study of a PCR based detection of genetically modified rice Oryza sativa commercially available in Saudi Arabia

    OpenAIRE

    Ibrahim, Mohamed; Alaraidh, Ibrahim; Amid, Azura; Farouk, Abd-El Aziem; Bazaid, Salih; Greiner, Ralf; Alghunaim, Abdullah

    2011-01-01

    A collaborative trial study has been conducted for validation of an extraction method and a subsequent PCR for  the detection of transgenic rice sold in Saudi Arabia. The tests were carried out in Saudi Arabia using Real-Time PCR and the positive samples were validated in another lab in Malaysia using PCR and agarose gel visualization.  The samples were tested for the existence of the NOS Terminator. A total of 150 samples were tested out of which three samples tested positi...

  9. Improvement of the decision efficiency of the accuracy profile by means of a desirability function for analytical methods validation. Application to a diacetyl-monoxime colorimetric assay used for the determination of urea in transdermal iontophoretic extracts.

    Science.gov (United States)

    Rozet, E; Wascotte, V; Lecouturier, N; Préat, V; Dewé, W; Boulanger, B; Hubert, Ph

    2007-05-22

    Validation of analytical methods is a widely used and regulated step for each analytical method. However, the classical approaches to demonstrate the ability to quantify of a method do not necessarily fulfill this objective. For this reason an innovative methodology was recently introduced by using the tolerance interval and accuracy profile, which guarantee that a pre-defined proportion of future measurements obtained with the method will be included within the acceptance limits. Accuracy profile is an effective decision tool to assess the validity of analytical methods. The methodology to build such a profile is detailed here. However, as for any visual tool it has a part of subjectivity. It was then necessary to make the decision process objective in order to quantify the degree of adequacy of an accuracy profile and to allow a thorough comparison between such profiles. To achieve this, we developed a global desirability index based on the three most important validation criteria: the trueness, the precision and the range. The global index allows the classification of the different accuracy profiles obtained according to their respective response functions. A diacetyl-monoxime colorimetric assay for the determination of urea in transdermal iontophoretic extracts was used to illustrate these improvements.

  10. Validation of an immunochemical assay for the detection of DNA damage as a tool for biological dosimetry of human exposure to ionizing radiation

    International Nuclear Information System (INIS)

    Schans, G.P. van der; Timmerman, A.J.; Wojewodzka, M.; Zaim, J.

    1997-01-01

    A method for biological dosimetry based on the immunochemical detection of DNA damage in human white blood cells has been validated. To this end the method developed at TNO (Rijswijk, the Netherlands) was also set up at INCT (Warsaw, Poland). Blood samples of 11 individuals were irradiated with 0 or 5 Gy of 170 kV X-rays at INCT and analyzed both at INCT and TNO. It appeared that in both laboratories damage could be detected to the same extent. The average background level of DNA damage amounted to 1.0 Gy-eq with an interindividual standard deviation of 0.25 Gy. The contribution of the sample variance to the total variance is only 14%. The radiosensitivity showed only a variation of about 10% and can, therefore, be neglected in estimating the radiation dose from the amount of DNA damage detected. (author)

  11. Validación del ensayo de disolución de las tabletas de propiltiuracilo 50 mg Validation of the dissolution assay of propylthiouracil 50 mg

    Directory of Open Access Journals (Sweden)

    Caridad M García Peña

    2008-04-01

    Full Text Available Se validó un método para evaluar la disolución de las tabletas de propiltiuracilo 50 mg por espectrofotometría, con detección ultravioleta a 274 nm. Se evaluaron los parámetros de especificidad, linealidad, precisión e influencia de la filtración. La curva de linealidad se realizó en el rango de 3,32 a 7,75 µg/mL con un coeficiente de correlación igual a 0,99984; la prueba estadística para el intercepto y la pendiente se consideró no significativa. En el estudio de la influencia de la filtración se demostró que en la filtración no se adsorbe el principio activo ni se aportan interferencias al filtrado, por lo que es posible su empleoA method to evaluate the dissolution of propylthiouracil tablets 50 mg by spectrophotometry, with ultraviolet detection of 274 nm, was validated. Specificity, linearity, precision and filtration influence were the evaluated parameters. The linearity curve was made at the range from 3.32 to 7.75 µg/mL with a correlation coefficient equal to 0.99984. The statistical test for the interval and the slope was not significant. In the study of filtration influence it was proved that the active principle was not absorbed in the filtration and that there were no interferences in the filtration, which made its use possible

  12. Streamlining Transportation Corridors Planning Processes and Validating the Application of Commercial Remote Sensing and Spatial Information (CRS-SI) Technologies for Environmental Impact Assessments

    Science.gov (United States)

    2008-02-05

    The new US DOT RITA program has selected MSU for addressing corridor planning and environmental assessment in new and innovative ways that can be compared to traditional approaches. Our primary focus is on the application and validation of new and in...

  13. Development and validation of the first assay method coupling liquid chromatography with chemiluminescence for the simultaneous determination of menadione and its thioether conjugates in rat plasma.

    Science.gov (United States)

    Elgawish, Mohamed Saleh; Shimomai, Chikako; Kishikawa, Naoya; Ohyama, Kaname; Wada, Mitsuhiro; Kuroda, Naotaka

    2013-09-16

    Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-l-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-l-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5-120 nM for MQ-GS and MQ-NAC and 10-240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.

  14. Development and validation of a high throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

    Science.gov (United States)

    Mawson, Deborah H; Jeffrey, Keon L; Teale, Philip; Grace, Philip B

    2018-06-19

    A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilises protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography - tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic run times, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high throughput sample analysis studies. This article is protected by copyright. All rights reserved.

  15. [Validation of a real time RT-PCR assay to detect foot-and-mouth disease virus and assessment of its performance in acute infection].

