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Sample records for ahr-calux assay comparison

  1. Comparison of Established and Emerging Biodosimetry Assays

    PubMed Central

    Rothkamm, K.; Beinke, C.; Romm, H.; Badie, C.; Balagurunathan, Y.; Barnard, S.; Bernard, N.; Boulay-Greene, H.; Brengues, M.; De Amicis, A.; De Sanctis, S.; Greither, R.; Herodin, F.; Jones, A.; Kabacik, S.; Knie, T.; Kulka, U.; Lista, F.; Martigne, P.; Missel, A.; Moquet, J.; Oestreicher, U.; Peinnequin, A.; Poyot, T.; Roessler, U.; Scherthan, H.; Terbrueggen, B.; Thierens, H.; Valente, M.; Vral, A.; Zenhausern, F.; Meineke, V.; Braselmann, H.; Abend, M.

    2014-01-01

    Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3–0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5–4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools. PMID:23862692

  2. Assay-dependent variability of serum insulin concentrations: a comparison of eight assays.

    PubMed

    Tohidi, Maryam; Arbab, Parvaneh; Ghasemi, Asghar

    2017-04-01

    Although insulin measurement is essential for both clinical and research purposes, there is currently no reference method for insulin assays. The aim of this study was to compare results of serum insulin determined by a number of commercially available assays. We compared eight insulin assays by analyzing 165 serum samples. Assays included two chemiluminescence (Roche and DiaSorin), four ELISA (Tosoh, Mercodia, Monobind, and Diametra), and two IRMA (Izotop and BioSource) methods. Each assay was compared with the mean of all assay methods and Bland-Altman difference plots were used to measure agreement between each assay and overall mean. Least squared perpendicular distance regression analysis (Deming's method) was used to calculate slope and intercept for bias and also for each assay vs. mean of eight assays. Findings showed that the lowest and highest median insulin concentrations varied by a factor of 1.8. Maximum and minimum correlations with mean of assays were observed for Roche (0.992) and BioSource (0.844), respectively. Significant bias was observed in six assays. In pairwise comparisons of different assays, the highest and least mean differences were 7.78 μU/mL and -0.14 μU/mL, respectively. In conclusion, serum insulin measurement with different assays showed a maximum of 1.8-fold difference, a point that should be taken into consideration in the interpretation of circulating insulin levels in both clinical and research fields.

  3. Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability.

    PubMed

    Wu, Xiang; Sills, Matthew A; Zhang, Ji-Hu

    2005-09-01

    High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

  4. Comparison of immunoturbidimetric and immunonephelometric assays for specific proteins.

    PubMed

    Mali, Bahera; Armbruster, David; Serediak, Ernie; Ottenbreit, Tammy

    2009-10-01

    Immunoturbidimetric assays for specific proteins are available on "open system" clinical chemistry analyzers. The analytical performance of nine immunoturbidimetric specific protein assays (C3, C4, CRP, Haptoglobin, IgA, IgG, IgM, RF, and Transferrin) was compared to immunonephelometry. Testing was performed on the Abbott ARCHITECT ci8200 and the Dade Behring BNII nephelometer and evaluated for precision, linearity, limit of detection, prozone phenomenon, method comparison, workflow, and proficiency testing survey comparison. Immunoturbidimetric assays performance was satisfactory for total precision, linearity, limit of detection and the prozone effect was not observed. Method comparison was acceptable for the immunoglobulins, CRP and transferrin but less favorable for the other assays, likely due to methodology and antibody specificity differences. Immunourbidimetric specific protein assays allow for efficient test consolidation on a general purpose clinical chemistry analyzer.

  5. Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture.

    PubMed Central

    Feorino, P; Forrester, B; Schable, C; Warfield, D; Schochetman, G

    1987-01-01

    We compared an antigen capture assay (Abbott Laboratories, North Chicago, Ill.) with a reverse transcriptase assay to identify and quantify human immunodeficiency virus (HIV) in culture. In direct comparisons of serial dilutions of lymphadenopathy-associated virus type 1, the antigen assay was 100-fold more sensitive than the reverse transcriptase assay in detecting the virus. The antigen assay reacted strongly with 60 different HIV isolates but did not cross-react with human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, cytomegalovirus, varicella-zoster virus, herpes simplex virus type 1, Epstein-Barr virus, adenovirus type 5, or poliovirus type 1 or with extracts from four different control human cell lines and eight different phytohemagglutinin-stimulated normal human lymphocytes. Peripheral blood lymphocyte samples from 50 individuals were evaluated by both the antigen assay and the reverse transcriptase assay. The cells from the 34 seropositive individuals were all positive by the antigen assay (range, 3 to 9 days; average time, 5.9 days) and the reverse transcriptase assay (range, 7 to 16 days; average time, 9.6 days). Cells from the 16 seronegative individuals were negative by both assays. These results indicate that the antigen assay is an important addition to the monitoring of HIV production in the lymphocytes of infected patients. PMID:2448334

  6. Highly Sensitive Protein Translation Assay for Trichothecene Toxicity in Airborne Particulates: Comparison with Cytotoxicity Assays

    PubMed Central

    Yike, Iwona; Allan, Terry; Sorenson, William G.; Dearborn, Dorr G.

    1999-01-01

    Screening assays for environmental mycotoxins in bulk samples currently use cytotoxicity in cell cultures, but their application to air particulate samples often lacks sensitivity and specificity for fungal spores. An assay based on inhibition of protein synthesis using translation of firefly luciferase in a rabbit reticulocyte system has been developed for the detection of trichothecene mycotoxins found in the spores of toxigenic fungi. Ethanol extracts of air particulates trapped on polycarbonate filters are ultrafiltered and applied at several dilutions to a translation reaction mixture. The activity of translated luciferase is measured directly in a luminometer, eliminating the need for radioisotopes and time-consuming sample processing. Parallel standard curves using a commercially available trichothecene provide for expression of the results in T-2 toxin equivalents per cubic meter of air. The assay can be completed in 2 h and is readily applicable to multiple samples. Comparison to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay indicates a 400-fold increase in sensitivity of trichothecene detection in addition to a much higher specificity for these toxins. Initial field testing indicates a strong correlation between the measured level of toxicity and the presence of toxigenic fungi detected with microbiological methods. In conclusion, this luciferase translation assay offers a rapid and highly sensitive and specific method for quantitative detection of trichothecene mycotoxin activity in air particulate samples. PMID:9872764

  7. Multisite comparison of high-sensitivity multiplex cytokine assays.

    PubMed

    Breen, Elizabeth Crabb; Reynolds, Sandra M; Cox, Christopher; Jacobson, Lisa P; Magpantay, Larry; Mulder, Candice B; Dibben, Oliver; Margolick, Joseph B; Bream, Jay H; Sambrano, Elise; Martínez-Maza, Otoniel; Sinclair, Elizabeth; Borrow, Persephone; Landay, Alan L; Rinaldo, Charles R; Norris, Philip J

    2011-08-01

    The concentrations of cytokines in human serum and plasma can provide valuable information about in vivo immune status, but low concentrations often require high-sensitivity assays to permit detection. The recent development of multiplex assays, which can measure multiple cytokines in one small sample, holds great promise, especially for studies in which limited volumes of stored serum or plasma are available. Four high-sensitivity cytokine multiplex assays on a Luminex (Bio-Rad, BioSource, Linco) or electrochemiluminescence (Meso Scale Discovery) platform were evaluated for their ability to detect circulating concentrations of 13 cytokines, as well as for laboratory and lot variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women's Interagency HIV Study (WIHS) and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples, interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha were detectable with at least three kits, while IL-1β was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (P < 0.001) between laboratories and/or lots with all kits. Nevertheless, the kits generally detected similar patterns of cytokine perturbation during primary HIV viremia. This multisite comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma concentrations of cytokines and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important.

  8. Determining Vitamin D Status: A Comparison between Commercially Available Assays

    PubMed Central

    Snellman, Greta; Melhus, Håkan; Gedeborg, Rolf; Byberg, Liisa; Berglund, Lars; Wernroth, Lisa; Michaëlsson, Karl

    2010-01-01

    Background Vitamin D is not only important for bone health but can also affect the development of several non-bone diseases. The definition of vitamin D insufficiency by serum levels of 25-hydroxyvitamin D depends on the clinical outcome but might also be a consequence of analytical methods used for the definition. Although numerous 25-hydroxyvitamin D assays are available, their comparability is uncertain. We therefore aim to investigate the precision, accuracy and clinical consequences of differences in performance between three common commercially available assays. Methodology/Principal Findings Serum 25-hydroxyvitamin D levels from 204 twins from the Swedish Twin Registry were determined with high-pressure liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS), a radioimmunoassay (RIA) and a chemiluminescent immunoassay (CLIA). High inter-assay disagreement was found. Mean 25-hydroxyvitamin D levels were highest for the HPLC-APCI-MS technique (85 nmol/L, 95% CI 81–89), intermediate for RIA (70 nmol/L, 95% CI 66–74) and lowest with CLIA (60 nmol/L, 95% CI 56–64). Using a 50-nmol/L cut-off, 8% of the subjects were insufficient using HPLC-APCI-MS, 22% with RIA and 43% by CLIA. Because of the heritable component of 25-hydroxyvitamin D status, the accuracy of each method could indirectly be assessed by comparison of within-twin pair correlations. The strongest correlation was found for HPLC-APCI-MS (r = 0.7), intermediate for RIA (r = 0.5) and lowest for CLIA (r = 0.4). Regression analyses between the methods revealed a non-uniform variance (p<0.0001) depending on level of 25-hydroxyvitamin D. Conclusions/Significance There are substantial inter-assay differences in performance. The most valid method was HPLC-APCI-MS. Calibration between 25-hydroxyvitamin D assays is intricate. PMID:20644628

  9. A multicenter comparison of whole blood vitamin B6 assays.

    PubMed

    van Zelst, Bertrand D; de Beer, Roseri J A C Roelofsen; Neele, Marjolein; Kos, Snježana; Kema, Ido P; Tegelaers, Frans P W; Cobbaert, Christa M; Weykamp, Cas W; de Jonge, Robert

    2016-04-01

    The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6. This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at -20 °C and one set was analyzed after lyophilization. In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants. The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.

  10. Genotyping of hepatitis C virus-comparison of three assays.

    PubMed

    Haushofer, Alexander C; Berg, Jörg; Hauer, René; Trubert-Exinger, Doris; Stekel, Herbert G; Kessler, Harald H

    2003-08-01

    Genotyping of hepatitis C virus (HCV) is clinically relevant to epidemiology, prognosis, and therapeutical management of HCV infection. Accuracy and specificity of three assays for HCV genotyping/subtyping were determined. The TruGene HCV 5'NC Genotyping Kit (TruGene), which is a direct sequencing test and two assays based on reversed hybridization, Inno-LiPA HCV II assay and ViennaLab HCV Strip Assay, were compared. Amplification products generated by the Cobas Amplicor HCV Test were used. A total of 100 consecutive HCV RNA positive samples derived from patients with chronic hepatitis C were examined for their genotypes/subtypes by the three assays. Identification of genotypes and subtypes by the TruGene assay as reference test for the Inno-LiPA HCV II assay and the ViennaLab HCV Strip Assay or Inno-LiPA HCV II assay as reference test for the TruGene and the ViennaLab HCV Strip Assay showed similar results for overall accuracies (TruGene as reference test for Inno-LiPA HCV II and ViennaLab HCV Strip Assay, genotypes/subtypes: 100%/95.5% and 97%/92%; Inno-LiPA HCV II as reference test for TruGene and ViennaLab HCV Strip Assay, genotypes/subtypes: 99%/85.9% and 97%/87.9%) and specificities (TruGene as reference test for Inno-LiPA HCV II and ViennaLab HCV Strip Assay, genotypes/subtypes: 100%/97.8% and 99%/97.7%; Inno-LiPA HCV II as reference test for TruGene and ViennaLab HCV Strip Assay, genotypes/subtypes: 100%/99.4% and 99.7%/98%). The three assays were found to be reliable for the detection and discrimination of all HCV genotypes common in Europe and in North America and to be suitable for the routine diagnostic laboratory.

  11. Comparison and standardization of soil enzyme assay for meaningful data interpretation.

    PubMed

    Deng, Shiping; Dick, Richard; Freeman, Christopher; Kandeler, Ellen; Weintraub, Michael N

    2017-02-01

    Data interpretation and comparison in enzyme assays can be challenging because of the complex nature of the environment and variations in methods employed. This letter provides an overview of common enzyme assays, the need for methods standardization, and solutions addressing some of the concerns in microplate fluorimetric assay approaches.

  12. Comparison of five assays for detection of Clostridium difficile toxin.

    PubMed

    Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

    2011-07-01

    Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  13. A comparison of protein quantitation assays for biopharmaceutical applications.

    PubMed

    Noble, J E; Knight, A E; Reason, A J; Di Matola, A; Bailey, M J A

    2007-10-01

    Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.

  14. Comparison of four assays for the detection of cryptococcal antigen.

    PubMed

    Binnicker, M J; Jespersen, D J; Bestrom, J E; Rollins, L O

    2012-12-01

    We compared the performance of four assays for the detection of cryptococcal antigen in serum samples (n = 634) and cerebrospinal fluid (CSF) samples (n = 51). Compared to latex agglutination, the sensitivity and specificity of the Premier enzyme immunoassay (EIA), Alpha CrAg EIA, and CrAg lateral flow assay (LFA) were 55.6 and 100%, 100 and 99.7%, and 100 and 99.8%, respectively, from serum samples. There was 100% agreement among the four tests for CSF samples, with 18 samples testing positive by each of the assays.

  15. A novel assay detecting recall response to Mycobacterium tuberculosis: Comparison with existing assays.

    PubMed

    Hsu, Denise C; Zaunders, John J; Plit, Marshall; Leeman, Craig; Ip, Susanna; Iampornsin, Thatri; Pett, Sarah L; Bailey, Michelle; Amin, Janaki; Ubolyam, Sasiwimol; Avihingsanon, Anchalee; Ananworanich, Jintanat; Ruxrungtham, Kiat; Cooper, David A; Kelleher, Anthony D

    2012-07-01

    A strategy to reduce the burden of active TB is isoniazid preventive therapy for latent TB infection (LTBI). However, current assays used to diagnose LTBI all have limitations. In these proof of concept studies, we compared the agreement of a novel flow cytometry assay detecting CD25/CD134 co-expression with QuantiFERON-TB Gold In-Tube (QFN-GIT) and Tuberculin skin test (TST) in the detection of recall immune response to TB. The CD25/CD134 assay, QFN-GIT and TST were performed on 74 participants referred for TB screening in Sydney and on 50 participants with advanced HIV infection (CD4 ≤ 350 × 10(6) cells/L) in Bangkok. The agreement between CD25/CD134 assay and QFN-GIT was 93.2% (Kappa 0.631 95% CI 0.336-0.926) in Sydney and 90% (Kappa 0.747 95% CI 0.541-0.954) in Bangkok. Discordant results occurred around the cut off of both tests. The agreement between CD25/CD134 assay and TST was 73.6% (Kappa 0.206 95% CI 0.004-0.409) in Sydney and 84% (Kappa 0.551 95% CI 0.296-0.806) in Bangkok. The CD25/CD134 assay showed good agreement with QFN-GIT in detecting recall response to TB both in well and less resourced setting as well as in persons with advanced HIV infection. Further study into the performance of this assay is thus warranted.

  16. Comparison of four multiplex PCR assays for the detection of viral pathogens in respiratory specimens.

    PubMed

    Anderson, Trevor P; Werno, Anja M; Barratt, Kevin; Mahagamasekera, Patalee; Murdoch, David R; Jennings, Lance C

    2013-08-01

    Multiplex PCR has become the test of choice for the detection of multiple respiratory viruses in clinical specimens. However, there are few direct comparisons of different PCR assays. This study compares 4 different multiplex PCR assays for the recovery of common respiratory viruses. We tested 213 respiratory specimens using four different multiplex PCR assays: the xTAG respiratory viral panel fast (Abbott Molecular Laboratories), Fast-track Respiratory Pathogen assay (Fast-track Diagnostics), Easyplex respiratory pathogen 12 kit (Ausdiagnostics), and an in-house multiplex real-time PCR assay. The performance of the four assays was very similar, with 93-100% agreement for all comparisons. Other issues, such as through-put, technical requirements and cost, are likely to be as important for making a decision about which of these assays to use given their comparative performance.

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

    PubMed

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

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

  18. Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase.

    PubMed

    Takagi, Toshimitsu; Shum, David; Parisi, Monika; Santos, Ruth E; Radu, Constantin; Calder, Paul; Rizvi, Zahra; Frattini, Mark G; Djaballah, Hakim

    2011-09-01

    Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.

  19. Multilaboratory Comparison of Hepatitis C Virus Viral Load Assays

    PubMed Central

    Caliendo, A. M.; Valsamakis, A.; Zhou, Y.; Yen-Lieberman, B.; Andersen, J.; Young, S.; Ferreira-Gonzalez, A.; Tsongalis, G. J.; Pyles, R.; Bremer, J. W.; Lurain, N. S.

    2006-01-01

    We report a multilaboratory evaluation of hepatitis C virus (HCV) viral load assays to determine their linear range, reproducibility, subtype detection, and agreement. A panel of HCV RNA samples ranging in nominal concentration from 1.0 to 7.0 log10 IU/ml was constructed by diluting a clinical specimen (genotype 1b). Replicates of the panel were tested in multiple laboratories using the Abbott TaqMan analyte-specific reagent (Abbott reverse transcription-PCR [RT-PCR]), Roche TaqMan RUO (Roche RT-PCR), Roche Amplicor Monitor HCV 2.0 (Roche Monitor), and Bayer VERSANT HCV RNA 3.0 (Bayer bDNA) assays. Bayer bDNA-negative specimens were tested reflexively using the Bayer VERSANT HCV RNA qualitative assay (Bayer TMA). Abbott RT-PCR and Roche RT-PCR detected all 28 replicates with a concentration of 1.0 log10 IU/ml and were linear to 7.0 log10 IU/ml. Roche Monitor and Bayer bDNA detected 27 out of 28 and 13 out of 28 replicates, respectively, of 3.0 log10 IU/ml. Bayer TMA detected all seven replicates with 1.0 log10 IU/ml. Bayer bDNA was the most reproducible of the four assays. The mean viral load values for panel members in the linear ranges of the assays were within 0.5 log10 for the different tests. Eighty-nine clinical specimens of various genotypes (1 through 4) were tested in the Bayer bDNA, Abbott RT-PCR, and Roche RT-PCR assays. For Abbott RT-PCR, mean viral load values were 0.61 to 0.96 log10 greater than the values for Bayer bDNA assay for samples with genotype 1, 2, or 3 samples and 0.08 log10 greater for genotype 4 specimens. The Roche RT-PCR assay gave mean viral load values that were 0.28 to 0.82 log10 greater than those obtained with the Bayer bDNA assay for genotype 1, 2, and 3 samples. However, for genotype 4 samples the mean viral load value obtained with the Roche RT-PCR assay was, on average, 0.15 log10 lower than that of the Bayer bDNA. Based on these data, we conclude that the sensitivity and linear range of the Abbott and Roche RT-PCR assays

  20. Comparison of rapid screening assays using organic chemicals

    SciTech Connect

    Beach, S.A.; Robideau, R.R.