    Science.gov (United States)

    Fondevila, Norberto; Compaired, Diego; Maradei, Eduardo; Duffy, Sergio

    2014-01-01

    A specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5-96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  16. The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Milena Rizzo

    2017-01-01

    Full Text Available miR-28-5p is an intragenic miRNA which is underexpressed in several tumor types showing a tumor suppressor (TS activity. Routinely, the known miR-28-5p targets are validated in specific tumor contexts but it is unclear whether these targets are also being regulated in other tumor types. To this end, we adopted the miRNA pull out assay to capture the miR-28-5p targets in DU-145 prostate cancer (PCa cells. Firstly, we demonstrated that miR-28-5p acts as a TS-miRNA in PCa, affecting cell proliferation, survival, and apoptosis. Secondly, we evaluated the enrichment of the 10 validated miR-28-5p targets in the pull out sample. We showed that E2F6, TEX-261, MAPK1, MPL, N4BP1, and RAP1B but not BAG1, OTUB1, MAD2L1, and p21 were significantly enriched, suggesting that not all the miR-28-5p targets are regulated by this miRNA in PCa. We then verified whether the miR-28-5p-interacting targets were regulated by this miRNA. We selected E2F6, the most enriched target in the pull out sample, and demonstrated that miR-28-5p downregulated E2F6 at the protein level suggesting that our approach was effective. In general terms, these findings support the miRNA pull out assay as a useful method to identify context-specific miRNA targets.

  17. Criterion-Validity of Commercially Available Physical Activity Tracker to Estimate Step Count, Covered Distance and Energy Expenditure during Sports Conditions

    Directory of Open Access Journals (Sweden)

    Yvonne Wahl

    2017-09-01

    Full Text Available Background: In the past years, there was an increasing development of physical activity tracker (Wearables. For recreational people, testing of these devices under walking or light jogging conditions might be sufficient. For (elite athletes, however, scientific trustworthiness needs to be given for a broad spectrum of velocities or even fast changes in velocities reflecting the demands of the sport. Therefore, the aim was to evaluate the validity of eleven Wearables for monitoring step count, covered distance and energy expenditure (EE under laboratory conditions with different constant and varying velocities.Methods: Twenty healthy sport students (10 men, 10 women performed a running protocol consisting of four 5 min stages of different constant velocities (4.3; 7.2; 10.1; 13.0 km·h−1, a 5 min period of intermittent velocity, and a 2.4 km outdoor run (10.1 km·h−1 while wearing eleven different Wearables (Bodymedia Sensewear, Beurer AS 80, Polar Loop, Garmin Vivofit, Garmin Vivosmart, Garmin Vivoactive, Garmin Forerunner 920XT, Fitbit Charge, Fitbit Charge HR, Xaomi MiBand, Withings Pulse Ox. Step count, covered distance, and EE were evaluated by comparing each Wearable with a criterion method (Optogait system and manual counting for step count, treadmill for covered distance and indirect calorimetry for EE.Results: All Wearables, except Bodymedia Sensewear, Polar Loop, and Beurer AS80, revealed good validity (small MAPE, good ICC for all constant and varying velocities for monitoring step count. For covered distance, all Wearables showed a very low ICC (<0.1 and high MAPE (up to 50%, revealing no good validity. The measurement of EE was acceptable for the Garmin, Fitbit and Withings Wearables (small to moderate MAPE, while Bodymedia Sensewear, Polar Loop, and Beurer AS80 showed a high MAPE up to 56% for all test conditions.Conclusion: In our study, most Wearables provide an acceptable level of validity for step counts at different

  18. A real-time polymerase chain reaction method for the identification of four commercially important salmon and trout species.

    Science.gov (United States)

    Feng, Junli; Wu, Zhigang; Xie, Xiao; Dai, Zhiyuan; Liu, Shasha

    2017-01-01

    A duplex quantitative real-time PCR (qPCR) assay was developed for rapid and accurate identification of four commercially important salmon and trout species (Oncorhynchus keta, Oncorhynchus nerka, Oncorhynchus mykiss, and Salmo salar) commonly used for production process of fish in China. The assays targeting the mitochondrial control region (CR) and 16S rRNA gene were able to simultaneously discriminate four target species and the family Salmonidae from processed as well as fresh fish. The qPCR efficiency of each reaction was calculated according to the standard curve, and the method was validated by amplification DNA extracted from single or artificial mixtures prepared with the reference salmon and trout species. Testing of 11 commercial salmon and trout products by the established qPCR assay demonstrated that it was really a useful and academic technique to identify four commercially important salmon and trout species.

  19. Improvement and validation of isotopic libraries of commercial gamma spectra evaluation packages. Report on task FIN A 955 of Finnish support programme to IAEA safeguards

    Energy Technology Data Exchange (ETDEWEB)

    Nikkinen, M.

    1997-06-01

    The Department of Safeguards at the International Atomic Energy Agency (IAEA) is running gamma spectroscopy analysis with various samples taken at various stages of the nuclear fuel cycle. It was found that the commercial gamma spectra analysis packages available do not include proper gamma-line libraries for the various tasks needed for the safeguards purposes because the libraries of these packages are often incomplete and outdated. New libraries were developed to satisfy the needs in the analysis tasks required for the safeguards purposes. These lines are limited by the number of gamma lines to avoid the problems with too many candidates for a single gamma peak. The work was carried out under the Task FIN A 955 Finnish Support Programme to IAEA Safeguards. (orig.) (18 refs.).

  20. Commercial Toilets

    Science.gov (United States)

    Whether you are looking to reduce water use in a new facility or replace old, inefficient toilets in commercial restrooms, a WaterSense labeled flushometer-valve toilet is a high-performance, water-efficient option worth considering.

  1. Analytical method for the identification and assay of 12 phthalates in cosmetic products: application of the ISO 12787 international standard "Cosmetics-Analytical methods-Validation criteria for analytical results using chromatographic techniques".

    Science.gov (United States)

    Gimeno, Pascal; Maggio, Annie-Françoise; Bousquet, Claudine; Quoirez, Audrey; Civade, Corinne; Bonnet, Pierre-Antoine

    2012-08-31

    Esters of phthalic acid, more commonly named phthalates, may be present in cosmetic products as ingredients or contaminants. Their presence as contaminant can be due to the manufacturing process, to raw materials used or to the migration of phthalates from packaging when plastic (polyvinyl chloride--PVC) is used. 8 phthalates (DBP, DEHP, BBP, DMEP, DnPP, DiPP, DPP, and DiBP), classified H360 or H361, are forbidden in cosmetics according to the European regulation on cosmetics 1223/2009. A GC/MS method was developed for the assay of 12 phthalates in cosmetics, including the 8 phthalates regulated. Analyses are carried out on a GC/MS system with electron impact ionization mode (EI). The separation of phthalates is obtained on a cross-linked 5%-phenyl/95%-dimethylpolysiloxane capillary column 30 m × 0.25 mm (i.d.) × 0.25 mm film thickness using a temperature gradient. Phthalate quantification is performed by external calibration using an internal standard. Validation elements obtained on standard solutions, highlight a satisfactory system conformity (resolution>1.5), a common quantification limit at 0.25 ng injected, an acceptable linearity between 0.5 μg mL⁻¹ and 5.0 μg mL⁻¹ as well as a precision and an accuracy in agreement with in-house specifications. Cosmetic samples ready for analytical injection are analyzed after a dilution in ethanol whereas more complex cosmetic matrices, like milks and creams, are assayed after a liquid/liquid extraction using ter-butyl methyl ether (TBME). Depending on the type of cosmetics analyzed, the common limits of quantification for the 12 phthalates were set at 0.5 or 2.5 μg g⁻¹. All samples were assayed using the analytical approach described in the ISO 12787 international standard "Cosmetics-Analytical methods-Validation criteria for analytical results using chromatographic techniques". This analytical protocol is particularly adapted when it is not possible to make reconstituted sample matrices. Copyright © 2012

  2. Space Commercialization

    Science.gov (United States)

    Martin, Gary L.

    2011-01-01

    A robust and competitive commercial space sector is vital to continued progress in space. The United States is committed to encouraging and facilitating the growth of a U.S. commercial space sector that supports U.S. needs, is globally competitive, and advances U.S. leadership in the generation of new markets and innovation-driven entrepreneurship. Energize competitive domestic industries to participate in global markets and advance the development of: satellite manufacturing; satellite-based services; space launch; terrestrial applications; and increased entrepreneurship. Purchase and use commercial space capabilities and services to the maximum practical extent Actively explore the use of inventive, nontraditional arrangements for acquiring commercial space goods and services to meet United States Government requirements, including measures such as public-private partnerships, . Refrain from conducting United States Government space activities that preclude, discourage, or compete with U.S. commercial space activities. Pursue potential opportunities for transferring routine, operational space functions to the commercial space sector where beneficial and cost-effective.

  3. A Stability-Indicating HPLC-DAD Method for Determination of Stiripentol: Development, Validation, Kinetics, Structure Elucidation and Application to Commercial Dosage Form

    Directory of Open Access Journals (Sweden)

    Hany W. Darwish

    2014-01-01

    Full Text Available A rapid, simple, sensitive, and accurate isocratic reversed-phase stability-indicating high performance liquid chromatography method has been developed and validated for the determination of stiripentol and its degradation product in its bulk form and pharmaceutical dosage form. Chromatographic separation was achieved on a Symmetry C18 column and quantification was achieved using photodiode array detector (DAD. The method was validated in accordance with the ICH requirements showing specificity, linearity (r2=0.9996, range of 1–25 μg/mL, precision (relative standard deviation lower than 2%, accuracy (mean recovery 100.08±1.73, limits of detection and quantitation (LOD = 0.024 and LOQ = 0.081 μg/mL, and robustness. Stiripentol was subjected to various stress conditions and it has shown marked stability under alkaline hydrolytic stress conditions, thermal, oxidative, and photolytic conditions. Stiripentol degraded only under acidic conditions, forming a single degradation product which was well resolved from the pure drug with significantly different retention time values. This degradation product was characterized by 1H-NMR and 13C-NMR spectroscopy as well as ion trap mass spectrometry. The results demonstrated that the method would have a great value when applied in quality control and stability studies for stiripentol.

  4. Assay strategies and methods for phospholipases

    International Nuclear Information System (INIS)

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

  5. Cross-validation of two commercial methods for volumetric high-resolution dose reconstruction on a phantom for non-coplanar VMAT beams

    International Nuclear Information System (INIS)

    Feygelman, Vladimir; Stambaugh, Cassandra; Opp, Daniel; Zhang, Geoffrey; Moros, Eduardo G.; Nelms, Benjamin E.

    2014-01-01

    Background and purpose: Delta 4 (ScandiDos AB, Uppsala, Sweden) and ArcCHECK with 3DVH software (Sun Nuclear Corp., Melbourne, FL, USA) are commercial quasi-three-dimensional diode dosimetry arrays capable of volumetric measurement-guided dose reconstruction. A method to reconstruct dose for non-coplanar VMAT beams with 3DVH is described. The Delta 4 3D dose reconstruction on its own phantom for VMAT delivery has not been thoroughly evaluated previously, and we do so by comparison with 3DVH. Materials and methods: Reconstructed volumetric doses for VMAT plans delivered with different table angles were compared between the Delta 4 and 3DVH using gamma analysis. Results: The average γ (2% local dose-error normalization/2mm) passing rate comparing the directly measured Delta 4 diode dose with 3DVH was 98.2 ± 1.6% (1SD). The average passing rate for the full volumetric comparison of the reconstructed doses on a homogeneous cylindrical phantom was 95.6 ± 1.5%. No dependence on the table angle was observed. Conclusions: Modified 3DVH algorithm is capable of 3D VMAT dose reconstruction on an arbitrary volume for the full range of table angles. Our comparison results between different dosimeters make a compelling case for the use of electronic arrays with high-resolution 3D dose reconstruction as primary means of evaluating spatial dose distributions during IMRT/VMAT verification

  6. Prospective evaluation of commercial antibody-based rapid tests in combination with a loop-mediated isothermal amplification PCR assay for detection of Orientia tsutsugamushi during the acute phase of scrub typhus infection.