    1994-12-31

    In a continuation of a study presented last year using metals, the sensitivity of short term toxicity tests is examined using common organic chemicals. In toxicity testing, the focus has shifted from the traditional long-term studies utilizing the mortality of complex, multicellular eukaryotic organisms as the endpoint towards short-term studies in which transformation of biochemical pathways are monitored. The relative sensitivity of aquatic screening techniques are compared to the standardized 48-hr Daphnia magna and Ceriodaphnia dubia, 96-hr fathead minnow and 96-hr algal acute assays. The short-term test procedures investigated are: dehydrogenase enzyme activity assays utilizing triphenyltetrazolium chloride (TTC) and resazurin as the calorimetric indicators; TOXI-Chromotest, inhibition of {beta}-galactosidase; reduction in bioluminescence output utilizing the Microtox{reg_sign} test; nitrification inhibition assays with a commercial preparation of nitrifying bacteria (Nitroseed{trademark}) and municipal activated sludge; respiration inhibition assays with a commercial preparation of heterotrophic bacteria (Polytox{reg_sign}) and activated sludge; inhibition of root growth in terrestrial plants; and galactosidase inhibition through the use of a fluorometrically tagged substrate with the Daphnia magna IQ{trademark} test. Toxicity values generated by this laboratory on commonly used organic chemicals are compared.

  1. Comparison of the Abbott Realtime HIV-1 and HCV viral load assays with commercial competitor assays.

    PubMed

    Schutten, Martin

    2008-07-01

    The introduction of commercially available quantitative HIV-1 RNA detection methods at the end of the last century has had a significant impact on the management of patients requiring treatment. Similarly for hepatitis C virus (HCV), clinical decision-making with respect to initiation and prolonging therapy is largely based on data from viral load assays. The methods developed in the early 1990s and further improved since then still have significant drawbacks. For example, they are labor intensive, have a small dynamic range and are contamination sensitive. The development of real-time detection techniques for reverse transcription PCR has in part solved these problems. In the present review the advantages and disadvantages of the recently marketed Abbott Realtime HCV and HIV-1 viral load assays relative to their competitors will be discussed.

  2. A comparison of assays measuring the viability of Legionella ...

    EPA Pesticide Factsheets

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubat

  3. A comparison of assays measuring the viability of Legionella ...

    EPA Pesticide Factsheets

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubat

  4. Comparison of bee products based on assays of antioxidant capacities

    PubMed Central

    Nakajima, Yoshimi; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Mishima, Satoshi; Hara, Hideaki

    2009-01-01

    Background Bee products (including propolis, royal jelly, and bee pollen) are popular, traditional health foods. We compared antioxidant effects among water and ethanol extracts of Brazilian green propolis (WEP or EEP), its main constituents, water-soluble royal jelly (RJ), and an ethanol extract of bee pollen. Methods The hydrogen peroxide (H2O2)-, superoxide anion (O2·-)-, and hydroxyl radical (HO·)- scavenging capacities of bee products were measured using antioxidant capacity assays that employed the reactive oxygen species (ROS)-sensitive probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) or aminophenyl fluorescein (APF). Results The rank order of antioxidant potencies was as follows: WEP > EEP > pollen, but neither RJ nor 10-hydroxy-2-decenoic acid (10-HDA) had any effects. Concerning the main constituents of WEP, the rank order of antioxidant effects was: caffeic acid > artepillin C > drupanin, but neither baccharin nor coumaric acid had any effects. The scavenging effects of caffeic acid were as powerful as those of trolox, but stronger than those of N-acetyl cysteine (NAC) or vitamin C. Conclusion On the basis of the present assays, propolis is the most powerful antioxidant of all the bee product examined, and its effect may be partly due to the various caffeic acids it contains. Pollen, too, exhibited strong antioxidant effects. PMID:19243635

  5. Comparison of two immunoradiometric assays for serum thyrotropin

    SciTech Connect

    Scheinin, B.; Drew, H.; La France, N.; Ladenson, P.; Wagner, H.N. Jr.

    1985-05-01

    An ultra-sensitive TSH assay capable of detecting subnormal TSH levels would be useful in confirming suppressed pituitary function as seen in hyperthyroidism. Two sensitive immunoradiometric TSH assays (IRMA's) were studied to determine how well they distinguished thyrotoxic patients from normal subjects. Serono Diagnostics' method employs three monoclonal antibodies specific for different regions of the TSH molecule with a minimum detectable dose (MDD) limit of 0.1 ..mu..IU/ml. Precision studies using a low TSH control in the 1.8 ..mu..IU/ml range gave CV's of 15.0%. Boots-Celltech Diagnostics method is a two site IRMA using two monoclonal antibodies. The MDD limit is 0.05 ..mu..IU/ml with precision CV's of 29.3% at a TSH control range of 0.62 ..mu..IU/ml. In 24 chemically thyrotoxic patients, the mean serum TSH concentration was significantly lower than in the normal control subjects: for Serono, 0.19 ..mu..IU/ml vs. 2.34 ..mu..IU/ml and for Boots Celltech, 0.18 IU/ml vs 2.06 ..mu..IU/ml. The range of TSH was 0 to 0.5 ..mu..IU/ml in thyrotoxic patients using Serono with the exception of one patient having a TSH value of 0.8 ..mu..IU/ml. The normal range was 0.6 to 6.0 ..mu..IU/ml. For Boots Celltech the thyrotoxic range was 0 to 0.2 ..mu..IU/ml with that same thyrotoxic patient giving a TSH value of 0.7 ..mu..IU/ml with a normal range of 0.6 to 5.0 IU/ml. Serum TSH measurements using both procedures are highly sensitive for distinguishing thyrotoxic patients from normal subjects and are useful to confirm suppressed pituitary function.

  6. Comparison of mouse strains using the local lymph node assay.

    PubMed

    Woolhiser, M R; Munson, A E; Meade, B J

    2000-05-05

    The local lymph node assay (LLNA), as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), only allows for the use of CBA mice. The objective of these studies was to begin to assess the response of chemical sensitizers in the LLNA across six strains of female mice (C57BL/6, SJL/J, BALB/c, B6C3F1, DBA/2 and CBA). The moderate sensitizer alpha-hexylcinnamaldehyde (HCA) was chosen as the test chemical, while toluene diisocyanate (TDI) and 2,4-dinitrofluorobenzene (DNFB) were evaluated at single concentrations as positive controls. Draining lymph node cell proliferation following acetone exposure varied across strains. SJL mice had a significantly higher degree of proliferation with 2111 d.p.m./2 nodes. The remaining five strains demonstrated responses which ranged from 345 to 887 dpm/2 nodes. DBA/2, B6C3F1, BALB/c and CBA mice had essentially equal levels of lymph node proliferation following exposure to the three chemicals. While C57BL/6 mice gave similar results as CBA mice following DNFB and HCA administration, the LLNA response to TDI was considerably lower. SJL mice provided low stimulation indexes (SI) values for all three chemicals evaluated. Regardless of the level of LLNA response, all six mouse strains identified the sensitization potential of HCA, TDI or DNFB. Based on these studies, DBA/2, B6C3F1 and BALB/c mice are good choices for continued evaluation as additional mouse strains for use in the LLNA.

  7. Interlaboratory comparison of dicentric chromosome assay using electronically transmitted images.

    PubMed

    García, O; Di Giorgio, M; Vallerga, M B; Radl, A; Taja, M R; Seoane, A; De Luca, J; Stuck Oliveira, M; Valdivia, P; Lamadrid, A I; González, J E; Romero, I; Mandina, T; Pantelias, G; Terzoudi, G; Guerrero-Carbajal, C; Arceo Maldonado, C; Espinoza, M; Oliveros, N; Martínez-López, W; Di Tomaso, M V; Méndez-Acuña, L; Puig, R; Roy, L; Barquinero, J F

    2013-04-01

    The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of ∼15 % was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CV on the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the

  8. Comparison of the Chiron Quantiplex branched DNA (bDNA) assay and the Abbott Genostics solution hybridization assay for quantification of hepatitis B viral DNA.

    PubMed

    Kapke, G E; Watson, G; Sheffler, S; Hunt, D; Frederick, C

    1997-01-01

    Several assays for quantification of DNA have been developed and are currently used in research and clinical laboratories. However, comparison of assay results has been difficult owing to the use of different standards and units of measurements as well as differences between assays in dynamic range and quantification limits. Although a few studies have compared results generated by different assays, there has been no consensus on conversion factors and thorough analysis has been precluded by small sample size and limited dynamic range studied. In this study, we have compared the Chiron branched DNA (bDNA) and Abbott liquid hybridization assays for quantification of hepatitis B virus (HBV) DNA in clinical specimens and have derived conversion factors to facilitate comparison of assay results. Additivity and variance stabilizing (AVAS) regression, a form of non-linear regression analysis, was performed on assay results for specimens from HBV clinical trials. Our results show that there is a strong linear relationship (R2 = 0.96) between log Chiron and log Abbott assay results. Conversion factors derived from regression analyses were found to be non-constant and ranged from 6-40. Analysis of paired assay results below and above each assay's limit of quantification (LOQ) indicated that a significantly (P < 0.01) larger proportion of observations were below the Abbott assay LOQ but above the Chiron assay LOQ, indicating that the Chiron assay is significantly more sensitive than the Abbott assay. Testing of replicate specimens showed that the Chiron assay consistently yielded lower per cent coefficients of variance (% CVs) than the Abbott assay, indicating that the Chiron assay provides superior precision.

  9. [Comparison of different molecular assays for the rapid detection of enterovirus 71 (EV71)].

    PubMed

    Wei, Hai-Yan; Huang, Xue-Yong; Xu, Yu-Ling; Ma, Hong; Chen, Hao-Min; Xu, Bian-Li

    2012-11-01

    Molecular detection of enterovirus (EV)71 RNA based on PCR methods is a quick and sensitive approach. At present, different PCR-based methods for EV71 RNA detection are available, but comparisons of results obtained using different approaches are limited. This study is to compare the analytical sensitivity and specificity of different real-time reverse transcription-polymerase chain reaction (rRT-PCR) and conventional reverse transcription-polymerase chain reaction (cRT-PCR) assays for enterovirus and EV71 detection, Altogether, three rRT-PCR assays and one cRT-PCR assay targeting the 5'UTR gene for universal detection of enterovirus; two rRT-PCR assays andone cRT-PCR assay targeting the VP1 gene for specific detection of EV 71 were examined. All assays showed good specificity. The detection sensitivity ranged from 8.19 x 10 to 8.19 x 10(5) copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All rRT-PCR assays showed 100% sensitivity for clinical specimens.

  10. Comparison of three assays for genetic effects of antineoplastic drugs on cancer patients and their nurses

    SciTech Connect

    Krepinsky, A. ); Bryant, D.W.; Davison, L.; McCalla, D.R. ); Young, B. ); Heddle, J. ); Douglas, G. ); Michalko, K. )

    1990-01-01

    Three assays have been compared for their ability to detect genetic damage caused by antineoplastic drugs in cancer patients and possible damage in the nurses who administered these drugs. The assays were sister chromatid exchanges (SCE) and chromosomal aberrations in peripheral blood lymphocytes, and the Salmonella/mammalian microsome assay on urine. Three comparisons were made: (1) patients before versus after treatment; (2) the administering nurses immediately after their work period versus after a few days off that followed (work and off-work); (3) the exposed nurses versus other nurses who did not administer antineoplastic drugs (controls). The SCE assay did not distinguish between the work and off-work samples in either the exposed or control nurses. Chromosomal aberration was the only assay which showed significant difference between the two samples of the exposed nurses and, consequently, between the exposed and control nurses. There is no evidence that the increase was connected to occupational exposure.

  11. International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II

    PubMed Central

    Heyndrickx, Leo; Heath, Alan; Sheik-Khalil, Enas; Alcami, Jose; Bongertz, Vera; Jansson, Marianne; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Ramaswamy, Meghna; Sattentau, Quentin; Tolazzi, Monica; Schuitemaker, Hanneke; Willems, Betty; Wrin, Terri; Fenyö, Eva Maria; Scarlatti, Gabriella

    2012-01-01

    Background Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). Methods In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Findings Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Conclusions Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus

  12. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    SciTech Connect

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer.

  13. A comparison of hemagglutination inhibition and neutralization assays for characterizing immunity to seasonal influenza A.

    PubMed

    Truelove, Shaun; Zhu, Huachen; Lessler, Justin; Riley, Steven; Read, Jonathan M; Wang, Shuying; Kwok, Kin On; Guan, Yi; Jiang, Chao Qiang; Cummings, Derek A T

    2016-11-01

    Serum antibody to influenza can be used to identify past exposure and measure current immune status. The two most common methods for measuring this are the hemagglutination inhibition assay (HI) and the viral neutralization assay (NT), which have not been systematically compared for a large number of influenza viruses. A total of 151 study participants from near Guangzhou, China, were enrolled in 2009 and provided serum. HI and NT assays were performed for 12 historic and recently circulating strains of seasonal influenza A. We compared titers using Spearman correlation and fit models to predict NT using HI results. We observed high positive mean correlation between HI and NT assays (Spearman's rank correlation, ρ=.86) across all strains. Correlation was highest within subtypes and within close proximity in time. Overall, an HI=20 corresponded to NT=10, and HI=40 corresponded to NT=20. Linear regression of log(NT) on log(HI) was statistically significant, with age modifying this relationship. Strain-specific area under a curve (AUC) indicated good accuracy (>80%) for predicting NT with HI. While we found high overall correspondence of titers between NT and HI assays for seasonal influenza A, no exact equivalence between assays could be determined. This was further complicated by correspondence between titers changing with age. These findings support generalized comparison of results between assays and give further support for use of the hemagglutination inhibition assay over the more resource intensive viral neutralization assay for seasonal influenza A, although attention should be given to the effect of age on these assays. © 2016 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  14. Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity.

    PubMed

    Sanghera, Jasbinder; Li, Rick; Yan, Jun

    2009-12-01

    Many assay technologies have been developed and utilized to efficiently assay and screen against protein kinase targets. The radiometric assay format for assaying the protein kinase targets has been considered the "Gold Standard" format since it allows the direct readout of kinase functional activity and is a universal assay that is highly sensitive. However, the hazardous nature of the radiometric assay together with the regulatory hurdles has led to the development of alternative assay formats for assessing protein kinase activity measurements. The luminescent ADP-Glo assay has been developed as an alternative to radiometric format for assaying protein kinase targets. This assay allows the measurement of the ADP product formed during the kinase reaction. Therefore, the luminescent ADP-Glo assay is similar to the radiometric format in that it measures the direct product of the protein kinase reaction. Furthermore, since the ADP product is generated by all protein kinase reactions, this is a universal format that can be used for assaying any given protein kinase target. Analysis of data generated with multiple protein kinase targets and the luminescent ADP-Glo technology shows comparable results to the radiometric assay format. Therefore, the luminescent ADP-Glo assay is a robust new technology for evaluating catalytic function of protein kinases as well as other ATPases.

  15. A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay.

    PubMed

    Moretti, Rocco; Thorson, Jon S

    2008-06-15

    A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate nucleotidyltransferase assay. The addition of a general phosphatase to nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate nucleotidyltransferase mechanistic and directed evolution/engineering studies.