    Science.gov (United States)

    Blacksell, Stuart D; Paris, Daniel H; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Teeratakul, Achara; Kantipong, Pacharee; Day, Nicholas P J

    2012-03-01

    Samples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection of Orientia tsutsugamushi IgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity.

  7. Performance of a commercial assay for the diagnosis of influenza A (H1N1 infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR

    Directory of Open Access Journals (Sweden)

    María G Barbás

    2012-03-01

    Full Text Available At the time of influenza A (H1N1 emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR. The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009 is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays.Durante la pandemia de influenza A (H1N1, la OMS recomendó algoritmos y protocolos de detección del virus mediante RT-PCR en tiempo real. El objetivo del presente estudio fue evaluar el desempeño del equipo que comercializa la empresa Roche, Real Time Ready Influenza A/H1N1 Detection Set (junio de 2009, en comparación con el protocolo de RT-PCR en tiempo real de los CDC. La sensibilidad global del ensayo de Roche para la detección del gen Inf A en presencia o ausencia del gen H1 fue 74,5 %. La sensibilidad para la detección de muestras positivas solo para el gen Inf A (ausencia del gen H1 fue 53,3 % y la sensibilidad para la detección de muestras positivas para H1N1 (presencia del gen Inf A y cualquier otro gen porcino fue 76,4 %. La especificidad fue 97,1 %. Existe una nueva versión del equipo (noviembre 2009 que, según se ha descrito, presenta buena sensibilidad en comparación con otros ensayos moleculares para detectar H1N1 pandémica.

  8. Commercial Banks

    Directory of Open Access Journals (Sweden)

    Abbas Asosheh

    2009-09-01

    Full Text Available Information systems outsourcing issues has been attracted in recent years because many information systems projects in organizations are done in this case. On the other hand, failure rate of this kind of projects is also high. The aim of this article is to find success factors in risk management of information systems outsourcing in commercial banks using these factors leads to increase the success rate of risk management of information systems outsourcing projects. Research methods in the present article based on purpose are applied and descriptive- survey. In addition, research tool is questionnaire which was used among commercial bank experts. For this purpose, First information systems outsourcing risks were identified and then ranked. In the next step, the information systems outsourcing reasons were surveyed and the most important reasons were identified. Then the risks which have not any relationship with the most important reasons were removed and success factors in managing residual risks were extracted.

  9. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

    Science.gov (United States)

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

  10. Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault-validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer.

    Science.gov (United States)

    Holtkötter, Hannah; Dias Filho, Claudemir Rodrigues; Schwender, Kristina; Stadler, Christian; Vennemann, Marielle; Pacheco, Ana Claudia; Roca, Gabriela

    2018-05-01

    Sexual assault is a serious offense and identification of body fluids originating from sexual activity has been a crucial aspect of forensic investigations for a long time. While reliable tests for the detection of semen and saliva have been successfully implemented into forensic laboratories, the detection of other body fluids, such as vaginal or menstrual fluid, is more challenging. Especially, the discrimination between peripheral and menstrual blood can be highly relevant for police investigations because it provides potential evidence regarding the issue of consent. We report the forensic validation of an immunochromatographic test that allows for such discrimination in forensic stains, the SERATEC PMB test, and its performance on real casework samples. The PMB test is a duplex test combining human hemoglobin and D-dimer detection and was developed for the identification of blood and menstrual fluid, both at the crime scene and in the laboratory. The results of this study showed that the duplex D-dimer/hemoglobin assay reliably detects the presence of human hemoglobin and identifies samples containing menstrual fluid by detecting the presence of D-dimers. The method distinguished between menstrual and peripheral blood in a swab from a historical artifact and in real casework samples of alleged sexual assaults. Results show that the development of the new duplex test is a substantial progress towards analyzing and interpreting evidence from sexual assault cases.

  11. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, Martin; Vieths, Stefan [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany); Holzhauser, Thomas, E-mail: holth@pei.de [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany)

    2011-01-24

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg{sup -1} almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg{sup -1}. We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg{sup -1} almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg{sup -1}. Further, between 100 and 100,000 mg kg{sup -1} spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a

  12. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

    2011-01-24

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(®) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(®) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

  13. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Roeder, Martin; Vieths, Stefan; Holzhauser, Thomas

    2011-01-01

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg -1 almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg -1 . We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg -1 almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg -1 . Further, between 100 and 100,000 mg kg -1 spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

  14. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  15. Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol.

    Science.gov (United States)

    Kayacelebi, Arslan Arinc; Schauerte, Celina; Kling, Katharina; Herbers, Jan; Beckmann, Bibiana; Engeli, Stefan; Jordan, Jens; Zoerner, Alexander A; Tsikas, Dimitrios

    2017-03-15

    2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d 8 -2AG; 10μM) as the MAGL substrate and measure deuterium-labeled AA (d 8 -AA; range 0-1μM) as the MAGL product. Unlabelled AA (d 0 -AA, 1μM) serves as the internal standard. d 8 -AA and d 0 -AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H] - →[M-H - CO 2 ] - , i.e., m/z 311→m/z 267 for d 8 -AA and m/z 303→m/z 259 for d 0 -AA. Prior to GC-MS analysis d 8 -AA and d 0 -AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB] - , i.e., m/z 311 for d 8 -AA and m/z 303 for d 0 -AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1μM) of d 8 -AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r 2 =0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination

  16. Comparison of two commercial assays for detection of human papillomavirus (HPV) in cervical scrape specimens: validation of the Roche AMPLICOR HPV test as a means to screen for HPV genotypes associated with a higher risk of cervical disorders.

    NARCIS (Netherlands)

    Ham, M.A.P.C. van; Bakkers, J.M.J.E.; Harbers, G.; Quint, W.G.V.; Massuger, L.F.A.G.; Melchers, W.J.G.

    2005-01-01

    Certain high-risk (HR) human papillomavirus (HPV) types are a necessary cause for the development of cervical disorders. Women with persistent HR HPV infections have an increased risk of developing high-grade cervical lesions, compared with those who have no or low-risk HPV infections. Therefore,

  17. 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.

  18. Rapid determination of flavonoids and phenolic acids in grape juices and wines by RP-HPLC/DAD: Method validation and characterization of commercial products of the new Brazilian varieties of grape.

    Science.gov (United States)

    Padilha, Carla Valéria da Silva; Miskinis, Gabriela Aquino; de Souza, Marcelo Eduardo Alves Olinda; Pereira, Giuliano Elias; de Oliveira, Débora; Bordignon-Luiz, Marilde Terezinha; Lima, Marcos Dos Santos

    2017-08-01

    A method for rapid determination of phenolic compounds by reversed-phase high-performance liquid chromatography (RP-HPLC), using a new column of faster resolution was validated and used to characterize commercial products produced with new grape Brazilian varieties of Northeast of Brazil. The in vitro antioxidant activity was also measured. The method showed linearity (R>0.9995), good precision (CV%<2.78), recovery (91.8-105.1%) and limits of detection (0.04-0.85mgL -1 ) and quantification (0.04-1.41mgL -1 ) according to other methods previously published with the difference of a run time of only 25min. The results obtained in the characterization of the samples differed for juices and wines from other world regions, mainly because of the high values of (-)-epigallocatechin and trans-caftaric acid. The products analyzed showed high antioxidant activity, especially the wine samples with values higher than those from wines of different regions of the world. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Validated assay for the simultaneous quantification of total vincristine and actinomycin-D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high-performance liquid chromatography coupled with tandem mass spectrometry

    NARCIS (Netherlands)

    Damen, Carola W. N.; Israëls, Trijn; Caron, Huib N.; Schellens, Jan H. M.; Rosing, Hilde; Beijnen, Jos H.

    2009-01-01

    A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin-D concentrations in human plasma and an assay for the determination of unbound vincristine are presented.

  20. Commercial applications

    International Nuclear Information System (INIS)

    Anon.

    1982-01-01

    The objective of this paper is to assess the near term (one-to-five-year) needs of domestic and foreign commercial suppliers of radiochemicals and radiopharmaceuticals for electromagnetically separated stable isotopes. Only isotopes purchased to make products for sale and profit are considered in this assessment. Radiopharmaceuticals produced from enriched stable isotopes supplied by the Calutron facility at ORNL are used in about 600,000 medical procedures each year in the United States. A temporary or permanent disruption of the supply of stable isotopes to the domestic radiopharmaceutical industry could curtail, if not eliminate, the use of such diagnostic procedures as the thallium heart scan, the gallium cancer scan, the gallium abscess scan, and the low-radiation-dose thyroid scan. The word could in the preceding sentence is underlined because an alternative source of enriched stable isotopes does exist in the USSR. Alternative starting materials could, in theory, eventually be developed for both the thallium and gallium scans. The development of a new technology for these purposes, however, would take at least five years and would be expensive. Hence, any disruption of the supply of enriched isotopes from ORNL and the resulting unavailability of critical nuclear medicine procedures would have a dramatic negative effect on the level of health care in the United States

  1. Commercial applications

    Science.gov (United States)

    The near term (one to five year) needs of domestic and foreign commercial suppliers of radiochemicals and radiopharmaceuticals for electromagnetically separated stable isotopes are assessed. Only isotopes purchased to make products for sale and profit are considered. Radiopharmaceuticals produced from enriched stable isotopes supplied by the Calutron facility at ORNL are used in about 600,000 medical procedures each year in the United States. A temporary or permanent disruption of the supply of stable isotopes to the domestic radiopharmaceutical industry could curtail, if not eliminate, the use of such diagnostic procedures as the thallium heart scan, the gallium cancer scan, the gallium abscess scan, and the low radiation dose thyroid scan. An alternative source of enriched stable isotopes exist in the USSR. Alternative starting materials could, in theory, eventually be developed for both the thallium and gallium scans. The development of a new technology for these purposes, however, would take at least five years and would be expensive. Hence, any disruption of the supply of enriched isotopes from ORNL and the resulting unavailability of critical nuclear medicine procedures would have a dramatic negative effect on the level of health care in the United States.

  2. Commercially available molecular tests for human papillomaviruses (HPV): 2015 update.

    Science.gov (United States)

    Poljak, Mario; Kocjan, Boštjan J; Oštrbenk, Anja; Seme, Katja

    2016-03-01

    Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular

  3. Microbiological assay for the analysis of certain macrolides in pharmaceutical dosage forms.

    Science.gov (United States)

    Mahmoudi, A; Fourar, R E-A; Boukhechem, M S; Zarkout, S

    2015-08-01

    Clarithromycin (CLA) and roxithromycin (ROX) are macrolide antibiotics with an expanded spectrum of activity that are commercially available as tablets. A microbiological assay, applying the cylinder-plate method and using a strain of Micrococcus luteus ATCC 9341 as test organism, has been used and validated for the quantification of two macrolide drugs; CLA and ROX in pure and pharmaceutical formulations. The validation of the proposed method was carried out for linearity, precision, accuracy and specificity. The linear dynamic ranges were from 0.1 to 0.5μg/mL for both compounds. Logarithmic calibration curve was obtained for each macrolide (r>0.989) with statistically equal slopes varying from 3.275 to 4.038, and a percentage relative standard deviation in the range of 0.24-0.92%. Moreover, the method was applied successfully for the assay of the studied drugs in pharmaceutical tablet dosage forms. Recovery from standard addition experiments in commercial products was 94.71-96.91% regarding clarithromycin and 93.94-98.12% regarding roxithromycin, with a precision (%RSD) 1.32-2.11%. Accordingly, this microbiological assay can be used for routine quality control analysis of titled drugs in tablet formulations. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

    Science.gov (United States)

    Miles, Timothy D; Martin, Frank N; Coffey, Michael D

    2015-02-01

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing

  5. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  6. Assay development status report for total cyanide

    International Nuclear Information System (INIS)

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN - ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation

  7. Radon investigations - Soil and commercial projects

    International Nuclear Information System (INIS)

    Goodwin, R.W.