  16. Comparison of Antibody Responses to Human Papillomavirus Vaccination as Measured by Three Assays

    PubMed Central

    Robbins, Hilary A.; Kemp, Troy J.; Porras, Carolina; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Gonzalez, Paula; Schiller, John; Lowy, Douglas; Poncelet, Sylviane; Esser, Mark; Matys, Katie; Hildesheim, Allan; Pinto, Ligia A.; Herrero, Rolando; Safaeian, Mahboobeh

    2014-01-01

    Background: Different assays, including the competitive Luminex immunoassay (cLIA), secreted alkaline phosphatase neutralization assay (SEAP-NA), and virus-like particle-based ELISA, are commonly used to measure antibody responses after human papillomavirus (HPV) vaccination. Direct assay comparisons aid interpretation of immunogenicity data evaluated by different assays. Methods: We compared cLIA to SEAP-NA and ELISA among 51 HPV16/18-vaccinated women enrolled in the Costa Rica Vaccine Trial. We tested replicate serum samples collected at months 0, 1, and 12 by HPV16/18 cLIA, SEAP-NA, and ELISA. For a subset (N = 10), we further tested month 6, 24 and 36 samples. We calculated seroprevalence estimates and Spearman rank correlation coefficients comparing cLIA to SEAP-NA and ELISA. Results: After one vaccine dose, seroprevalence by SEAP-NA and ELISA was 100% (both HPV16 and HPV18), and by cLIA was 96% (95% CI 87–100%) for HPV16 and 71% (95% CI 56–83%) for HPV18. Seroprevalence was 100% by all assays after three doses. Correlation between assays was high after one vaccine dose [cLIA/SEAP-NA ρ = 0.91 (HPV16) and ρ = 0.86 (HPV18); cLIA/ELISA ρ = 0.84 (HPV16) and ρ = 0.74 (HPV18); all p < 0.001] and remained high through month 36. Ratios of mean antibody levels to seropositivity cutoffs at month 36 were lower for cLIA than for SEAP-NA or ELISA, particularly for HPV18 (HPV18 ratio for cLIA 1.9, SEAP-NA 3.5, ELISA 3.4). Conclusion: Though correlation between cLIA and SEAP-NA/ELISA is high and stable after vaccination, the assays differ in scale and sensitivity, with notable differences after one vaccine dose and for HPV18. Our results demonstrate that comparisons of antibody responses to HPV vaccination measured by different assays are approximate, and must consider biological and technical differences between assays. PMID:24455487

  17. Comparison of antibody responses to human papillomavirus vaccination as measured by three assays.

    PubMed

    Robbins, Hilary A; Kemp, Troy J; Porras, Carolina; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Gonzalez, Paula; Schiller, John; Lowy, Douglas; Poncelet, Sylviane; Esser, Mark; Matys, Katie; Hildesheim, Allan; Pinto, Ligia A; Herrero, Rolando; Safaeian, Mahboobeh

    2014-01-13

    Different assays, including the competitive Luminex immunoassay (cLIA), secreted alkaline phosphatase neutralization assay (SEAP-NA), and virus-like particle-based ELISA, are commonly used to measure antibody responses after human papillomavirus (HPV) vaccination. Direct assay comparisons aid interpretation of immunogenicity data evaluated by different assays. We compared cLIA to SEAP-NA and ELISA among 51 HPV16/18-vaccinated women enrolled in the Costa Rica Vaccine Trial. We tested replicate serum samples collected at months 0, 1, and 12 by HPV16/18 cLIA, SEAP-NA, and ELISA. For a subset (N = 10), we further tested month 6, 24 and 36 samples. We calculated seroprevalence estimates and Spearman rank correlation coefficients comparing cLIA to SEAP-NA and ELISA. After one vaccine dose, seroprevalence by SEAP-NA and ELISA was 100% (both HPV16 and HPV18), and by cLIA was 96% (95% CI 87-100%) for HPV16 and 71% (95% CI 56-83%) for HPV18. Seroprevalence was 100% by all assays after three doses. Correlation between assays was high after one vaccine dose [cLIA/SEAP-NA ρ = 0.91 (HPV16) and ρ = 0.86 (HPV18); cLIA/ELISA ρ = 0.84 (HPV16) and ρ = 0.74 (HPV18); all p < 0.001] and remained high through month 36. Ratios of mean antibody levels to seropositivity cutoffs at month 36 were lower for cLIA than for SEAP-NA or ELISA, particularly for HPV18 (HPV18 ratio for cLIA 1.9, SEAP-NA 3.5, ELISA 3.4). Though correlation between cLIA and SEAP-NA/ELISA is high and stable after vaccination, the assays differ in scale and sensitivity, with notable differences after one vaccine dose and for HPV18. Our results demonstrate that comparisons of antibody responses to HPV vaccination measured by different assays are approximate, and must consider biological and technical differences between assays.

  18. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    PubMed

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  19. Comparison between S+L- assay and LacZ marker rescue assay for detecting replication-competent gammaretroviruses.

    PubMed

    Hashimoto-Gotoh, A; Yoshikawa, R; Miyazawa, T

    2015-09-01

    To avoid contamination of adventitious gammaretroviruses in biological products such as vaccines, it is necessary to check the master seed cells for manufacturing. There are several assays to detect infectious gammaretroviruses. Among these, sarcoma-positive, leukemia-negative (S+L-) assay is a classical infectivity assay, which is often recommended in governmental guidelines. The S+L- cells used in S+L- assay generate unique focus upon the infection of replication-competent gammaretroviruses. Although S+L- assay is well recognized for the detection, their applicability is questionable in some cases. On the other hand, LacZ marker rescue (LMR) assay detects infectious gammaretroviruses by transducing LacZ marker gene to the target cells, which shows lacZ-positive foci if the infectious virus is present. In this study, we compared LMR and S+L- assays for detection of a variety of endogenous and exogenous gammaretroviruses. As results, LMR assay could detect all gammaretroviruses examined. On the other hand, S+L- assay using feline S+L- cells, termed QN10S, could not detect porcine endogenous retrovirus (PERV) subgroups A/B. Further, S+L- mink cells could not detect feline leukemia virus subgroups B in addition to PERV-A/B. These data indicate that LMR assay is better suited to detect wider range of gammaretroviruses. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  20. Development of an Internally Controlled Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Comparison of Four Molecular Dengue Virus Detection Assays

    PubMed Central

    Waggoner, Jesse J.; Abeynayake, Janaki; Sahoo, Malaya K.; Gresh, Lionel; Tellez, Yolanda; Gonzalez, Karla; Ballesteros, Gabriela; Balmaseda, Angel; Karunaratne, Kumudu; Harris, Eva

    2013-01-01

    A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log10 cDNA equivalents/μl and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μl, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P ≤ 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided. PMID:23637298

  1. Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae

    PubMed Central

    Ratliff, Amy E.; Duffy, Lynn B.

    2014-01-01

    A loop-mediated isothermal amplification (LAMP) system, the illumigene Mycoplasma DNA amplification assay (Meridian Bioscience, Inc., Cincinnati, OH) was evaluated to determine its analytical sensitivity, specificity, and clinical application in comparison to historic culture in a collection of archived respiratory specimens. The illumigene limit of detection was ≤88 CFU/reaction for 10 Mycoplasma pneumoniae reference strains. This assay correctly identified 36 M. pneumoniae reference strains and clinical isolates from various geographic origins, including both of the main subtypes. No cross-reactions were detected with other mycoplasmas, ureaplasmas, other bacterial species, viruses, yeasts, or human DNA. Among 214 respiratory specimens previously cultured for M. pneumoniae, when real-time PCR with bidirectional sequencing of the PCR products was used to resolve discrepancies, the sensitivity was 22 of 22 (100%) and the specificity was 190 of 192 (99%). This commercial LAMP assay is a useful rapid method for detecting M. pneumoniae in clinical specimens. Additional prospective clinical trials with direct comparison to culture and PCR are warranted. PMID:24430454

  2. A comparison of in-house real-time LAMP assays with a commercial assay for the detection of pathogenic bacteria

    USDA-ARS?s Scientific Manuscript database

    Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. ...

  3. Comparison of total protein concentration in skeletal muscle as measured by the Bradford and Lowry assays.

    PubMed

    Seevaratnam, Rajini; Patel, Barkha P; Hamadeh, Mazen J

    2009-06-01

    The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland-Altman analysis revealed considerable bias (15.8 +/- 29.7%). Both Lowry reagents and its protein-reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05-0.50 mg/ml) than the Bradford (0-2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein-reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.

  4. Comparison between fibroblast wound healing and cell random migration assays in vitro.

    PubMed

    Ascione, Flora; Vasaturo, Angela; Caserta, Sergio; D'Esposito, Vittoria; Formisano, Pietro; Guido, Stefano

    2016-09-10

    Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Antiproliferative effects of selenium compounds in colon cancer cells: comparison of different cytotoxicity assays.

    PubMed

    Schröterová, Ladislava; Králová, Vera; Vorácová, Adéla; Hasková, Pavlína; Rudolf, Emil; Cervinka, Miroslav

    2009-10-01

    A number of cytotoxicity assays are currently available, each of them using specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. In this study we compared the potential of five commonly employed cytotoxicity assays (WST-1, XTT, MTT, Brilliant blue and Neutral red assay) to detect antiproliferative effects of three selenium compounds, sodium selenite, seleno-L-methionine (SeMet) and Se-(Methyl)selenocysteine (SeMCys) on three colorectal cancer cell lines in vitro. Cells were exposed to the selected selenium compounds in the concentration range of 0-256 microM during 48 h. WST-1 and XTT failed to detect cytotoxic effect, with the exception of the highest concentration of selenium compounds tested. Conversely, the metabolic activity of selenium treated cells measured by WST-1 and XTT significantly increased in comparison to untreated controls. MTT, Neutral red and Brilliant blue assays were more sensitive and yielded mutually comparable results, with significant decrease of measured parameters in a concentration-dependent manner. To a smaller extent, the results were affected by the different chemical nature of the selenium compounds tested as well as by the biological properties of individual cell lines.

  6. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with INNO-LiPA HPV genotyping extra assay.

    PubMed

    Else, Elizabeth A; Swoyer, Ryan; Zhang, Yuhua; Taddeo, Frank J; Bryan, Janine T; Lawson, John; Van Hyfte, Inez; Roberts, Christine C

    2011-05-01

    Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.

  7. A comprehensive comparison of assays for detection and identification of Ralstonia solanacearum race 3 biovar 2.

    PubMed

    Li, X; Nie, J; Hammill, D L; Smith, D; Xu, H; De Boer, S H

    2014-10-01

    To determine the reliable combination of protocols for specific detection and identification of R. solanacearum race 3 biovar 2 (R3bv2) through a comprehensive comparison among currently available techniques. Sensitivity and specificity of the conventional isolation, bioassay, serological assays, conventional and real-time PCR and multiplex PCR were assessed for the detection of 25 strains of R. solanacearum biovars 1, 2 and 3 (Phylotypes I, II, III and IV) in spiked potato saps. Results indicated that all assays evaluated varied in complexity and sensitivity and should be applied strategically in indexing schemes to maximize efficiency of testing without compromising accuracy of the results. The TaqMan PCR assay, with an internal reaction control and confirmation by melting curve and electrophoretic analysis, achieved best sensitivity at 10(2) -10(3 ) CFU ml(-1) for all eighteen strains of R. solanacearum R3bv2. Selective enrichment on mSMSA medium plates enhanced the detection sensitivity up to 10-100 CFU ml(-1) for the conventional PCR-based assays. This is the first time nine different assays were compared side by side for their sensitivity and specificity in detection and identification of R. solanacearum R3bv2. The data accumulated here will provide basis for regulatory applications for low level detection and rapid identification of latently infections caused by R. solanacearum R3bv2. © 2014 Her Majesty the Queen in Right of Canada © 2014 Society for Applied Microbiology Reproduced with the permission of the Minister of Canadian Food inspection Agency.

  8. Dose estimation using dicentric chromosome assay and cytokinesis block micronucleus assay: comparison between manual and automated scoring in triage mode.

    PubMed

    De Amicis, Andrea; De Sanctis, Stefania; Di Cristofaro, Sara; Franchini, Valeria; Regalbuto, Elisa; Mammana, Giacomo; Lista, Florigio

    2014-06-01

    In cases of an accidental overexposure to ionizing radiation, it is essential to estimate the individual absorbed dose of a potentially radiation-exposed person. For this purpose, biological dosimetry can be performed to confirm, complement or even replace physical dosimetry when this proves to be unavailable. The most validated biodosimetry techniques for dose estimation are the dicentric chromosome assay, the "gold standard" for individual dose assessment, and cytokinesis-block micronucleus assay. However, both assays are time consuming and require skilled scorers. In case of large-scale accidents, different strategies have been developed to increase the throughput of cytogenetic service laboratories. These are the decrease of cell numbers to be scored for triage dosimetry; the automation of procedures including the scoring of, for example, aberrant chromosomes and micronuclei; and the establishment of laboratory networks in order to enable mutual assistance if necessary. In this study, the authors compared the accuracy of triage mode biodosimetry by dicentric chromosome analysis and the cytokinesis block micronucleus assay performing both the manual and the automated scoring mode. For dose estimation using dicentric chromosome assay of 10 blind samples irradiated up to 6.4 Gy of x-rays, a number of metaphase spreads were analyzed ranging from 20 up to 50 cells for the manual and from 20 up to 500 cells for the automatic scoring mode. For dose estimation based on the cytokinesis block micronucleus assay, the micronucleus frequency in both 100 and 200 binucleated cells was determined by manual and automatic scoring. The results of both assays and scoring modes were compared and analyzed considering the sensitivity, specificity, and accuracy of dose estimation with regard to the discrimination power of clinically relevant binary categories of exposure doses.

  9. Actual fusion efficiency in the lipid mixing assay - Comparison between nanodiscs and liposomes

    NASA Astrophysics Data System (ADS)

    François-Martin, Claire; Pincet, Frédéric

    2017-03-01

    Lipid exchange occurs between membranes during fusion or active lipid transfer. These processes are necessary in vivo for the homeostasis of the cell at the level of the membranes, the organelles and the cell itself. They are also used by the cell to interact with the surrounding medium. Several assays have been developed to characterize in vitro these processes on model systems. The most common one, relying on fluorescence dequenching, measures lipid mixing between small membranes such as liposomes or nanodiscs in bulk. Usually, relative comparisons of the rate of lipid exchange are made between measurements performed in parallel. Here, we establish a quantitative standardization of this assay to avoid any bias resulting from the temperatures, the chosen fluorescent lipid fractions and from the various detergents used to normalize the measurements. We used this standardization to quantitatively compare the efficiency of SNARE-induced fusion in liposome-liposome and liposome-nanodisc configurations having similar collision frequency. We found that the initial yield of fusion is comparable in both cases, 1 per 2-3 million collisions in spite of a much larger dequenching signal with nanodiscs. Also, the long-term actual fusion rate is slightly lower with nanodiscs than in the liposome-liposome assay.

  10. Actual fusion efficiency in the lipid mixing assay - Comparison between nanodiscs and liposomes

    PubMed Central

    François-Martin, Claire; Pincet, Frédéric

    2017-01-01

    Lipid exchange occurs between membranes during fusion or active lipid transfer. These processes are necessary in vivo for the homeostasis of the cell at the level of the membranes, the organelles and the cell itself. They are also used by the cell to interact with the surrounding medium. Several assays have been developed to characterize in vitro these processes on model systems. The most common one, relying on fluorescence dequenching, measures lipid mixing between small membranes such as liposomes or nanodiscs in bulk. Usually, relative comparisons of the rate of lipid exchange are made between measurements performed in parallel. Here, we establish a quantitative standardization of this assay to avoid any bias resulting from the temperatures, the chosen fluorescent lipid fractions and from the various detergents used to normalize the measurements. We used this standardization to quantitatively compare the efficiency of SNARE-induced fusion in liposome-liposome and liposome-nanodisc configurations having similar collision frequency. We found that the initial yield of fusion is comparable in both cases, 1 per 2–3 million collisions in spite of a much larger dequenching signal with nanodiscs. Also, the long-term actual fusion rate is slightly lower with nanodiscs than in the liposome-liposome assay. PMID:28266607

  11. Telomere length measurement by a novel Luminex-based assay: a blinded comparison to Southern blot.

    PubMed

    Pierce, Brandon L; Jasmine, Farzana; Roy, Shantanu; Zhang, Chenan; Aviv, Abraham; Hunt, Steven C; Ahsan, Habibul; Kibriya, Muhammad G

    2016-01-01

    Telomere length (TL) is a potential biomarker of aging and age-related disease risk. We recently published a novel Luminex-based method for high-throughput, low-cost TL measurement. Here we describe a blinded comparison of the Luminex method to Southern blot, the most precise TL measurement method. Luminex and Southern blot measurements for the same 50 DNA samples were taken in two independent laboratories; each sample was measured twice, several months apart. The inter-assay CV for Luminex ranged from 5.5 to 9.1 (depending on CV estimation method), and Southern blot CV from 1.0 to 1.7. Both measures were inversely associated with age. The correlation between the repeated measurements was 0.66 for Luminex and 0.97 for Southern blot. The correlation between Southern blot and Luminex was 0.65 in round 1 and 0.75 in round 2, and the relationship showed no evidence of non-linearity. Our results demonstrate that the Luminex assay is a valid and reproducible method for high-throughput TL measurement. The Luminex assay involves no DNA amplification, which may make Luminex an attractive alternative to PCR-based TL measurement.

  12. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

  13. A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

    PubMed

    Chan, E L; Sidaway, F; Horsman, G B

    1996-03-01

    The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

  14. Actual fusion efficiency in the lipid mixing assay - Comparison between nanodiscs and liposomes.

    PubMed

    François-Martin, Claire; Pincet, Frédéric

    2017-03-07

    Lipid exchange occurs between membranes during fusion or active lipid transfer. These processes are necessary in vivo for the homeostasis of the cell at the level of the membranes, the organelles and the cell itself. They are also used by the cell to interact with the surrounding medium. Several assays have been developed to characterize in vitro these processes on model systems. The most common one, relying on fluorescence dequenching, measures lipid mixing between small membranes such as liposomes or nanodiscs in bulk. Usually, relative comparisons of the rate of lipid exchange are made between measurements performed in parallel. Here, we establish a quantitative standardization of this assay to avoid any bias resulting from the temperatures, the chosen fluorescent lipid fractions and from the various detergents used to normalize the measurements. We used this standardization to quantitatively compare the efficiency of SNARE-induced fusion in liposome-liposome and liposome-nanodisc configurations having similar collision frequency. We found that the initial yield of fusion is comparable in both cases, 1 per 2-3 million collisions in spite of a much larger dequenching signal with nanodiscs. Also, the long-term actual fusion rate is slightly lower with nanodiscs than in the liposome-liposome assay.

  15. Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

    PubMed Central

    Oeyen, Merel; Schols, Dominique

    2017-01-01

    The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV) entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in in vitro experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of in vitro CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, i.e. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid) showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors. PMID:28410420

  16. Comparison of the cytotoxicity of high-level disinfectants by the MTT assay and direct contact assay.

    PubMed

    Ryu, Mizuyuki; Matsumura, Reiko; Quan, Glenlelyn; Furuta, Taro

    2013-01-01

    Most critical instruments are not designed for heat sterilization and autoclaving. These items are usually treated with chemical agents such as peracetic acid(PAA), glutaraldehyde (GA) and ortho-phthalaldehyde (OPA). MTT assay is often used to evaluate the in vitro cytotoxicity of these chemical agents. In this study, disinfectants were allowed to come in direct contact with cells. Their cytotoxicity was evaluated based on cell viability and adhesive properties. The results obtained from the direct contact method were compared with those obtained from the conventional MTT assay wherein the disinfectants were added into a nutrient medium. It was found that the two methods yielded very different results, especially when aldehyde- and halogen-containing disinfectants were tested, and that toxicity may be underestimated in the MTT assay. Hence, it can be assumed that the direct contact assay is more accurate when evaluating the cytotoxicity of residual chemicals. It was also observed that the cytotoxicity of PAA was lower than that of GA and OPA.

  17. Comparison of measles virus-specific antibody titres as measured by enzyme-linked immunosorbent assay and virus neutralisation assay.

    PubMed

    van den Hof, Susan; van Gageldonk-Lafeber, Arianne B; van Binnendijk, Robert S; van Gageldonk, Pieter G M; Berbers, Guy A M

    2003-10-01

    We assessed whether measles virus-specific antibody levels in the Dutch population as estimated by an enzyme-linked immunosorbent assay (ELISA) were comparable with estimates by virus neutralisation assay (NT), prompted by a relatively low ELISA seroprevalence in the 10-21-year-old group. We tested 791 sera from individuals aged 2-49 years both in ELISA and NT. Seroprevalence in the 10-21-year-old group was 93.4% (95% confidence interval (CI) 89.5-97.2%) in ELISA versus 97.2% (CI 94.7-99.6%) in NT. There was good agreement between NT and ELISA seroprevalences in the vaccinated 2-9-year-olds and the unvaccinated 22-49-year-olds.

  18. A standardized comparison of commercially available prion decontamination reagents using the Standard Steel-Binding Assay

    PubMed Central

    Edgeworth, Julie Ann; Sicilia, Anita; Linehan, Jackie; Brandner, Sebastian; Jackson, Graham S.; Collinge, John

    2011-01-01

    Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of mammals, such as variant Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. Prions pose significant public health concerns through contamination of blood products and surgical instruments, and can resist conventional hospital sterilization methods. Prion infectivity binds avidly to surgical steel and can efficiently transfer infectivity to a suitable host, and much research has been performed to achieve effective prion decontamination of metal surfaces. Here, we exploit the highly sensitive Standard Steel-Binding Assay (SSBA) to perform a direct comparison of a variety of commercially available decontamination reagents marketed for the removal of prions, alongside conventional sterilization methods. We demonstrate that the efficacy of marketed prion decontamination reagents is highly variable and that the SSBA is able to rapidly evaluate current and future decontamination reagents. PMID:21084494

  19. Interlaboratory comparison of the dicentric chromosome assay for radiation biodosimetry in mass casualty events.

    PubMed

    Wilkins, Ruth C; Romm, Horst; Kao, Tzu-Cheg; Awa, Akio A; Yoshida, Mitsuaki A; Livingston, Gordon K; Jenkins, Mark S; Oestreicher, Ursula; Pellmar, Terry C; Prasanna, Pataje G S

    2008-05-01

    This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose-effect calibration curve for the yield of dicentrics for (60)Co gamma rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + alphaD + betaD(2). For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.

  20. Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    PubMed

    Kösters, K; Riffelmann, M; Dohrn, B; von König, C H

    2000-05-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.

  1. A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.

    PubMed

    Wang, Deguo; Wang, Yongzhen; Xiao, Fugang; Guo, Weiyun; Zhang, Yongqing; Wang, Aiping; Liu, Yanhong

    2015-05-25

    Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145 and Streptococcus agalactiae. False-positive results were observed in all 12 in-house real-time LAMP assays, while all the negative controls of Isothermal Master Mix remained negative after amplification. The detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg, whereas the sensitivity of the commercialized kit for E. coli O145 was 100 pg. In conclusion, the 12 in-house real-time LAMP assays were impractical to use, while the commercialized kit Isothermal Master Mix was useful for the detection of most bacterial pathogens.

  2. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    PubMed

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng

    2016-05-01

    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  3. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  4. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  5. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY.
    MC Cardon, PC Hartig,LE Gray, Jr. and VS Wilson.
    U.S. EPA, ORD, NHEERL, RTD, Research Triangle Park, NC, USA.
    Typically, in vitro hazard assessments for ...

  6. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    EPA Science Inventory

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay
    Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. Wilson
    U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  7. Comparison of representative strains of infectious hematopoietic necrosis virus by serological neutralization and cross protection assays

    SciTech Connect

    Engelking, H.M.; Leong, J.C. ); Harry, J.B. )

    1991-05-01

    Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. An extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.

  8. Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison.

    PubMed

    Ruberu, Shryamalie R; Liu, Yun-Gang; Wong, Carolyn T; Perera, S Kusum; Langlois, Gregg W; Doucette, Gregory J; Powell, Christine L

    2003-01-01

    A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

  9. Comparison of the sensitivities of common in vitro and in vivo assays of estrogenic activity: application of chemical toxicity distributions.

    PubMed

    Dobbins, Laura L; Brain, Richard A; Brooks, Bryan W

    2008-12-01

    A number of contaminants in municipal effluent discharges are estrogen agonists to fish. Whereas several in vitro and in vivo techniques have been developed to assess the estrogenic activity of these compounds or ambient environmental samples, previous comparisons of the relative sensitivities of these approaches remain inconclusive. We employed a probabilistic hazard assessment approach using chemical toxicity distributions (CTDs) to perform a novel evaluation of relative sensitivities of six common in vitro and in vivo assays. We predicted that there was an 8.3% (human breast ademocarcinoma cell line, MCF-7, assay), 6.3% (yeast estrogen screen assay), or 1.9% (fish hepatocyte vitellogenin, VTG, assay) probability of detecting a compound in aquatic systems that will elicit an estrogenic response at concentrations at or below 0.1 microg/L, suggesting that the MCF-7 assay was the most sensitive in vitro assay evaluated in this study. The probabilities of eliciting the estrogenic response of VTG induction at a concentration less than 0.1 microg/L in rainbow trout, fathead minnow, and Japanese medaka were determined at 29.9, 26.2, and 18.8%, respectively. Thus, rainbow trout VTG induction was the most sensitive in vivo assay assessed. Subsequently, CTDs may provide a useful technique for hazard assessment of chemical classes for which exposure data are limited and for chemicals with common toxicological mechanisms and modes of action.

  10. Comparison of JEV neutralization assay using pseudotyped JEV with the conventional plaque-reduction neutralization test.

    PubMed

    Lee, Hee-Jung; Min, Kyung-Il; Park, Ki Hoon; Choi, Hyo Jung; Kim, Min-Kyoung; Ahn, Chi-Young; Hong, Young-Jin; Kim, Young Bong

    2014-05-01

    We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV-PV). JEV-PV-based neutralization assay offers several advantages compared with the current standard plaque-reduction neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque reduction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.

  11. Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity

    PubMed Central

    Jennings, Cheryl; Johnson, Victoria A.; Coombs, Robert W.; McKinnon, John E.; Bremer, James W.; Cobb, Bryan R.; Cloherty, Gavin A.; Mellors, John W.; Ribaudo, Heather J.

    2015-01-01

    Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice. PMID:26063861

  12. Preliminary report of the comparison of multiple non-destructive assay techniques on LANL Plutonium Facility waste drums

    SciTech Connect

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

    1999-03-01

    Prior to disposal, nuclear waste must be accurately characterized to identify and quantify the radioactive content. The DOE Complex faces the daunting task of measuring nuclear material with both a wide range of masses and matrices. Similarly daunting can be the selection of a non-destructive assay (NDA) technique(s) to efficiently perform the quantitative assay over the entire waste population. In fulfilling its role of a DOE Defense Programs nuclear User Facility/Technology Development Center, the Los Alamos National Laboratory Plutonium Facility recently tested three commercially built and owned, mobile nondestructive assay (NDA) systems with special nuclear materials (SNM). Two independent commercial companies financed the testing of their three mobile NDA systems at the site. Contained within a single trailer is Canberra Industries segmented gamma scanner/waste assay system (SGS/WAS) and neutron waste drum assay system (WDAS). The third system is a BNFL Instruments Inc. (formerly known as Pajarito Scientific Corporation) differential die-away imaging passive/active neutron (IPAN) counter. In an effort to increase the value of this comparison, additional NDA techniques at LANL were also used to measure these same drums. These are comprised of three tomographic gamma scanners (one mobile unit and two stationary) and one developmental differential die-away system. Although not certified standards, the authors hope that such a comparison will provide valuable data for those considering these different NDA techniques to measure their waste as well as the developers of the techniques.

  13. Measurement of urinary histamine: comparison of fluorometric and radioisotopic-enzymatic assay procedures

    SciTech Connect

    Warren, K.; Dyer, J.; Merlin, S.; Kaliner, M.

    1982-02-01

    Assessment of urinary histamine may prove useful in determining the role of histamine in human health and disease. Urinary histamine may be accurately estimated by a modified fluorometric assay employing diamine oxidase (DAO) digestion and cation-exchange chromatography. However, the radioisotopic-enzymatic assay is less expensive, easier to perform, and possibly more sensitive. Therefore the two procedures were compared. The radioenzyme assay was found to be affected by factors in urine (possibly salt concentrations) requiring extraction of histamine from urine by butanol-heptane. Moreover, it was found to be necessary to compare DAO-digested samples with undigested samples to accurately estimate histamine levels and to run the standard curve of histamine in DAO-digested urine. Even with these modifications, the radioenzyme assay was not as accurate as the fluorometric assay for urine samples having histamine values about 60 ng/ml. Therefore we recommend utilization of the modified fluorometric assay for the measurement of urinary histamine levels.

  14. Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals.

    PubMed Central

    Körner, Wolfgang; Vinggaard, Anne Marie; Térouanne, Béatrice; Ma, Risheng; Wieloch, Carise; Schlumpf, Margret; Sultan, Charles; Soto, Ana M

    2004-01-01

    We evaluated and compared four in vitro assays to detect androgen agonists and antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation assay (assay 1) with human mammary carcinoma cells stably transfected with human androgen receptor. The other laboratories used reporter gene assays, two based on stably transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells (assay 4), and the third based on transient transfection of Chinese hamster ovary cells (assay 3). Four laboratories received four coded compounds and two controls: two steroidal androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT), and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both compounds, the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol 10(-7) M to 4.7 times symbol 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed cytotoxicity for this compound at or above 1 times symbol 10(-5) M. In summary, all assays proved sensitive screening tools to detect and quantify androgen receptor-mediated androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of variation between 8 and 90%. PMID

  15. Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica

    PubMed Central

    Waters, Patrick; Reindl, Markus; Saiz, Albert; Schanda, Kathrin; Tuller, Friederike; Kral, Vlastimil; Nytrova, Petra; Sobek, Ondrej; Nielsen, Helle Hvilsted; Barington, Torben; Lillevang, Søren T; Illes, Zsolt; Rentzsch, Kristin; Berthele, Achim; Berki, Tímea; Granieri, Letizia; Bertolotto, Antonio; Giometto, Bruno; Zuliani, Luigi; Hamann, Dörte; van Pelt, E Daniëlle; Hintzen, Rogier; Höftberger, Romana; Costa, Carme; Comabella, Manuel; Montalban, Xavier; Tintoré, Mar; Siva, Aksel; Altintas, Ayse; Deniz, Günnur; Woodhall, Mark; Palace, Jacqueline; Paul, Friedemann; Hartung, Hans-Peter; Aktas, Orhan; Jarius, Sven; Wildemann, Brigitte; Vedeler, Christian; Ruiz, Anne; Leite, M Isabel; Trillenberg, Peter; Probst, Monika; Saschenbrecker, Sandra; Vincent, Angela; Marignier, Romain

    2016-01-01

    Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0–1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5–100%) of all 21 assays. The specificities (85.8–100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology. PMID:27113605

  16. Comparison of enzyme immunoassay–based assays for environmental Alternaria alternata

    PubMed Central

    Barnes, Charles; Portnoy, Jay; Sever, Michelle; Arbes, Samuel; Vaughn, Ben; Zeldin, Darryl C.

    2007-01-01

    Background Alternaria alternata–derived allergenic materials are causes of human disease. Several immunoassays exist to quantify these materials. Objective To compare methods for evaluating Alternaria content. Methods Four methods, including 1 monoclonal antibody (MAb)–based assay specific for recombinant Alt a 1, 1 MAb-based assay for chromatographically purified Alt a 1, 1 polyclonal antibody (PAb)–based assay for chromatographically purified Alt a 1, and 1 PAb-based assay for whole Alternaria extract, were evaluated. Environmental samples collected as part of the National Survey of Lead and Allergens in Housing were examined. Alternaria spore counts were determined in dust by observation. Results The MAb-based assay for recombinant Alt a 1 detected Alternaria in few samples (25%); the PAb-based assay for whole Alternaria proteins detected antigen in 97% of the samples. The PAb- and MAb-based assays for purified Alt a 1 detected antigen in 100% of the samples. There was a significant positive correlation between the 2 assays directed against purified Alt a 1. There was a positive correlation between the PAb-based assay for whole Alternaria and the PAb-based assay for Alt a 1. Nearly all the dust samples contained Alternaria spores, and there was a strong positive correlation between counts and all assays. Conclusion Because of the multifaceted nature of Alternaria, the disparities between methods for quantifying Alternaria, the cross-reactivity between fungal allergens, and the documented genetic promiscuity of this fungus, enzyme immunoassays using PAbs against a range of Alternaria proteins will probably produce the most reliable estimation of overall Alternaria exposure in house dust. PMID:17042141

  17. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

    PubMed Central

    Maslanka, S E; Gheesling, L L; Libutti, D E; Donaldson, K B; Harakeh, H S; Dykes, J K; Arhin, F F; Devi, S J; Frasch, C E; Huang, J C; Kriz-Kuzemenska, P; Lemmon, R D; Lorange, M; Peeters, C C; Quataert, S; Tai, J Y; Carlone, G M

    1997-01-01

    A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines

  18. Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses.

    PubMed

    Leonardi, Gary P; Wilson, Adele M; Zuretti, Alejandro R

    2013-05-01

    Sofia, a novel, fluorescent lateral-flow immunoassay was compared with two conventional colorimetric assays, Quickvue Influenza A+B and Directigen FLU A+B, to identify influenza viral antigen from patient nasopharyngeal specimens. A total of 118 frozen original influenza-positive specimens and 57 prospective specimens were examined. Using rt-PCR as a referee assay, sensitivity values (%) for influenza A/B of 80.0/74.8, 73.3/59.3 and 73.3/40.7 were obtained using the Sofia, Quickvue and Directigen assays, respectively. All assays demonstrated reduced sensitivity for influenza B as compared with influenza A virus. With respect to the Sofia assay, the sensitivity of influenza B for the Directigen assay was significantly diminished. False positive results were not observed in the Sofia and Directigen assays. The Quickvue assay produced 3 false-positive results (2 influenza A and 1 influenza B) resulting in a specificity (%) of 96 and 98 for influenza A and B, respectively. Cross-reactivity to other respiratory viruses was not observed among immunoassays. A sensitivity rank (highest to low) of rt-PCR>culture>Sofia>Quickvue>Directigen was established using dilutions of influenza A and B. Sofia provides enhanced sensitivity and objective result interpretation over conventional colorimetric immunoassays.

  19. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  20. A COMPARISON OF THREE ASSAY PROCEDURES FOR DETERMINING CHLORINE INACTIVATION OF WATERBORNE PATHOGENIC BACTERIA

    EPA Science Inventory

    One criterion on which chlorine treatment of water may be based is the concentration (C) in mg/l multiplied by the time (t) in min of exposure or Ct values. We compared different Ct values on waterborne pathogenic bacteria by cultural assay for viability and 2 assays that mea...

  1. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  2. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  3. Chromogenic assay of human coagulation factor VIII: statistical comparison of 2 working dilution procedures.

    PubMed

    Alonso, C; Gonzalez, A; Frutos, G

    2005-08-01

    The effect of 2 different practices for preparation of working dilutions in the chromogenic substrate method for potency assay of factor VIII was evaluated. In this study the potency of several concentrate materials was shown to be statistically equivalent, whether performing the assay with independent or serial working dilutions.

  4. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  5. Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

  6. A COMPARISON OF THREE ASSAY PROCEDURES FOR DETERMINING CHLORINE INACTIVATION OF WATERBORNE PATHOGENIC BACTERIA

    EPA Science Inventory

    One criterion on which chlorine treatment of water may be based is the concentration (C) in mg/l multiplied by the time (t) in min of exposure or Ct values. We compared different Ct values on waterborne pathogenic bacteria by cultural assay for viability and 2 assays that mea...

  7. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products

    PubMed Central

    Carballo, José Luis; Hernández-Inda, Zaira L; Pérez, Pilar; García-Grávalos, María D

    2002-01-01

    Background The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity. Material and Methods The bioactivity of the isopropanolic (2-PrOH) extracts of 14 species of marine invertebrates and 6 species of macroalgae was evaluated with the shrimp lethality assay (lethality assay), as well as with another assay based on the inhibition of hatching of the cyst (hatchability assay). The extracts were also assayed for cytotoxicity against two human cell lines, lung carcinoma A-549 and colon carcinoma HT-29, in order to assess the sensitivity of the shrimp assays to detect cytotoxic activity. Results Two sponges (Hyatella sp, Dysidea sp.), two gorgonians (Pacifigorgia adamsii, Muricea sp.), one tunicate (Polyclinum laxum), and three echinoderms (Holothuria impatiens, Pseudoconus californica and Pharia pyramidata) showed a strong cytostatic (growth inhibition) and cytotoxic effect. The hatchability assay showed a strong activity in 4 of the species active against the two human cell lines tested (Hyatella sp, Dysidea sp., Pacifigorgia adamsii and Muricea sp.), and the lethality assay also showed a high lethality in 4 of them (Pacifigorgia adamsii, Muricea sp., Polyclinum laxum, and Pharia pyramidata). Each bioassay detected activity in 50% of the species that were considered active against the two human cell lines tested. However, the simultaneous use of both bioassays increased the percentage to 75%. Conclusions Our results seem consistent with the correlation previously established between cytotoxicity and brine shrimp lethality in plant extracts. We suggest using both bioassays simultaneously to test natural marine products for pharmacological

  8. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products.

    PubMed

    Carballo, José Luis; Hernández-Inda, Zaira L; Pérez, Pilar; García-Grávalos, María D

    2002-09-23

    The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity. The bioactivity of the isopropanolic (2-PrOH) extracts of 14 species of marine invertebrates and 6 species of macroalgae was evaluated with the shrimp lethality assay (lethality assay), as well as with another assay based on the inhibition of hatching of the cyst (hatchability assay). The extracts were also assayed for cytotoxicity against two human cell lines, lung carcinoma A-549 and colon carcinoma HT-29, in order to assess the sensitivity of the shrimp assays to detect cytotoxic activity. Two sponges (Hyatella sp, Dysidea sp.), two gorgonians (Pacifigorgia adamsii, Muricea sp.), one tunicate (Polyclinum laxum), and three echinoderms (Holothuria impatiens, Pseudoconus californica and Pharia pyramidata) showed a strong cytostatic (growth inhibition) and cytotoxic effect. The hatchability assay showed a strong activity in 4 of the species active against the two human cell lines tested (Hyatella sp, Dysidea sp., Pacifigorgia adamsii and Muricea sp.), and the lethality assay also showed a high lethality in 4 of them (Pacifigorgia adamsii, Muricea sp., Polyclinum laxum, and Pharia pyramidata). Each bioassay detected activity in 50% of the species that were considered active against the two human cell lines tested. However, the simultaneous use of both bioassays increased the percentage to 75%. Our results seem consistent with the correlation previously established between cytotoxicity and brine shrimp lethality in plant extracts. We suggest using both bioassays simultaneously to test natural marine products for pharmacological activity.