    1987-01-01

    The liability issues of radon exposure have prompted potential purchasers of vacant land for commercial/industrial development, and commercial landlords, renting large commercial buildings, to determine the radon gas levels at such sites. This paper deals with both pre-construction sites subject to freezing conditions and to large commercial structures. A correlation of radon gas levels within a commercial building and a sister pre-construction site confirms the validity of using activated charcoal canisters as a cost effective means to combating radon in large structures

  8. Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident

    Science.gov (United States)

    Semenova, Vera A.; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

    2017-01-01

    To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from −4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = −0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. PMID:27814939

  9. Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

    Science.gov (United States)

    Semenova, Vera A; Steward-Clark, Evelene; Maniatis, Panagiotis; Epperson, Monica; Sabnis, Amit; Schiffer, Jarad

    2017-01-01

    To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r 2 ), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r 2  = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate. Published by Elsevier Ltd.

  10. Detection of a Giardia lamblia coproantigen by using a commercially available immunoenzymatic assay, in Belo Horizonte, Brazil Avaliação do ProSpeT Giardia ensaio em microplaca na detecção de coproantígenos de Giardia lamblia, em fezes de pacientes de Belo Horizonte, Brasil

    Directory of Open Access Journals (Sweden)

    Míriam Oliveira e ROCHA

    1999-05-01

    Full Text Available It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRÁS to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0% by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%. The assay identified 27 of the 30 positive patients (sensitivity = 90.0% and was negative in 59 of the 60 negatives (specificity = 98.3% in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.O diagnóstico da giardíase através da pesquisa de trofozoítos ou cistos do parasita nas fezes apresenta uma elevada percentagem de resultados falso-negativos. No presente trabalho foi feita, pela primeira vez no Brasil, uma avaliação do ProSpecT Giardia Ensaio em Microplaca (Alexon, Inc., BIOBRÁS, utilizando amostras fecais de pacientes de Belo Horizonte, Brasil. Um total de 90 amostras fecais foram testadas, primeiramente como fezes frescas e, então, preservadas em formalina 10%, para um novo teste 2 meses após. Com as amostras frescas, o ensaio imunoenzimático, através de leitura visual ou no espectrofotômetro, foi capaz de

  11. Use of Molecular Assays in Diagnosis and Monitoring of Cytomegalovirus Disease following Renal Transplantation

    OpenAIRE

    Aitken, Celia; Barrett-Muir, Winsome; Millar, Colin; Templeton, Kate; Thomas, Janice; Sheridan, Fran; Jeffries, Donald; Yaqoob, Magdi; Breuer, Judith

    1999-01-01

    We compared two commercial molecular assays (the Murex Hybrid Capture CMV DNA assay [HCA], version 2, and the Roche Amplicor plasma PCR assay) with a standard shell vial assay in detecting and predicting cytomegalovirus (CMV) disease in a group of renal transplant patients and assessed the role of viral load measurements (using the HCA) in their management. The sensitivity of the HCA and Amplicor assay in terms of disease detection was 100%, compared to 71% for the shell vial assay. Both the ...

  12. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

    OpenAIRE

    Bezeaud, A; Rosenswajg, M; Guillin, M C

    1989-01-01

    Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar,...

  13. Evaluation of a molybdenum assay canister

    International Nuclear Information System (INIS)

    Yoshizumi, T.T.; Keener, S.J.

    1988-01-01

    The performance characteristics of a commercial molybdenum assay canister were evaluated. The geometrical variation of the technetium-99m (/sup 99m/Tc) activity reading was studied as a function of the elution volume for the standard vials. It was found that the /sup 99m/Tc canister activity reading was ∼ 5% lower than that of the standard method. This is due to attenuation by the canister wall. However, the effect of the geometric variation on the clinical dose preparation was found to be insignificant. The molybdenum-99 ( 99 Mo) contamination level was compared by two methods: (1) the commercial canister and (2) the standard assay kit. The 99 Mo contamination measurements with the canister indicated consistently lower readings than those with the standard 99 Mo assay kit. The authors conclude that the canister may be used in the clinical settings. However, the user must be aware of the problems and the limitations associated with this canister

  14. Evaluation and optimization of a commercial blocking ELISA for detecting antibodies to influenza A virus for research and surveillance of mallards.

    Science.gov (United States)

    Shriner, Susan A; VanDalen, Kaci K; Root, J Jeffrey; Sullivan, Heather J

    2016-02-01

    The availability of a validated commercial assay is an asset for any wildlife investigation. However, commercial products are often developed for use in livestock and are not optimized for wildlife. Consequently, it is incumbent upon researchers and managers to apply commercial products appropriately to optimize program outcomes. We tested more than 800 serum samples from mallards for antibodies to influenza A virus with the IDEXX AI MultiS-Screen Ab test to evaluate assay performance. Applying the test per manufacturer's recommendations resulted in good performance with 84% sensitivity and 100% specificity. However, performance was improved to 98% sensitivity and 98% specificity by increasing the recommended cut-off. Using this alternative threshold for identifying positive and negative samples would greatly improve sample classification, especially for field samples collected months after infection when antibody titers have waned from the initial primary immune response. Furthermore, a threshold that balances sensitivity and specificity reduces estimation bias in seroprevalence estimates. Published by Elsevier B.V.