  9. Comparison of three different anti-Xa assays in major orthopedic surgery patients treated with fondaparinux.

    PubMed

    Ikejiri, Makoto; Wada, Hideo; Yamaguchi, Toshio; Miyazaki, Shinichi; Hasegawa, Masahiro; Wakabayashi, Hiroki; Asanuma, Kunihiro; Sakaguchi, Akane; Matsumoto, Takeshi; Ohishi, Kohshi; Fujimoto, Naoki; Yamada, Norikazu; Ito, Masaaki; Katayama, Naoyuki; Sudo, Akihiro

    2016-05-01

    Anti-Xa assays are useful for monitoring the effects of selective anti-Xa drugs, such as fondaparinux, in the prophylaxis of deep vein thrombosis. In the present study, anti-Xa activity was measured using three different assays, Testzym(®) Heparin S, STA(®)-Liquid Anti-Xa and HemosIL(®) Liquid Heparin. Anti-Xa activity in each assay gradually increased from day one after administration to day eight, and still remained on day 15. Although there were significant differences in anti-Xa activity among the three assays, the activity showed significant correlation across assays. There were no significant differences in the anti-Xa activity between patients with and without DVT or between patients with and without massive bleeding on day one before and after administration, day four, day eight and day 15. Anti-Xa activity in each assay was weakly correlated with antithrombin (AT) activity. The AT activity in patients were significantly higher on days four, eight and 15 compared with day one before and after administration, suggesting that AT activity increases following the administration of fondaparinux. The three anti-Xa assay kits tested are useful for monitoring fondaparinux treatment in orthopedic surgery patients.

  10. Measurement of urinary histamine: comparison of fluorometric and radioisotopic-enzymatic assay procedures

    SciTech Connect

    Warren, K.; Dyer, J.; Merlin, S.; Kaliner, M.

    1982-02-01

    Assessment of urinary histamine may prove useful in determining the role of histamine in human health and disease. Urinary histamine may be accurately estimated by a modified fluorometric assay employing diamine oxidase (DAO) digestion and cation-exchange chromatography. Normal urine histamine values obtained by this assay are: arithmetic means (+/- SEM), 8.6 +/- 0.6 ng/ml and 10.5 +/- 0.7 micrograms/24 hr; geometric means (+/- SEM), 6.2 +/- 1.1 ng/ml and 10.0 +/- 1.3 micrograms/24 hr. However, the radioisotopic-enzymatic assay is less expensive, easier to perform, and possibly more sensitive. Therefore the two procedures were compared. The radioenzyme assay was found to be affected by factors in urine (possibly salt concentrations) requiring extraction of histamine from urine by butanol-heptane. Moreover, it was found to be necessary to compare DAO-digested samples with undigested samples to accurately estimate histamine levels and to run the standard curve of histamine in DAO-digested urine. Even with these modifications, the radioenzyme assay was not as accurate as the fluorometric assay for urine samples having histamine values about 60 ng/ml. Therefore we recommend utilization of the modified fluorometric assay for the measurement of urinary histamine levels.

  11. Serum Sulfonamide Concentration After Oral Administration: Comparison of Results of Chemical Assay with Two Bioassay Techniques

    PubMed Central

    Berman, Harris A.; Barza, Michael; Weinstein, Louis

    1972-01-01

    Sulfonamide concentrations were studied in 210 serum samples from 10 volunteers after ingestion of sulfacytine, sulfisoxazole, and sulfadiazine. Results obtained by chemical assay were compared with bioassay values determined by two techniques: twofold broth dilution and agar diffusion. Although the correlation for individual samples was rather poor for the twofold broth-dilution method, the agar-diffusion bioassay correlated well with chemical determinations. The agar-diffusion assay tended to “underread” the chemical assay by an amount characteristic of each drug. PMID:5045472

  12. Comparison of cross-sectional HIV incidence assay results from dried blood spots and plasma

    PubMed Central

    Schlusser, Katherine E.; Pilcher, Christopher; Kallas, Esper G.; Santos, Breno R.; Deeks, Steven G.; Facente, Shelley; Keating, Sheila M.; Busch, Michael P.; Murphy, Gary; Welte, Alex; Quinn, Thomas; Eshleman, Susan H.

    2017-01-01

    Background Assays have been developed for cross-sectional HIV incidence estimation using plasma samples. Large scale surveillance programs are planned using dried blood spot (DBS) specimens for incidence assessment. However, limited information exists on the performance of HIV cross-sectional incidence assays using DBS. Methods The assays evaluated were: Maxim HIV-1 Limiting Antigen Avidity EIA (LAg-Avidity), Sedia HIV-1 BED-Capture EIA (BED-CEIA), and CDC modified BioRad HIV-1/2 Plus O Avidity-based Assay (CDC-BioRad Avidity) using pre-determined cutoff values. 100 matched HIV-1 positive plasma and DBS samples, with known duration of infection, from the Consortium for the Evaluation and Performance of HIV Incidence Assays repository were tested. All assays were run in duplicate. To examine the degree of variability within and between results for each sample type, both categorical and continuous results were analyzed. Associations were assessed with Bland Altman, R2 values and Cohen’s kappa coefficient (ĸ). Results Intra-assay variability using the same sample type was similar for all assays (R2 0.96 to 1.00). The R2 values comparing DBS and plasma results for LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity were 0.96, 0.94, and 0.84, respectively. The concordance and ĸ values between DBS and plasma for all three assays were >87% and >0.64, respectively. The Bland-Altman analysis showed significant differences between plasma and DBS samples. For all three assays, a higher number of samples were classified as recent infections using DBS samples. Conclusions DBS and plasma sample results were highly correlated. However, when compared to plasma, each assay performed somewhat differently in DBS at the lower and higher ends of the dynamic range. DBS samples were more likely to be classified as recently infected by all three assays, which may lead to overestimation of incidence in surveys using performance criteria derived for plasma samples. PMID:28231277

  13. [Comparison of two commercial molecular assays for quantitative measurement of hepatitis B viral DNA].

    PubMed

    Zalewska, Małgorzata; Domagała, Małgorzata; Gładysz, Andrzej

    2003-12-01

    The detection and quantification of hepatitis B virus (HBV) genomes appear to be the most reliable method for monitoring HBV infection and assessing responses to antiviral treatment. For quantitative determination of HBV viremia molecular biology-based assays are used. The aim of this study was to compare and evaluate the performance of two HBV DNA detection and quantification commercial assays: hybrid-capture Digene Hybrid Capture HBV DNA assays and based on competitive polymerase chain reaction (PCR) Cobas Amplicor HBV Monitor Roche Diagnostics. Reproducibility, linearity, sensitivity were determined with 2-fold dilution series of high-titers samples and with 113 sera samples from patients with chronic HBV infection. Within-run and between-run coefficients of variation ranged from 2.4-9.7% for hybrid-capture and from 3.7-15% for PCR-based Monitor. The hybrid-capture and PCR Monitor assays appeared to be linear throughout their range of quantification: 5-2000 pg/ml and 2 x 10(2)-2 x 10(5) copies/ml respectively. The HBV DNA units used in the two assays were not comparable. Hybrid-capture and Monitor gave concordant results with 87 (82.1%) of 106 samples. The assays were both positive with 79 (74.5%) samples and were negative in 7 (7.5%) cases. Hybrid-capture and Monitor gave discordant results in 17 (17.9%) cases. The Monitor Assay was positive in 13 (61.9%) of the 21 samples negative in hybrid-capture. The competitive PCR-based Monitor assay appear to be significantly more sensitive but slightly less reproducible than the hybrid-capture. In the group of patients with seroconversion to anti-HBe PCR method should be used for measurement of viral load. In the presence of HBe antigen concentration of HBV DNA may be tested by hybrid-capture assay. Also these two assays may be used in complementary fashion in the management of HBV infected patients. It seems reasonable to use a hybrid-capture assay first, because its linear range of quantification is extended to high

  14. Comparison of the ames assay and mutatox in assessing the mutagenic potential of contaminated dredged material. Final technical report

    SciTech Connect

    Jarvis, A.S.

    1995-04-01

    The Ames assay and Mutatox were evaluated to compare their ability to identify the genotoxic potential of dredged sediments. The Ames assay has been used extensively in the testing of environmental contaminants. Mutatox, a bacterial bioluminescence test, was developed as a genotoxicity bioassay. Ten sediments with varying degrees of contamination were soxhlet extracted. These extracts were divided into crude and clean samples. Cleaned samples were prepared using silica-gel chromatography resulting in 20 extract samples. Both the Ames test (TA98 and TAl00) and Mutatox were conducted with and without S9 metabolic activation. TA98+S9 and TA1OO+S9 indicated a positive mutagenic response in 80 and 50 percent, respectively, of the sediment extracts. Half of the extracts indicated a positive mutagenic response with TA98-S9, while only 10 percent did so with TAlOO-S9. Mutatox indicated a positive mutagenic response with S9 activation in 75 percent of the extracts and no mutagenic response in any of the sediment extracts without metabolic activation. In a side-by-side comparison of the Ames assay (TA98+S9) and Mutatox, 80 percent of the sediment extracts had similar responses, both positive and negative. Fifty percent of the sediment extracts had similar responses when tested with TAlOO+S9 and Mutatox. Mutatox compared favorably with the Ames assay and shows promise as a screening tool to assess sediment genotoxicity when used with Ames assay as a confirmation.

  15. Multicenter Comparison Study of both Analytical and Clinical Performance across Four Roche Hepatitis C Virus RNA Assays Utilizing Different Platforms.

    PubMed

    Vermehren, Johannes; Stelzl, Evelyn; Maasoumy, Benjamin; Michel-Treil, Veronique; Berkowski, Caterina; Marins, Ed G; Paxinos, Ellen E; Marino, Enrique; Wedemeyer, Heiner; Sarrazin, Christoph; Kessler, Harald H

    2017-04-01

    The efficacy of antiviral treatment for chronic hepatitis C virus (HCV) infection is determined by measuring HCV RNA at specific time points throughout therapy using highly sensitive and accurate HCV RNA assays. This study compared the performances of two recently developed real-time PCR HCV RNA assays, cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and cobas HCV for use on the cobas 4800 system (cobas 4800 HCV), with those of two established assays, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2 (CAP/CTM v2) and the Cobas TaqMan HCV test, version 2 for use with the High Pure system (HPS/CTM v2). The limits of detection (LODs) and linearity at lower concentrations (5 to 1000 IU/ml) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1 to 4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1 to GT 4. Results from cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (<0.3 log10 IU/ml difference between expected and observed results) with LODs of 8.2 IU/ml and 11.7 IU/ml, respectively, for GT 1. The new assays showed excellent agreement with results from CAP/CTM v2 and HPS/CTM v2 in samples with quantifiable viral loads. The concordances using the 6 million IU/ml cutoff were high among all four assays (90 to 94%). In conclusion, the cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear and correlate well with the established Roche assays used in clinical practice. Copyright © 2017 Vermehren et al.

  16. Multicenter Comparison Study of both Analytical and Clinical Performance across Four Roche Hepatitis C Virus RNA Assays Utilizing Different Platforms

    PubMed Central

    Vermehren, Johannes; Stelzl, Evelyn; Maasoumy, Benjamin; Michel-Treil, Veronique; Berkowski, Caterina; Marins, Ed G.; Paxinos, Ellen E.; Marino, Enrique; Wedemeyer, Heiner; Sarrazin, Christoph

    2017-01-01

    ABSTRACT The efficacy of antiviral treatment for chronic hepatitis C virus (HCV) infection is determined by measuring HCV RNA at specific time points throughout therapy using highly sensitive and accurate HCV RNA assays. This study compared the performances of two recently developed real-time PCR HCV RNA assays, cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and cobas HCV for use on the cobas 4800 system (cobas 4800 HCV), with those of two established assays, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2 (CAP/CTM v2) and the Cobas TaqMan HCV test, version 2 for use with the High Pure system (HPS/CTM v2). The limits of detection (LODs) and linearity at lower concentrations (5 to 1000 IU/ml) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1 to 4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1 to GT 4. Results from cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (<0.3 log10 IU/ml difference between expected and observed results) with LODs of 8.2 IU/ml and 11.7 IU/ml, respectively, for GT 1. The new assays showed excellent agreement with results from CAP/CTM v2 and HPS/CTM v2 in samples with quantifiable viral loads. The concordances using the 6 million IU/ml cutoff were high among all four assays (90 to 94%). In conclusion, the cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear and correlate well with the established Roche assays used in clinical practice. PMID:28122870

  17. Evaluation and comparison of two assays for detection of immunity to rubella infection.

    PubMed Central

    Brody, J P; Binkley, J H; Harding, S A

    1979-01-01

    Two commercially available rapid screening tests, Rubacell (Abbott Laboratories; passive hemagglutination) and FIAX (International Diagnostic Technology; indirect immunofluorescence) were compared with a standard hemagglutination inhibition assay for detection of immunity to rubella infection. In tests of approximately 300 sera, both rapid assays were specific and sensitive and showed a high predictive value of a positive result. Within-run reproducibility studies were excellent for both tests; however, Rubacell was superior to FIAX with respect to time-cost analysis. PMID:397225

  18. Multicenter Comparison of PCR Assays for Detection of Human Herpesvirus 6 DNA in Serum▿

    PubMed Central

    Flamand, Louis; Gravel, Annie; Boutolleau, David; Alvarez-Lafuente, Roberto; Jacobson, Steve; Malnati, Mauro S.; Kohn, Debra; Tang, Yi-Wei; Yoshikawa, Tetsushi; Ablashi, Dharam

    2008-01-01

    Human herpesvirus 6 (HHV-6) is a ubiquitous virus with which infections have been associated with pathologies ranging from delayed bone marrow engraftment to a variety of neurological diseases. The lack of a standardized assay that can be used to detect and estimate HHV-6 DNA contents in various clinical specimens can lead and has led to discordant results among investigators and on the potential association of HHV-6 to diseases. To identify the most reliable and sensitive assays, an identical set of 11 coded serum samples spiked with various quantities of the HHV-6A variant (range, 4 to 400,000 genome copies/ml) was sent to eight independent laboratories around the world. Each laboratory was asked to estimate the HHV-6 DNA content by use of its own protocols and assays. Among the various assays, three TaqMan-based real-time PCR assays yielded quantities that were closest to the quantity of HHV-6 that had been spiked. To provide better homogeneity between the results from the different laboratories working on HHV-6, we propose that investigators interested in quantifying HHV-6 in clinical samples adopt one of these assays. PMID:18550745

  19. A comparison of the rabbit scarification technique with titrations in cell cultures for the potency assay of smallpox vaccine.

    PubMed

    KOLB, R W; CUTCHINS, E C; JONES, W P; AYLOR, H T

    1961-01-01

    Effective tissue culture methods have been developed for the titration of smallpox vaccines, and it seemed appropriate to study their application as methods for assaying the potency of commercial smallpox vaccines. Thirty-four commercial smallpox vaccines were assayed for potency by titration in both rabbit and monkey kidney cell test systems and by the traditional rabbit scarification method.Approximately 85% of the vaccines showed acceptable potency by the rabbit scarification method. Comparative studies between all three test systems showed that consistent results could be obtained with 79% of all vaccines tested. A similar degree of correlation with the same vaccines was shown by a direct comparison of infectivity titres between the two animal tissue cell systems.Certain discrepancies in the potency of individual vaccines and difficulties inherent in cell culture assay were noted. In view of these deficiencies, it was concluded that further comparison of the rabbit scarification and cell culture methods with vaccines of borderline potency should be studied, and that, until the properties of smallpox vaccines have been compared in the rabbit test, cell culture and man, the cell culture methods should be neither accepted nor rejected.

  20. A comparison of the rabbit scarification technique with titrations in cell cultures for the potency assay of smallpox vaccine*

    PubMed Central

    Kolb, Robert W.; Cutchins, Ernest C.; Jones, William P.; Aylor, Harry T.

    1961-01-01

    Effective tissue culture methods have been developed for the titration of smallpox vaccines, and it seemed appropriate to study their application as methods for assaying the potency of commercial smallpox vaccines. Thirty-four commercial smallpox vaccines were assayed for potency by titration in both rabbit and monkey kidney cell test systems and by the traditional rabbit scarification method. Approximately 85% of the vaccines showed acceptable potency by the rabbit scarification method. Comparative studies between all three test systems showed that consistent results could be obtained with 79% of all vaccines tested. A similar degree of correlation with the same vaccines was shown by a direct comparison of infectivity titres between the two animal tissue cell systems. Certain discrepancies in the potency of individual vaccines and difficulties inherent in cell culture assay were noted. In view of these deficiencies, it was concluded that further comparison of the rabbit scarification and cell culture methods with vaccines of borderline potency should be studied, and that, until the properties of smallpox vaccines have been compared in the rabbit test, cell culture and man, the cell culture methods should be neither accepted nor rejected. PMID:14457962

  1. Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions.

    PubMed Central

    Takimoto, S; Grandien, M; Ishida, M A; Pereira, M S; Paiva, T M; Ishimaru, T; Makita, E M; Martinez, C H

    1991-01-01

    Nasopharyngeal secretions obtained from 94 children with acute respiratory illness were examined for the presence of respiratory syncytial virus (RSV), adenovirus, and influenza virus type A by virus culturing (virus isolation technique [VIT]), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Similar results were obtained in at least two tests for RSV, influenza virus type A, and adenovirus in 92 (97.9%), 88 (93.6%), and 88 (93.6%) cases, respectively. Both rapid virus detection methods showed good specificity for the diagnosis of these virus infections (greater than or equal to 90.7%) and were more sensitive than was VIT for RSV detection. In a more accurate statistical analysis, the indexes of agreement between VIT and ELISA were substantial for RSV (kappa = 0.69; zeta = 5.5; P less than 0.0001), influenza virus type A (kappa = 0.67; zeta = 5.3; P less than 0.0001), and adenovirus (kappa = 0.71; zeta = 6.0; P less than 0.0001), while it was almost perfect for RSV when ELISA was compared with IFA (kappa = 0.88; zeta = 5.7; P less than 0.0001). Although the observed agreement was good in the comparison of these two tests for these three viruses (89%0, the indexes of agreement were moderate in the comparison of IFA and VIT for RSV (K = 0.55; Z = 2.0; P < 0.05), influenza virus type A (K = 0.42; Z = 9.7; P < 0.0001), and adenovirus (K = 0.41; Z = 6.5; P < 0.0001) and of ELISA and IFA for influenza virus type A (K = 0.55; Z = 7.0; P < 0.0001) and adenovirus (K = 0.59; Z = 6.8; P < 0.0001). All of the statistical evaluations demonstrated better agreement between ELISA and VIT for influenza virus type A and adenovirus. PMID:2037663

  2. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride.

    PubMed

    Fotakis, George; Timbrell, John A

    2006-01-05

    The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events. Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0-300 microM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay. In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl(2) for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl(2) exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl(2), toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay. In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.

  3. Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

    PubMed

    Plappert-Helbig, Ulla; Guérard, Melanie

    2015-12-01

    To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories.

  4. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

    PubMed

    Keid, L B; Soares, R M; Vasconcellos, S A; Salgado, V R; Megid, J; Richtzenhain, L J

    2010-07-17

    The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

  5. An enzymatic method for assaying sulbactam in human serum: comparison with high performance liquid chromatography.

    PubMed

    Sotto, A; Peray, P; Geny, F; Brunschwig, C; Carrière, C; Galtier, M; Ramuz, M; Jourdan, J

    1995-03-01

    An enzymatic method using nitrocefin as substrate was developed to assay sulbactam in human serum. Serum containing sulbactam was incubated with purified titrated TEM-1 beta-lactamase and nitrocefin was then added to the mixture to determine the remaining beta-lactamase activity and consequently the concentration of sulbactam. Assays were carried out on five patients with pulmonary infections receiving sulbactam plus amoxycillin iv. The values for serum sulbactam concentrations determined by the enzymatic method were compared with those determined by high performance liquid chromatography (HPLC). The correlation coefficient was 0.990 for serum sulbactam concentrations below 15 mg/L.

  6. Comparison between a second and a third generation parathyroid hormone assay in hemodialysis patients.

    PubMed

    Gannagé-Yared, Marie-Hélène; Farès, Chirine; Ibrahim, Tony; Rahal, Zeina Abou; Elias, Michele; Chelala, Dania

    2013-10-01

    Third generation parathyroid hormone (PTH) assays are new generation assays that do not recognize the PTH7-84 fragment whereas second generation assays detect both PTH1-84 and PTH7-84 fragments. Despite the excellent correlation between both assays in chronic renal failure (CRF) subjects, the mean PTH levels are typically 50% lower with the third compared to the second generation assays. The assessment of third generation PTH assays has not been extensively studied in hemodialysis subjects. The purpose of our study was to compare a third generation PTH assay to a second generation one in a population of hemodialysis subjects. 92 haemodialysis subjects (36 women and 56 men) with a mean age of 67±12.9 years were included in this study. Anthropometric and clinical parameters (Body Mass Index (BMI) and blood pressure) were measured. Second and third generation PTH assays (Cis biomedical and Diasorin respectively) were performed in each subject. In addition, the following biochemical tests were measured: 25-hydroxyvitamin D (25-(OH)D), 1,25-hydroxyvitamin D (1,25-(OH)2D), crosslaps and alkaline phosphatase. The mean second and third generation PTHs are respectively 211±205 pg/ml and 151±164 pg/ml. The mean third generation PTH values are 28.4% lower compared to the second generation ones. Both methods are strongly correlated (r=0.923, p<0.001). This correlation persisted without any significant difference after controlling for gender, age, BMI and Blood Pressure. However, the difference between both methods increases when baseline PTH increases. Each of the second and third generation method is significantly correlated with hemodialysis duration (p<0.01), crosslaps (p<0.001), alkaline phosphatase (p<0.05), but not with age, BMI, Blood Pressure, 25-(OH)D or 1,25-(OH) 2D levels. Our results show that both second and third generation PTH methods are strongly correlated in hemodialysis patients mainly when PTH values are low. However, the difference between both methods

  7. A European multicentre study on the comparison of HCV viral loads between VERIS HCV assay and COBAS(®) TaqMan(®) HCV Test and RealTime HCV Assay.

    PubMed

    Braun, Patrick; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Micheli, Valeria; Sauné, Karine; Trimoulet, Pascale; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-05-01

    Beckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Molecular Diagnostic System(¥) for HCV viral load monitoring. Evaluate the clinical performance of the new quantitative VERIS HCV Assay. Comparison was performed on 279 plasma specimens from HCV infected patients tested with the VERIS HCV Assay and COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test and 369 specimens tested with the VERIS HCV Assay and RealTime HCV Assay. Patient monitoring sample results from four time points were also compared. The average bias between the VERIS HCV Assay and the COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test was 0.04 log10IU/mL, while between the VERIS HCV Assay and the RealTime HCV Assay average bias was 0.21 log10IU/mL. Bias, however, was not consistent across the measuring range. Analysis at the lower end of quantification levels 50, 100, and 1000IU/mL showed a predicted bias for VERIS HCV Assay versus COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test between -0.42 and -0.22 log10IU/mL and for VERIS HCV Assay versus RealTime HCV Assay between 0.00 and 0.13 log10IU/mL. Patient monitoring of HCV viral load over time demonstrated similar levels between VERIS HCV Assay results and COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test (52 samples from 13 patients) and RealTime HCV Assay (112 samples from 28 patients). VERIS HCV Assay for use on the DxN VERIS Molecular Diagnostic System represents a reliable new tool for easy sample to result HCV RNA viral load monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins

    DTIC Science & Technology

    2016-04-01

    assay HIV human immunodeficiency virus LPS lipopolysaccharide NIDA National Institute on Drug Abuse PE phycoerythrin TBI traumatic brain injury...Whitaker Human Research and Engineering Directorate, ARL Approved for public release; distribution is unlimited. FOR OFFICIAL...SUPPLEMENTARY NOTES 14. ABSTRACT The complex injury cascades associated with mild traumatic- brain injury partly involve inflammation in the brain that

  9. Antioxidant Generation during Coffee Roasting: A Comparison and Interpretation from Three Complementary Assays.

    PubMed

    Opitz, Sebastian E W; Smrke, Samo; Goodman, Bernard A; Keller, Marco; Schenker, Stefan; Yeretzian, Chahan

    2014-11-12

    Coffee is a major source of dietary antioxidants; some are present in the green bean, whereas others are generated during roasting. However, there is no single accepted analytical method for their routine determination. This paper describes the adaption of three complementary assays (Folin-Ciocalteu (FC), ABTS and ORAC) for the routine assessment of antioxidant capacity of beverages, their validation, and use for determining the antioxidant capacities of extracts from coffee beans at different stages in the roasting process. All assays showed a progressive increase in antioxidant capacity during roasting to a light roast state, consistent with the production of melanoidins having a higher antioxidant effect than the degradation of CGAs. However, the three assays gave different numbers for the total antioxidant capacity of green beans relative to gallic acid (GA), although the range of values was much smaller when chlorogenic acid (CGA) was used as reference. Therefore, although all three assays indicated that there was an increase in antioxidant activity during coffee roasting, and the large differences in responses to GA and CGA illustrate their different sensitivities to different types of antioxidant molecule.

  10. GENOTOXICITY OF TEN CIGARETTE SMOKE CONDENSATES IN FOUR TEST SYSTEMS: COMPARISONS BETWEEN ASSAYS AND CONDENSATES

    EPA Science Inventory

    What is the study?
    This the first assessment of a set of cigarette smoke condensates from a range of cigarette types in a variety (4) of short-term genotoxicity assays.
    Why was it done?
    No such comparative study of cigarette smoke condensates has been reported. H...

  11. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  12. Antioxidant Generation during Coffee Roasting: A Comparison and Interpretation from Three Complementary Assays

    PubMed Central

    Opitz, Sebastian E. W.; Smrke, Samo; Goodman, Bernard A.; Keller, Marco; Schenker, Stefan; Yeretzian, Chahan

    2014-01-01

    Coffee is a major source of dietary antioxidants; some are present in the green bean, whereas others are generated during roasting. However, there is no single accepted analytical method for their routine determination. This paper describes the adaption of three complementary assays (Folin-Ciocalteu (FC), ABTS and ORAC) for the routine assessment of antioxidant capacity of beverages, their validation, and use for determining the antioxidant capacities of extracts from coffee beans at different stages in the roasting process. All assays showed a progressive increase in antioxidant capacity during roasting to a light roast state, consistent with the production of melanoidins having a higher antioxidant effect than the degradation of CGAs. However, the three assays gave different numbers for the total antioxidant capacity of green beans relative to gallic acid (GA), although the range of values was much smaller when chlorogenic acid (CGA) was used as reference. Therefore, although all three assays indicated that there was an increase in antioxidant activity during coffee roasting, and the large differences in responses to GA and CGA illustrate their different sensitivities to different types of antioxidant molecule. PMID:28234339

  13. A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays

    PubMed Central

    Polacchini, Alessio; Metelli, Giuliana; Francavilla, Ruggiero; Baj, Gabriele; Florean, Marina; Mascaretti, Luca Giovanni; Tongiorgi, Enrico

    2015-01-01

    Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKineTM, Promega-Emax®, R&D-System-Quantikine®) and one multiplexing assay (Millipore-Milliplex®). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications. PMID:26656852

  14. Comparison of two commercial radial immunodiffusion assays for detection of bovine immunoglobulin G in newborn calves.

    PubMed

    Ameri, Mehrdad; Wilkerson, Melinda J

    2008-05-01

    Bovine failure of passive transfer (FPT), defined as inadequate transfer of colostral immunoglobulins from the dam to the calf, has been associated with increased risk in neonatal mortality. Currently, radial immunodiffusion (RID) assay is considered to be the gold standard in determining FPT in serum samples from calves. There are 2 commercial RID assays routinely used for serodiagnosis of FPT in calves: VET-RID and SRID. Discrepancies between results of these RID assays were observed in the authors' laboratory. The objective of this study was to compare 2 commercial RID assays by testing a paired panel of 30 blood samples collected from newborn Holsteins at birth before, and 24 hr after, ingestion of colostrum, a commercial bovine reference serum, and a panel of different concentrations of 2 purified bovine immunoglobulin G (IgG) products. Overall, the results of this study showed a high level of discrepancy and poor agreement between the 2 RID kits. The interassay precision study revealed lower between-run coefficients of variation for the VET-RID kit compared with the SRID kit. The spiking and recovery study using purified bovine IgG products demonstrated that the VET-RID kit more closely approximates the expected concentrations of the purified bovine IgG products, whereas the SRID kit consistently overestimates the concentration of purified bovine IgG products. It was concluded that this may be due to inaccuracies in the internal standards of the SRID kit.

  15. GENOTOXICITY OF TEN CIGARETTE SMOKE CONDENSATES IN FOUR TEST SYSTEMS: COMPARISONS BETWEEN ASSAYS AND CONDENSATES

    EPA Science Inventory

    What is the study?
    This the first assessment of a set of cigarette smoke condensates from a range of cigarette types in a variety (4) of short-term genotoxicity assays.
    Why was it done?
    No such comparative study of cigarette smoke condensates has been reported. H...

  16. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  17. Genetic comparison of Brucella canis isolates by the MLVA assay in South Korea.

    PubMed

    Kang, Sung-Il; Heo, Eun Jeong; Cho, Donghee; Kim, Jong Wan; Kim, Ji-Yeon; Jung, Suk Chan; Her, Moon

    2011-06-01

    The multiple-locus VNTR analysis (MLVA) assay is a method frequently employed as a molecular epidemiological tool for Brucella genetic fingerprinting. The purpose of this study was to assess the genotyping of 77 B. canis isolates from 14 different dog breeding farms in Korea by the MLVA assay and to compare the epidemiological relationships between the Korean isolates and foreign ones. Simpson's diversity index for 17 loci showed a range from 0 to 0.846 in 77 B. canis isolates. B. canis isolates in Korea were observed to have high genetic diversity at the most variable loci and were divided into 30 distinct genotypes by phylogenetic analysis. Some B. canis isolates were closely related to previously typed isolates in other countries. The MLVA assay can be helpful to analyze the epidemiological correlation of B. canis isolates in domestic pet animals and to track the geographic origin by comparing the genetic patterns with foreign isolates. Therefore, the MLVA assay will be useful as a tool for control and preventive measures of canine brucellosis.

  18. Comparison of five commercial anti-tetanus toxoid immunoglobulin G enzyme-linked immunosorbent assays.

    PubMed

    Perry, A L; Hayes, A J; Cox, H A; Alcock, F; Parker, A R

    2009-12-01

    Five commercially available enzyme-linked immunosorbent assays for the measurement of anti-tetanus toxoid immunoglobulin G (IgG) antibodies were evaluated for performance. The data suggest that there are manufacturer-dependent differences in sensitivity and accuracy for the determination of tetanus toxoid IgG antibodies that could result in different diagnostic interpretations.

  19. Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

    PubMed

    Wu, Nan; Dacres, Helen; Anderson, Alisha; Trowell, Stephen C; Zhu, Yonggang

    2014-01-01

    Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now. We used a thrombin bioprobe based on a form of BRET (BRET(H)), which uses the BRET(1) substrate, native coelenterazine, with the typical BRET(2) donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H) ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay. The BRET(H) thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET(1)-based version but it is substantially brighter than the BRET(2) version. The normalised BRET(H) ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays. These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H) provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

  20. Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting.

    PubMed

    Strydom, K; Ismail, F; Matabane, M M Z; Onwuegbuna, O; Omar, S V; Ismail, N

    2015-09-01

    In a head-to-head comparison of the MTBDRplus version 2.0 (Hain Lifescience), the Xpert MTB/RIF (Cepheid), and the Anyplex MTB/NTM (Seegene) assays, we demonstrated equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampin resistance with further analysis of discordances. The Xpert assay does not detect isoniazid resistance while the Anyplex assay showed high false positivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Comparison between Indirect Immunofluorescence Assay and Shell Vial Culture for Detection of Mumps Virus from Clinical Samples

    PubMed Central

    Reina, Jordi; Ballesteros, Francisca; Ruiz de Gopegui, Enrique; Munar, Maria; Mari, Margarita

    2003-01-01

    We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (P > 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy. PMID:14605158

  2. Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA▿

    PubMed Central

    Ginocchio, C. C.; Barth, D.; Zhang, F.

    2008-01-01

    This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay). PMID:18367578

  3. Comparison of the Freelite serum free light chain (SFLC) assay with serum and urine electrophoresis/immunofixation and the N Latex FLC assay.

    PubMed

    Sasson, S C; McGill, K; Wienholt, L; Carr, A; Brown, D A; Kelleher, A D; Breit, S N; Sewell, W A

    2015-10-01

    Few reports have compared available serum free light chain (SFLC) assays. Here, a retrospective audit of the Freelite SFLC assay compared results to electrophoresis (EP)/immunofixation (IFX) and the N Latex FLC assay.A total of 244 samples collected over 3.5 months were studied using the Freelite and N Latex FLC nephelometry assays. Results were compared with serum and/or urine EP/IFX. The precision and linearity of the N Latex FLC assay was examined.Detectable paraprotein by serum or urine EP/IFX was present in 94% of samples with kappa and 100% with lambda FLC restriction. The correlation between the assays was higher for kappa (rho = 0.97) than lambda (rho = 0.89) especially when lambda results were above the upper limit of normal (rho = 0.62). Agreement in the categorical diagnosis as measured by the Cohen's kappa statistic was good (0.70). The N Latex FLC assay displayed good precision and linearity. In discordant samples the Freelite and N Latex FLC assays had equivalent agreement with IFX.Traditional methods of EP/IFX detected paraproteins in the majority of cases. Correlation between the Freelite and N Latex FLC assay is better for kappa than lambda FLC. The two assays are not entirely equivalent. Care should be taken by interpreting physicians and laboratories considering switching assays.

  4. Comparison between ELISA and gel-filtration assay for the quantitation of airway mucins.

    PubMed

    Shin, C Y; Kang, S J; Kim, K C; Ko, K H

    1998-06-01

    In this study, we developed immunoassay methods for the more convenient and effective detection of rat tracheal mucin and the results were compared with those of [3H]glucosamine based gel-filtration method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically recognizes rat tracheal mucins, was used throughout in this study. To induce mucin secretion, varying concentrations of ATP (0-2 mM) were applied to the primary rat tracheal surface epithelial (RTSE) cell culture which had been metabolically radiolabeled with [3H]glucosamine and the secretion of mucin was analyzed both by the immunoassay and the gel-filtration chromatography methods. For the immunoassay, the following two procedures were employed. 1) Simple ELISA; the culture spent media were directly coated onto the assay plate and the immunoreactivity with mAbRT03 was assessed from the standard curve generated with the purified rat mucin. 2) Inhibition ELISA; A known amount of the purified rat mucin was coated onto the assay plate and then ATP-stimulated culture spent media were added to inhibit the immunoreactivity with mAbRT03. The contents of mucin in the sample were calculated from the standard inhibition curve which was generated with the purified rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present result indicates that ELISA can be substituted for the laborious, time-consuming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.

  5. Evaluation of the Haemoglobin Colour Scale and comparison with the HemoCue haemoglobin assay.

    PubMed Central

    Paddle, J. J.