  15. Harmonization of radiobiological assays: why and how?

    International Nuclear Information System (INIS)

    Prasanna, Pataje G.

    2014-01-01

    The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

  16. Validated high-performance liquid chromatographic method for the standardisation of Ptychopetalum olacoides Benth., Olacaceae, commercial extracts Validação de método de cromatografia líquida de alta performance para padronização de extratos comerciais de Ptychopetalum olacoides Benth., Olacaceae

    Directory of Open Access Journals (Sweden)

    Renata Colombo

    2010-11-01

    Full Text Available Ptychopetalum olacoides Benth., Olacaceae, popularly known as marapuama or muirapuama or miriantã, is a species native to the Amazonian region of Brazil. Extracts of the bark of the plant have been used traditionally for its stimulating and aphrodisiac properties and currently commercialised by the herbal industry as constituents in a wide range of phytomedicines. Fractionation by open column chromatography followed by preparative HPLC-UV/PAD of the stem bark and of three commercial extracts of P. olacoides allowed the isolation of three components that were common to all extracts analysed, and these were identified by NMR to be vanillic acid, protocatechuic acid and theobromine. Vanillic acid, which has been proposed as a phytochemical marker for P. olacoides, was employed as an external standard in the development and validation of a rapid qualitative and quantitative HPLC assay for the analyte. The recoveries values of the developed method were 99.02% and the LOD and LOQ values were 0.033 and 0.11 mg.L-1, respectively. The described method may be applied to the standardisation of herbs, extracts or phytomedicines commercialised as marapuama.Ptychopetalum olacoides Benth., Olacaceae, popularmente conhecida como marapuama, muirapuama ou miriantã, é uma espécie nativa da região da Amazônia do Brasil. Extratos das cascas da planta são tradicionalmente usados por suas propriedades estimulantes e afrodisíacas, e frequentemente comercializados como constituinte de uma grande variedade de formulações fitoterápicas. O fracionamento por coluna cromatográfica aberta seguida por CLAE-UV/PAD das cascas do caule de três extratos comerciais de P. olacoides permitiram o isolamento de três substâncias comuns em todos os extratos analisados. Os compostos foram identificados por RMN como ácido vanílico, ácido protocatecuíco e teobromina. O ácido vanílico foi utilizado como marcador fitoquímico para P. olacoides e empregado como padr

  17. Evaluation of a Chlamydia trachomatis-specific, commercial, real-time PCR for use with ocular swabs.

    Science.gov (United States)

    Pickering, Harry; Holland, Martin J; Last, Anna R; Burton, Matthew J; Burr, Sarah E

    2018-02-20

    Trachoma, the leading infectious cause of blindness worldwide, is caused by conjunctival Chlamydia trachomatis infection. Trachoma is diagnosed clinically by observation of conjunctival inflammation and/or scarring; however, there is evidence that monitoring C. trachomatis infection may be required for elimination programmes. There are many commercial and 'in-house' nucleic acid amplification tests for the detection of C. trachomatis DNA, but the majority have not been validated for use with ocular swabs. This study evaluated a commercial assay, the Fast-Track Vaginal swab kit, using conjunctival samples from trachoma-endemic areas. An objective, biostatistical-based method for binary classification of continuous PCR data was developed, to limit potential user-bias in diagnostic settings. The Fast-Track Vaginal swab assay was run on 210 ocular swab samples from Guinea-Bissau and Tanzania. Fit of individual amplification curves to exponential or sigmoid models, derivative and second derivative of the curves and final fluorescence value were examined for utility in thresholding for determining positivity. The results from the Fast-Track Vaginal swab assay were evaluated against a commercial test (Amplicor CT/NG) and a non-commercial test (in-house droplet digital PCR), both of whose performance has previously been evaluated. Significant evidence of exponential amplification (R 2  > 0.99) and final fluorescence > 0.15 were combined for thresholding. This objective approach identified a population of positive samples, however there were a subset of samples that amplified towards the end of the cycling protocol (at or later than 35 cycles), which were less clearly defined. The Fast-Track Vaginal swab assay showed good sensitivity against the commercial (95.71) and non-commercial (97.18) tests. Specificity was lower against both (90.00 and 96.55, respectively). This study defined a simple, automated protocol for binary classification of continuous, real-time q

  18. Development and validation of a thallium flux-based functional assay for the sodium channel NaV1.7 and its utility for lead discovery and compound profiling.

    Science.gov (United States)

    Du, Yu; Days, Emily; Romaine, Ian; Abney, Kris K; Kaufmann, Kristian; Sulikowski, Gary; Stauffer, Shaun; Lindsley, Craig W; Weaver, C David

    2015-06-17

    Ion channels are critical for life, and they are targets of numerous drugs. The sequencing of the human genome has revealed the existence of hundreds of different ion channel subunits capable of forming thousands of ion channels. In the face of this diversity, we only have a few selective small-molecule tools to aid in our understanding of the role specific ion channels in physiology which may in turn help illuminate their therapeutic potential. Although the advent of automated electrophysiology has increased the rate at which we can screen for and characterize ion channel modulators, the technique's high per-measurement cost and moderate throughput compared to other high-throughput screening approaches limit its utility for large-scale high-throughput screening. Therefore, lower cost, more rapid techniques are needed. While ion channel types capable of fluxing calcium are well-served by low cost, very high-throughput fluorescence-based assays, other channel types such as sodium channels remain underserved by present functional assay techniques. In order to address this shortcoming, we have developed a thallium flux-based assay for sodium channels using the NaV1.7 channel as a model target. We show that the assay is able to rapidly and cost-effectively identify NaV1.7 inhibitors thus providing a new method useful for the discovery and profiling of sodium channel modulators.

  19. Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food--validation and proof-of-concept study with caper buds.

    Science.gov (United States)

    Wiese, Stefanie; Wubshet, Sileshi G; Nielsen, John; Staerk, Dan

    2013-12-15

    This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Fluorescence lifetime assays: current advances and applications in drug discovery.