    2002-01-01

    OBJECTIVE: To evaluate the Haemoglobin Colour Scale developed by WHO for estimating haemoglobin concentration and to compare the results obtained using it and the HemoCue assay with those determined using a reference method, the Technicon H3 analyser. METHODS: The Colour Scale and HemoCue assay were used to test 408 blood samples. Subsequently, Bland-Altman plots were determined and the proximity of the test results to those obtained using the reference method was determined. FINDINGS: The mean difference between the Haemoglobin Colour Scale and the reference method was 0.19 g/dl (95% confidence interval: 3.50 g/dl below to 3.11 g/dl above); the corresponding value for the HemoCue assay was 0.50 g/dl (1.16 g/dl below to 0.16 g/dl above). Only 46.08% of the results obtained by means of the Colour Scale were within 1.0 g/dl of the reference method, whereas 95.34% of the HemoCue results fell within this limit; 22.79% of the Colour Scale results but none of the HemoCue results lay more than 2.0 g/dl from the reference method. CONCLUSION: The Haemoglobin Colour Scale test is too inaccurate for general use, particularly if devices such as the HemoCue are available. PMID:12471402

  6. Comparison of the PRNT and an immune fluorescence assay in yellow fever vaccinees receiving immunosuppressive medication.

    PubMed

    Wieten, Rosanne W; Jonker, Emile F F; Pieren, Daan K J; Hodiamont, Caspar J; van Thiel, Pieter P A M; van Gorp, Eric C M; de Visser, Adriëtte W; Grobusch, Martin P; Visser, Leo G; Goorhuis, Abraham

    2016-03-04

    The 17D-yellow fever (YF) vaccination is considered contraindicated in immune-compromised patients; however, accidental vaccination occurs. In this population, measuring the immune response is useful in clinical practice. In this study we compare two antibody tests (the Immune Fluorescence Assay and the Plaque Reduction Neutralization Test) in a group of Dutch immune-compromised travellers with a median of 33 days (IQR [28-49]) after primary YF vaccination. We collected samples of 15 immune-compromised vaccinees vaccinated with the 17D yellow fever vaccine between 2004 and 2012. All samples measured in the plaque reduction neutralization test yielded positive results (>80% virus neutralization with a 1:10 serum dilution). Immune Fluorescence Assay sensitivity was 28% (95% CI [0.12-0.49]). No adverse events were reported. All immune-compromised patients mounted an adequate response with protective levels of virus neutralizing antibodies to the 17-D YF vaccine. No adverse effects were reported. Compared to the plaque reduction neutralization test, the sensitivity of the Immune Fluorescence Assay test was low. Further research is needed to ascertain that 17D vaccination in immune-compromised patients is safe. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Comparison of various assays to quantitate macrophage activation by biological response modifiers

    SciTech Connect

    Schultz, R.M.; Nanda, S.; Altom, M.G.

    1984-01-01

    Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), and poly I.poly C. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effector cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O/sub 2/- release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and poly I.poly C rendered macrophages cytolytic for P815 target cells at concentrations greater than or equal to 1 microgram/ml. In contrast, significant cytolysis was observed with MDP only at 100 micrograms/ml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.

  8. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.

    PubMed

    Bae, Hi-Gung; Nitsche, Andreas; Teichmann, Anette; Biel, Stefan S; Niedrig, Matthias

    2003-06-30

    Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).

  9. Accuracies of Leuconostoc phenotypic identification: a comparison of API systems and conventional phenotypic assays.

    PubMed

    Kulwichit, Wanla; Nilgate, Sumanee; Chatsuwan, Tanittha; Krajiw, Sunisa; Unhasuta, Chudaachhara; Chongthaleong, Anan

    2007-07-02

    Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria. Clinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-beta hemolysis in our institute during 1997-2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCR's, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005-2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays. Out of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed. The current API systems cannot accurately identify Leuconostoc. Identification of vancomycin

  10. Comparison of microbial community assays for the assessment of stream biofilm ecology.

    PubMed

    Vinten, A J A; Artz, R R E; Thomas, N; Potts, J M; Avery, L; Langan, S J; Watson, H; Cook, Y; Taylor, C; Abel, C; Reid, E; Singh, B K

    2011-06-01

    We investigated a range of microbiological community assays performed on scrapes of biofilms formed on artificial diffusing substrates deployed in 8 streams in eastern Scotland, with a view to using them to characterize ecological response to stream water quality. The assays considered were: Multiplex Terminal Restriction Fragment Length Polymorphism or M-TRFLP (a molecular method), Phospholipid Fatty Acid or PLFA analysis (a biochemical method) and MICRORESP™ (a physiological method) alongside TDI, diatom species, and chlorophyll a content. Four of the streams were classified as of excellent status (3-6μg/L Soluble Reactive Phosphorus (SRP)) with respect to soluble P content under the EU Water Framework Directive and four were of borderline good/moderate or moderate status (43-577μg/L SRP). At each site, 3 replicates of 3 solute diffusion treatments were deployed in a Latin square design. Solute diffusion treatments were: KCl (as a control solute), N and P (to investigate the effect of nutrient enrichment), or the herbicide isoproturon (as a "high impact" control, which aimed to affect biofilm growth in a way detectable by all assays). Biofilms were sampled after 4weeks deployment in a low flow period of early summer 2006. The chlorophyll a content of biofilms after 4weeks was 2.0±0.29mg/m(2) (mean±se). Dry matter content was 16.0±13.1g/m(2). The M-TRFLP was successfully used for generating community profiles of cyanobacteria, algae and bacteria and was much faster than diatom identification. The PFLA and TDI were successful after an increase in the sample size, due to low counts. The MICRORESP(™) assays were often below or near detection limit. We estimated the per-sample times for the successful assays as follows: M-TRFLP: 20min, PLFA 40min, TDI 90min. Using MANOVA on the first 5 principal co-ordinates, all the assays except MICRORESP(™) showed significant differences between sites, but none of the assays showed a significant effect of either initial

  11. Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4.

    PubMed Central

    Koelle, M; Bartholomew, W R

    1982-01-01

    Measurements of patient serum complement components C3 and C4 are useful indicators of complement consumption in immune complex diseases. A fluorometric quantitative immunofluorescence system was evaluated in terms of measuring these complement components, and the results were compared with those of radial immunodiffusion assays. For comparison of the two systems, 232 patient sera were evaluated for C3, and 202 specimens were tested for C4. Analysis of the data by linear regression indicated a proportional difference between the methods. C3 and C4 concentrations measured by the fluorometric method were lower than those measured by radial immunodiffusion, especially concentrations exceeding the normal ranges. In detecting lower concentrations (less than 120 mg/dl for C3 and less than 25 mg/dl for C4), the two methods showed better agreement. Each assay system was reproducible and could be used to evaluate changes that occur in concentrations of complement components during therapeutic treatment. However, the ease in processing a large volume of specimens and the short time needed to complete the assay are advantages that make the fluorometric method more suitable than radial immunodiffusion for use in a large clinical laboratory. PMID:6811611

  12. Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4.

    PubMed

    Koelle, M; Bartholomew, W R

    1982-08-01

    Measurements of patient serum complement components C3 and C4 are useful indicators of complement consumption in immune complex diseases. A fluorometric quantitative immunofluorescence system was evaluated in terms of measuring these complement components, and the results were compared with those of radial immunodiffusion assays. For comparison of the two systems, 232 patient sera were evaluated for C3, and 202 specimens were tested for C4. Analysis of the data by linear regression indicated a proportional difference between the methods. C3 and C4 concentrations measured by the fluorometric method were lower than those measured by radial immunodiffusion, especially concentrations exceeding the normal ranges. In detecting lower concentrations (less than 120 mg/dl for C3 and less than 25 mg/dl for C4), the two methods showed better agreement. Each assay system was reproducible and could be used to evaluate changes that occur in concentrations of complement components during therapeutic treatment. However, the ease in processing a large volume of specimens and the short time needed to complete the assay are advantages that make the fluorometric method more suitable than radial immunodiffusion for use in a large clinical laboratory.

  13. Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: comparison of assay methods and strains.

    PubMed

    Burkholder, Joann M; Gordon, Andrew S; Moeller, Peter D; Law, J Mac; Coyne, Kathryn J; Lewitus, Alan J; Ramsdell, John S; Marshall, Harold G; Deamer, Nora J; Cary, S Craig; Kempton, Jason W; Morton, Steven L; Rublee, Parke A

    2005-03-01

    Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays.

  14. Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains

    PubMed Central

    Burkholder, JoAnn M.; Gordon, Andrew S.; Moeller, Peter D.; Law, J. Mac; Coyne, Kathryn J.; Lewitus, Alan J.; Ramsdell, John S.; Marshall, Harold G.; Deamer, Nora J.; Cary, S. Craig; Kempton, Jason W.; Morton, Steven L.; Rublee, Parke A.

    2005-01-01

    Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays. PMID:15728353

  15. Comparison of electrochemiluminescence assay and ELISA for the detection of Clostridium botulinum type B neurotoxin.

    PubMed

    Guglielmo-Viret, V; Attrée, O; Blanco-Gros, V; Thullier, P

    2005-06-01

    We compared the ELISA and electrochemiluminescence (ECL) immunoassay technologies for the detection of botulinum type B neurotoxin (BotNT B), which requires highly sensitive techniques due to its potent biological activity. BotNT B complexes are the naturally secreted form of the toxin, approximately a third of which consists of the neurotoxin itself; they were aliquoted and frozen for this study. Results of both techniques were interpreted with the same standard statistical tests (ANOVA and Tukey). We first compared two commercial assays for BotNT B: the detection limit of the colorimetric ELISA was 1.56 ng/ml BotNT B complexes versus 0.39-0.78 ng/ml in the ECL test. We then used the same monoclonal antibody and the same polyclonal antibody, respectively purified by protein A and protein G chromatography, to optimize an in-house ELISA test and an in-house ECL test, making it possible to directly compare the two technologies without interference due to the properties of the antibodies used in the two tests. The colorimetric in-house ELISA had a detection threshold of 3.12 ng/ml versus the in-house ECL test whose detection threshold was 0.78-1.56 ng/ml. Thus, in both cases, the ECL assay was two to four times more sensitive than the colorimetric ELISA. The ECL assay was also more rapid (2.5 h for the in-house ECL versus 5 h for in-house ELISA with precoated wells). Overall, these elements can be used to compare the qualities of the two technologies, at least for the detection of protein antigens such as toxins.

  16. Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots.

    PubMed

    Williams, Y; Byrne, S; Bashir, M; Davies, A; Whelan, A; Gun'ko, Y; Kelleher, D; Volkov, Y

    2008-10-01

    Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots. Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence. This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.

  17. Biofilm removal by medical device cleaners: comparison of two bioreactor detection assays.

    PubMed

    Hadi, R; Vickery, K; Deva, A; Charlton, T

    2010-02-01

    Currently there are no standards for testing efficacy of medical device cleaners. With fears of prion transmission, residual protein on medical devices needs to be minimised. A bioreactor model was used to grow Pseudomonas aeruginosa biofilm on polytetrafluoroethylene coupons. The biofilm was subjected to various cleaners and residual biofilm was detected either by Crystal Violet assay (CrV) or a commercially available protein assay (PA) following hydrolysis of the biofilm. Percentage reduction of biofilm was compared with untreated controls in three independent tests. There was no significant difference in percentage biofilm reduction irrespective of whether the CrV or PA was used to detect residual biofilm. Processing of coupons attached to the bioreactor rod and position of coupon within the rod had no significant effect on cleaning efficiency or detection of residual biofilm. Both within-run and between-run variation was very low for good cleaners such as 10g/L NaOH, Zen, and 3M Rapid Multi-Enzyme Cleaner (RMEC) 70500 but was higher for poor cleaners such as Tween 20 which removed less than 20% of the biofilm. Confocal microscopy and electron microscopy provided visual confirmation of the assay results. We propose that this method is suitable as a test method for evaluating the efficacy of surgical instrument cleaners in removing biofilm, as both within-run and between-run variation was low, detection of residual biofilm can be done using either CrV or PA, and the apparatus is easy to use, cheap and readily available.

  18. Comparison of Type I, Type III, and Type VI Collagen Binding Assays in Diagnosis of VWD

    PubMed Central

    Flood, Veronica H.; Gill, Joan Cox; Christopherson, Pamela A.; Wren, Jeffrey S.; Friedman, Kenneth D.; Haberichter, Sandra L.; Hoffmann, Raymond G.; Montgomery, Robert R.

    2013-01-01

    Summary Background Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Collagen binding assays (VWF:CB), however, are not part of the routine workup for von Willebrand disease (VWD). Objectives This study presents data on collagen binding for healthy controls and VWD subjects to compare three different collagens. Patients/Methods VWF antigen (VWF:Ag), VWF ristocetin cofactor activity, and VWF:CB with types I, III, and VI collagen were examined for samples obtained from the Zimmerman Program. Results Mean VWF:CB in healthy controls was similar and highly correlated for types I, III, and VI collagen. The mean VWF:CB/VWF:Ag ratios for types I, III, and VI collagen were 1.31, 1.19, and 1.21 respectively. In type 1 VWD subjects, VWF:CB was similar to VWF:Ag with mean VWF:CB/VWF:Ag ratios for types I, III, and VI collagen of 1.32, 1.08, and 1.1 respectively. For type 2A and 2B subjects, VWF:CB was uniformly low, with mean ratios of 0.62 and 0.7 for type I collagen, 0.38 and 0.4 for type III collagen, and 0.5 and 0.47 for type VI collagen. Conclusions Normal ranges for type I, III, and VI collagen are correlated, but higher values were obtained with type I collagen as compared to types III and VI. The low VWF:CB in type 2A and 2B subjects suggests that VWF:CB may also supplement analysis of multimer distribution. However, these results reflect only one set of assay conditions per collagen type and therefore may not be generalizable to all collagen assays. PMID:22507643

  19. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

    PubMed

    Ku, Hyung-Keun; Lim, Hyuk-Min; Oh, Kyong-Hwa; Yang, Hyo-Jin; Jeong, Ji-Seon; Kim, Sook-Kyung

    2013-03-01

    The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    PubMed Central

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  1. Comparison of the prognostic impact of serum anti-EBV antibody and plasma EBV DNA assays in nasopharyngeal carcinoma

    SciTech Connect

    Twu, C.-W.; Wang, W.-Y.; Liang, W.-M.; Jan, J.-S.; Jiang, R.-S.; Chao, Jeffrey; Jin, Y.-T.; Lin, J.-C. . E-mail: jclin@vghtc.gov.tw

    2007-01-01

    Purpose: Nasopharyngeal carcinoma (NPC) has been proven as an Epstein-Barr virus (EBV)-associated cancer. Serum anti-EBV antibodies and plasma EBV DNA have been investigated as surrogate markers for NPC. A comparison of the prognostic impacts of both assays has never been reported. Methods and Materials: Paired serum and plasma samples from 114 previously untreated NPC patients were collected and subjected to an immunofluorescence assay for immunoglobulin (Ig)A and IgG antibodies against the viral capsid antigen (VCA) and a real-time quantitative polymerase chain reaction assay for EBV DNA measurement. The effects of both assays on patient prognosis were thoroughly investigated. Results: Relapsed patients had significantly higher pretreatment EBV DNA concentration than patients without relapse (p 0.0006). No associations of VCA-IgA (p = 0.9669) or VCA-IgG (p = 0.6125) were observed between patients with and without relapse. The 4-year overall survival (60.3% vs. 93.1%, p < 0.0001) and relapse-free survival rates (54.4% vs. 77.9%, p = 0.0009) were significantly lower in patients with higher pretreatment EBV DNA load than in those with lower EBV DNA load. Patients with persistently detectable EBV DNA after treatment had significantly worse 4-year overall (30.8% vs. 84.6%, p < 0.0001) and relapse-free survival rates (15.4% vs. 74.0%, p < 0.0001) than those with undetectable EBV DNA. The VCA-IgA and VCA-IgG titer could not predict survivals (all p > 0.1). Cox multivariate analyses also showed the same results. Conclusion: Plasma EBV DNA is superior to serum EBV VCA antibodies in prognostic predictions for NPC.

  2. Comparison of three different techniques of human sperm DNA isolation for methylation assay.

    PubMed

    Yuan, Hong-fang; Kuete, Martin; Su, Li; Yang, Fan; Hu, Zhi-yong; Tian, Bo-zhen; Zhang, Hui-ping; Zhao, Kai

    2015-12-01

    Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (P<0.01). PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 (DDX4) and copy number variations (CNVs) than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.

  3. Comparison of immunodiffusion techniques with standard complement fixation assay for quantitation of coccidioidal antibodies.

    PubMed Central

    Wieden, M A; Galgiani, J N; Pappagianis, D

    1983-01-01

    Quantitative immunodiffusion (QID) and complement fixation (CF) methods were compared for their agreement in detecting coccidioidal antibodies. For these studies, we assayed 719 sera from 181 patients with coccidioidomycosis. Over 60% of the specimens had CF results of 1:2 to 1:256. A total of 43 patients had five or more specimens obtained over periods of between 1 and 8 years. The QID method, as originally performed, agreed within a twofold dilution of the CF titer in 191 of 267 sera (71.5%). Modification of QID by repeated filling of the antigen and serum wells improved agreement to 84.7% (383 of 452 sera). The degree of CF titer change in patients over time periods was more closely matched by the modified than by the original QID method. Discrepancies between the CF and QID methods appeared not to be due to a subpopulation of patients. QID measurement of coccidioidal antibodies may be a useful substitute for the CF assay in certain clinical laboratories. Images PMID:6415091

  4. Comparison of Innovative Molecular Approaches and Standard Spore Assays for Assessment of Surface Cleanliness ▿

    PubMed Central

    Cooper, Moogega; La Duc, Myron T.; Probst, Alexander; Vaishampayan, Parag; Stam, Christina; Benardini, James N.; Piceno, Yvette M.; Andersen, Gary L.; Venkateswaran, Kasthuri

    2011-01-01

    A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces. PMID:21652744

  5. Comparison of two assays in the diagnosis of toxoplasmosis: immunological and molecular.

    PubMed

    Hashoosh, D A; Majeed, I A

    2014-02-11

    Serological tests for Toxoplasma gondii are inadequate because antibody production either fails or is significantly delayed. This study in eastern Iraq investigated the IgG-avidity ELISA test for detecting recent T. gondii infections among pregnant women and compared immunological methods and PCR as molecular assays in the diagnosis of T. gondii. Serums samples were taken from 130 pregnant women at risk of toxoplasmosis and a control group of 25 women with normal pregnancy. Of 50 IgM- and/or IgG-positive samples, only 15 showed low IgG-avidity antibodies. PCR was performed on 25 selected samples. Toxoplasma DNA was detected in 15/15 IgM-positive with low IgG-avidity and 1/3 IgM-positive with high IgG-avidity. None of the IgM-negative with high IgG-avidity showed any Toxoplasma DNA. ELISA IgG-avidity when used in combination with ELISA IgG/IgM is a valuable assay for the exclusion of ongoing or recently acquired T. gondii infection in pregnant women.