    Science.gov (United States)

    Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

    2011-06-01

    Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

  2. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  3. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  4. 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.

  5. 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.

  6. Infection among Commercial Sex Workers in Jos

    African Journals Online (AJOL)

    A study of HHV8 seropositivity was conducted among commercial sex workers in Jos aimed at determining the prevalence in relation to history of STD, duration of prostitution, age and number of sexual partners per day. Antibodies to HHV8 were detected by enzyme linked - immunosorbent assay (ELISA) (Advanced ...

  7. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay.

    Directory of Open Access Journals (Sweden)

    Gregory M Laird

    Full Text Available HIV-1 persists in infected individuals in a stable pool of resting CD4(+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

  8. The Relationship between Different Assays for Detection and Quantification of Amyloid Beta 42 in Human Cerebrospinal Fluid

    Directory of Open Access Journals (Sweden)

    Teresa A. Ellis

    2012-01-01

    Full Text Available Alzheimer's disease (AD, which is characterized by a degeneration of neurons and their synapses, is one of the most common forms of dementia. CSF levels of amyloid 42 (A42 have been recognized as a strong candidate to serve as an AD biomarker. There are a number of commercial assays that are routinely employed for measuring A42; however, these assays give diverse ranges for the absolute levels of CSF A42. In order to employ CSF A42 as a biomarker across multiple laboratories, studies need to be performed to understand the relationship between the different platforms. We have analyzed CSF samples from both diseased and nondiseased subjects with two different widely used assay platforms. The results showed that different values for the levels of CSF A42 were reported, depending on the assay used. Nonetheless, both assays clearly demonstrated statistically significant differences in the levels of A42 in CSF from AD relative to age-matched controls (AMC. This paper provides essential data for establishing the relationship between these assays and provides an important step towards the validation of A42 as a biomarker for AD.

  9. A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

    Science.gov (United States)

    Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

    2013-11-13

    A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (assay variability assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

  10. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  11. Who and How: Comprehensive RNA-Based BodyfluID Assay to Provide Context to a Recovered DNA Profile

    Science.gov (United States)

    2015-08-25

    capillary electrophoresis (CE) and high resolution melt ( HRM ) assays 3 were developed and fully validated. The results of this study demonstrate...Multiplex High Resolution Melt ( HRM ) Assays ................................ 11  D. Developmental Validation of the CE and HRM Assays...Sequences (Top) and Primer Mix Composition (Bottom) for the Blood-Menstrual Blood HRM Assay

  12. Commercial Buildings Characteristics, 1992

    Energy Technology Data Exchange (ETDEWEB)

    1994-04-29

    Commercial Buildings Characteristics 1992 presents statistics about the number, type, and size of commercial buildings in the United States as well as their energy-related characteristics. These data are collected in the Commercial Buildings Energy Consumption Survey (CBECS), a national survey of buildings in the commercial sector. The 1992 CBECS is the fifth in a series conducted since 1979 by the Energy Information Administration. Approximately 6,600 commercial buildings were surveyed, representing the characteristics and energy consumption of 4.8 million commercial buildings and 67.9 billion square feet of commercial floorspace nationwide. Overall, the amount of commercial floorspace in the United States increased an average of 2.4 percent annually between 1989 and 1992, while the number of commercial buildings increased an average of 2.0 percent annually.

  13. The validation of the analytical method (HPLC, use for identification and assay of the pharmaceutical active ingredient, colistine sulphate and the finished product Colidem 50 – hydrosoluble powder, in SC DELOS impex ‘96 SRL

    Directory of Open Access Journals (Sweden)

    Maria Neagu,

    2011-06-01

    Full Text Available In SC DELOS IMPEX ’96 SRL the quality of the active pharmaceutical ingredient (API for the finished product Colidem 50 - hydrosoluble powder is make according to European Pharmacopoeia, curent edition. The method for analysis use in this purpose is the compendial method „Colistine sulphate” in E.P. in current edition and represent a optimized variant, developed and validated „in house”.The parameters which was included in the methodology validation for chromatographic method are the follow: Selectivity/Specificity, Linearity, Range of Linearity, Limit of Detection and Limit of Quantification, Precision (Repeatability - intra day, inter-Day Reproducibility, Accuracy, Robustness, Stability Solutions and System Suitability.

  14. OpenComet: An automated tool for comet assay image analysis

    Directory of Open Access Journals (Sweden)

    Benjamin M. Gyori

    2014-01-01

    Full Text Available Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

  15. Solid phase assays

    International Nuclear Information System (INIS)

    Reese, M.G.; Johnson, L.R.; Ransom, D.K.

    1980-01-01

    In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

  16. Validation of quality control tests of a multi leaf collimator using electronic portal image devices and commercial software; Validacion de unas pruebas de control de calidad del colimador multilamina utilizando dispositivos electronicos de imagen portal y una aplicacion comercial

    Energy Technology Data Exchange (ETDEWEB)

    Latorre-Musoll, A.; Jornet Sala, N.; Carrasco de Fez, P.; Edualdo Puell, T.; Ruiz Martinez, A.; Ribas Morales, M.

    2013-07-01

    We describe a daily quality control procedure of the multi leaf collimator (MLC) based on electronic portal image devices and commercial software. We designed tests that compare portal images of a set of static and dynamic MLC configurations to a set of reference images using commercial portal dosimetry software. Reference images were acquired using the same set of MLC configurations after the calibration of the MLC. To assess the sensitivity to detect MLC under performances, we modified the MLC configurations by inserting a range of leaf position and speed errors. Distance measurements on portal images correlated with leaf position errors down to 0.1 mm in static MLC configurations. Dose differences between portal images correlated both with speed errors down to 0.5% of the nominal leaf velocities and with leaf position errors down to 0.1 mm in dynamic MLC configurations. The proposed quality control procedure can assess static and dynamic MLC configurations with high sensitivity and reliability. (Author)

  17. Factor IX assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  18. Factor VIII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  19. Factor II assay