  6. Comparison of SNP-based detection assays for food analysis: Coffee authentication.

    PubMed

    Spaniolas, Stelios; Bazakos, Christos; Tucker, Gregory A; Bennett, Malcolm J

    2014-01-01

    Recently, DNA-based authentication methods were developed to serve as complementary approaches to analytical chemistry techniques. The single nucleotide polymorphism (SNP)-based reaction chemistries, when combined with the existing detection methods, could result in numerous analytical approaches, all with particular advantages and disadvantages. The dual aim of this study was (a) to develop SNP-based analytical assays such as the single-base primer extension (SNaPShot) and pyrosequencing in order to differentiate Arabica and Robusta varieties for the authentication of coffee beans and (b) to compare the performances of SNaPshot, pyrosequencing and the previously developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using an Agilent 2100 Bioanalyzer on the basis of linearity (R2) and LOD, expressed as percentage of the adulterant species, using green coffee beans (Arabica and Robusta) as a food model. The results showed that SNaPshot analysis exhibited the best LOD, whereas pyrosequencing revealed the best linearity (R2 = 0.997). The PCR-RFLP assay using the Agilent 2100 Bioanalyzer could prove to be a very useful method for a laboratory that lacks sequencing facilities but it can be used only if a SNP creates/deletes a restriction site.

  7. Quantitative human chorionic gonadotropin analysis. I. Comparison of an immunoradiometric assay and a radioimmunoassay

    SciTech Connect

    Shapiro, A.I.; Wu, T.F.; Ballon, S.C.; Lamb, E.J.

    1984-01-01

    An immunoradiometric assay (IRMA) for the quantitative analysis of human chorionic gonadotropin (hCG) was evaluated for specificity, sensitivity, accuracy and precision. The results were compared with those of the conventional radioimmunoassay (RIA) used in our laboratory. The IRMA is a solid-phase, double-antibody immunoassay that sandwiches the intact hCG molecule between the two antibodies. It has specificity, accuracy, and precision which are similar to those of the RIA. The RIA is based upon the assumptions that the antigenicity of the tracer is not altered by the iodination process and that the antibody reacts equally with all of the antigens, including the radiolabeled antigen. The IRMA does not use radiolabeled antigens and thus is free of the assumptions made in the conventional RIA. The IRMA may be more accurate at the lower limits of the assay because it does not require logarithmic transformations. Since the IRMA does not measure the free beta-subunit of hCG, it cannot be endorsed as the sole technique to quantitate hCG in patients with gestational trophoblastic neoplasia until the significance of the free beta-subunit in these patients is determined.

  8. Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction.

    PubMed

    Eggeling, C; Jäger, S; Winkler, D; Kask, Peet

    2005-10-01

    We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

  9. Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays.

    PubMed

    Baumann, Martin J; Murphy, Leigh; Lei, Nina; Krogh, Kristian B R M; Borch, Kim; Westh, Peter

    2011-03-01

    In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates. 2010 Elsevier Inc. All rights reserved.

  10. [Comparison of five commercial assays for the detection of Legionella pneumophila antigens in urine].

    PubMed

    de Ory, Fernando; Minguito, Teodora

    2009-02-01

    Antigenuria detection is the main approach for diagnosing Legionella infections. The aim of this study was to compare 5 commercially available methods for detecting Legionella pneumophila soluble antigens in urine. Seventy-one urine samples were tested, 62 from patients with bacterial infection and 9 from patients with respiratory syncytial virus infection. All samples were assayed for the presence of L. pneumophila by immunoenzymatic (ELISA) (Binax and Bartels), and immunochromatographic (IC) (Binax, SAS and Uni-Gold) methods. Identical results (35 positive and 17 negative) were obtained by the 5 assays in 52 samples (73.2%). Samples showing discrepant results were classified by the majority criterion, and/or other laboratory results (serology), and/or epidemiological findings. On this basis, 51 samples were ultimately classified as positive, and 20 as negative. Sensitivity values of ELISA-Binax, ELISA-Bartels, IC-Binax, IC-SAS and IC-Uni-Gold were 80.4, 100, 82.4, 86.3, and 70.6%, respectively. Corresponding values for specificity were 90, 95, 100, 95 and 100%. The results indicate that the methods compared are all adequate for diagnosing Legionella infection, although some have certain limitations regarding sensitivity.

  11. Comparison of automated treponemal and nontreponemal test algorithms as first-line syphilis screening assays.

    PubMed

    Huh, Hee Jin; Chung, Jae Woo; Park, Seong Yeon; Chae, Seok Lae

    2016-01-01

    Automated Mediace Treponema pallidum latex agglutination (TPLA) and Mediace rapid plasma reagin (RPR) assays are used by many laboratories for syphilis diagnosis. This study compared the results of the traditional syphilis screening algorithm and a reverse algorithm using automated Mediace RPR or Mediace TPLA as first-line screening assays in subjects undergoing a health checkup. Samples from 24,681 persons were included in this study. We routinely performed Mediace RPR and Mediace TPLA simultaneously. Results were analyzed according to both the traditional algorithm and reverse algorithm. Samples with discordant results on the reverse algorithm (e.g., positive Mediace TPLA, negative Mediace RPR) were tested with Treponema pallidum particle agglutination (TPPA). Among the 24,681 samples, 30 (0.1%) were found positive by traditional screening, and 190 (0.8%) by reverse screening. The identified syphilis rate and overall false-positive rate according to the traditional algorithm were lower than those according to the reverse algorithm (0.07% and 0.05% vs. 0.64% and 0.13%, respectively). A total of 173 discordant samples were tested with TPPA by using the reverse algorithm, of which 140 (80.9%) were TPPA positive. Despite the increased false-positive results in populations with a low prevalence of syphilis, the reverse algorithm detected 140 samples with treponemal antibody that went undetected by the traditional algorithm. The reverse algorithm using Mediace TPLA as a screening test is more sensitive for the detection of syphilis.

  12. Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness.

    PubMed

    Cooper, Moogega; La Duc, Myron T; Probst, Alexander; Vaishampayan, Parag; Stam, Christina; Benardini, James N; Piceno, Yvette M; Andersen, Gary L; Venkateswaran, Kasthuri

    2011-08-01

    A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

  13. Comparison of the Capillary Tube and Explant Methods for the In Vitro Assay of Tuberculin

    PubMed Central

    Heilman, Dorothy H.; Affronti, Lewis F.; Leichner, John P.

    1973-01-01

    Purified antigens prepared from mycobacteria were tested for nonspecific toxicity and tuberculin activity by two in vitro methods. One technique utilized measurements of macrophage migration in 4-day-old cultures of spleen from normal and tuberculin-sensitive rabbits. The other method was a modification of the capillary tube technique. To obtain enough peritoneal macrophages for good quantitation, changes were made in the method of harvesting cells, and in some instances exudates from two or more Wright strain no. 13 guinea pigs were combined. The capillary tube method was as sensitive as the explant method for detecting nonspecific toxicity. Each tuberculin assay included a group of control cultures of cells from sensitive animals, test groups containing two widely spaced concentrations of a standard tuberculin, PPD-S, and one or more concentration of the tuberculin to be tested. Macrophages from tuberculin-sensitive animals were regularly inhibited by 0.25 μg of PPD-S per ml with both in vitro methods. The potency of the test tuberculins relative to that of PPD-S was somewhat greater in capillary tube assays than in explant cultures. A purified tuberculopolysaccharide was equally inhibitory for both normal and tuberculin-sensitive cells in both culture systems. PMID:4202957

  14. Comparison of amino acid digestibility of feedstuffs determined with the precision-fed cecectomized rooster assay and the standardized ileal amino acid digestibility assay

    USDA-ARS?s Scientific Manuscript database

    The objective of this study was to evaluate and compare amino acid digestibility of several feedstuffs using 2 commonly accepted methods: the precision-fed cecectomized rooster assay (PFR) and the standardized ileal amino acid assay (SIAAD). Six corn, 6 corn distillers dried grains with or without s...

  15. A comparison of quantitative-competitive and realtime PCR assays using an identical target sequence to detect Epstein-Barr virus viral load in the peripheral blood.

    PubMed

    Xu, Shushen; Green, Michael; Kingsley, Laurence; Webber, Steven; Rowe, David

    2006-11-01

    Monitoring the load of Epstein-Barr virus (EBV) in the peripheral blood by quantitative PCR has been accepted as a useful tool for predicting the onset of EBV related diseases, confirming an EBV disease diagnosis and following the response to treatment interventions. In the present study, the use of a realtime polymerase chain reaction (rt-PCR) assay developed for unpurified cell preparations was examined and the results of the realtime assay were compared to an EBV quantitative-competitive PCR assay (QC-PCR). Both assays use the same target sequence and the same method for determining the standard value for the copy number of EBV genomes present. A comparison of 572 PCR results reveals that the realtime assay gave 5-10-fold higher values than the QC-PCR. Fifty-one results (8.9%) were discordant between the two sets of data. The most commonly encountered discordant result was detection of low amounts of EBV DNA by the rt-PCR assay that were not detected in specimens by QC-PCR. The two assays had a high degree of correlation across the range of load detection allowing clinically relevant threshold values determined in the QC-PCR assay to be inferred for the rt-PCR assay. External normalization of the rt-PCR assay was determined to be an important tool for monitoring the quality and/or quantity of human DNA in the starting material. rt-PCR assays with unpurified cell lysates compare favorably with quantitative-competitive assays and when normalized offer real advantages in specimen preparation, assay manipulations and reproducibility over both quantitative-competitive assays and realtime assays that require purified nucleic acid inputs.

  16. Comparison of immunodiffusion and enzyme linked immunosorbent assay for antibodies to four Aspergillus species.

    PubMed Central

    Froudist, J H; Harnett, G B; McAleer, R

    1989-01-01

    Antigenic extracts were prepared from Aspergillus fumigatus, A niger, A flavus and A terreus for use in enzyme linked immunosorbent assay (ELISA) and immunodiffusion (ID) tests for Aspergillus antibodies to determine whether the use of antigenic extracts from species other than A fumigatus increased the sensitivity of the ELISA. ELISA titres correlated well with positive ID tests. Patient titres by ELISA were significantly higher than control titres for all species. Patient titres to A niger were also significantly higher than titres to the other species. Total number of ID bands to A fumigatus correlated significantly with anti-A fumigatus ELISA titres. It is concluded that the use of antigenic extracts from species other than A fumigatus improves the sensitivity of the ELISA. PMID:2511230

  17. Comparisons of ELISA and Western blot assays for detection of autophagy flux.

    PubMed

    Oh, Sung-Hee; Choi, Yong-Bok; Kim, June-Hyun; Weihl, Conrad C; Ju, Jeong-Sun

    2017-08-01

    We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3).

  18. Comparison of DNA Fragmentation Assay in Frozen-Thawed Cat Epididymal Sperm.

    PubMed

    Kunkitti, P; Sjödahl, A; Bergqvist, A-S; Johannisson, A; Axnér, E

    2016-08-01

    DNA fragmentation of frozen-thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA(®) ), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis. © 2016 Blackwell Verlag GmbH.

  19. Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): A methodological comparison

    PubMed Central

    2009-01-01

    The present study applied the comet assay to erythrocytes of Oreochromis niloticus with the aim of improving protocols to detect DNA damage in these cells, by using two distinct pHs (pH = 12.1 and pH > 13) and evaluating whether there is a correspondence between silver and ethidium bromide staining. Comets were visually examined and, the frequency of cells with and without damage was obtained, as well as the distribution of classes and scores. By using the Kruskal-Wallis test, our results revealed that pH 12.1 is more effective, although both pHs can be used. Our findings also suggest that silver staining can substitute ethidium bromide, an expensive and highly toxic stain that requires specific equipment for examination. PMID:21637662

  20. Comparison between Urinary Oestrogen Assay and Serial Ultrasonic Cephalometry in Assessment of Fetal Growth Retardation

    PubMed Central

    Campbell, Stuart; Kurjak, Asim

    1972-01-01

    Urinary oestrogen assay and serial ultrasonic cephalometry were performed on 284 patients who were considered on clinical grounds to be at risk of having a growth-retarded fetus. It was found that ultrasonic cephalometry was significantly better than oestrogens in diagnosing the small-for-dates baby, but that there was no significant difference between the two methods in predicting perinatal asphyxia. Of the 14 stillbirths, three were in the normal ultrasonic growth rate group and five had normal oestrogen excretion. Both methods were found to be of value in the diagnosis of fetal growth-retardation, although cephalometry would seem to have some advantages, especially in distinguishing between fetal growth-retardation and mistaken maturity. PMID:4673993

  1. The E-screen assay: a comparison of different MCF7 cell stocks.

    PubMed Central

    Villalobos, M; Olea, N; Brotons, J A; Olea-Serrano, M F; Ruiz de Almodovar, J M; Pedraza, V

    1995-01-01

    MCF7 human breast cancer cells have been studied extensively as a model for hormonal effects on breast cancer cell growth and specific protein synthesis. Because the proliferative effect of natural estrogen is considered the hallmark of estrogen action, it was proposed that this property be used to determine whether a substance is an estrogen. The E-screen assay, developed for this purpose, is based on the ability of MCF7 cells to proliferate in the presence of estrogens. The aim of our study was to characterize the response of four MCF7 cell stocks (BUS, ATCC, BB, and BB104) and determine which of them performed best in the E-screen test. The four stocks assayed were distinguishable by their biological behavior. In the absence of estrogen, MCF7 BUS cells stopped proliferating and accumulated in the G0/G1 phase of the cell cycle; estrogen receptors increased, progesterone receptors decreased, and small amounts of pS2 protein were secreted. Of all the MCF7 stocks tested, MCF7 BUS cells showed the highest proliferative response to estradiol-17 beta: cell yields increased up to sixfold over those of nontreated cells in a 144-hr period. The differences between estrogen-supplemented and nonsupplemented MCF7 BUS cells were due mostly to G0/G1 proliferative arrest mediated by charcoal dextran-stripped serum. MCF7 BUS cell stocks and others showing a similar proliferative pattern should be chosen for use in the E-screen test, or whenever a proliferative effect of estrogen is to be demonstrated. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 2. A Figure 2. B Figure 2. C Figure 2. D Figure 3. A Figure 3. B Figure 4. A Figure 4. B Figure 5. A Figure 5. B Figure 5. C Figure 5. D PMID:7498097

  2. Comparison of GMT presto assay and Roche cobas® 4800 CT/NG assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in dry swabs.

    PubMed

    de Waaij, Dewi J; Dubbink, Jan Henk; Peters, Remco P H; Ouburg, Sander; Morré, Servaas A

    2015-11-01

    Urogenital Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the most prevalent bacterial STIs worldwide. Molecular tests are the standard for the detection of CT and NG, as these are difficult to culture. The recently introduced CE-IVD marked GMT Presto assay promises to be a valuable addition in CT and NG diagnostics. The advantage of the Presto assay is that it works on many PCR systems and the DNA can be isolated by any system.We compared the Presto assay to the widely used Roche cobas® 4800 CT/NG test for the detection of CT and NG in 612 vaginal and rectal dry collected swabs. Discrepant samples were tested by the TIB MOLBIOL Lightmix Kit 480 HT CT/NG assay. The alloyed gold standard was defined as two concurring Presto and cobas® 4800 results, or, with discrepant Presto and cobas® results, two concurring results of either test together with the Lightmix Kit 480 HT CT/NG assay. For the Presto assay,we observed 77 CT positive (13%) and 22 NG positive (3,6%) vaginal samples, and 41 CT positive (6,7%) and 11 NG positive (1,8%) rectal samples. For the cobas® 4800 assay,we observed 77 CT positive (13%) and 21NG positive (3,4%) vaginal samples, and 39 CT positive (6,4%) and 11 NG positive (1,8%) rectal samples. Ten CT samples were discrepant between Presto and cobas® 4800 CT/NG assays, while two NG samples were discrepant. CT sensitivity in both assays was 100% compared to the alloyed gold standard. The sensitivity was 100% for both vaginal and rectal dry swabs, underlining the suitability of these sample types for detection of CT and NG. The Presto assay is therefore valuable for molecular detection of CT and NG in dry vaginal and rectal swabs.

  3. Comparison of NIR FRET pairs for quantitative transferrin-based assay

    NASA Astrophysics Data System (ADS)

    Sinsuebphon, Nattawut; Bevington, Travis; Zhao, Lingling; Ken, Abe; Barroso, Margarida; Intes, Xavier

    2014-02-01

    Transferrin (Tfn) is commonly used as a drug delivery carrier for cancer treatment. Tfn cellular internalization can be observed by Förster resonance energy transfer (FRET), which occurs when two fluorophores - donor and acceptor - are a few nanometers apart. Donor fluorescence lifetime can be used to sense and quantify FRET occurrence. In FRET state, the donor is quenched leading to a significant reduction in its lifetime. In this study, donor and acceptor near-infrared (NIR) fluorophore-labeled Tfn were used to quantify cellular internalization in breast cancer cell line (T47D). Based on donor lifetime, quantum yield and spectral data, seven NIR FRET pairs were chosen for this comparison. Performance of the different NIR FRET pairs was evaluated in vitro in multiwell plate settings and by analyzing the relationship between quenched donor fraction and acceptor:donor ratio. Additionally, we performed brightness comparison between each pairs. Several parameters, such as brightness, lifetime, R0 and FRET donor population values are used to identify the most suitable NIR FRET pair for in vivo studies in preclinical settings.

  4. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection.

    PubMed

    Roberts, Christine C; Swoyer, Ryan; Bryan, Janine T; Taddeo, Frank J

    2011-05-01

    Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.

  5. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

    PubMed

    Rochelle, Paul A; Marshall, Marilyn M; Mead, Jan R; Johnson, Anne M; Korich, Dick G; Rosen, Jeffrey S; De Leon, Ricardo

    2002-08-01

    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all

  6. Comparison of Mycoplasma arthritidis strains by enzyme-linked immunosorbent assay, immunoblotting, and DNA restriction analysis.

    PubMed Central

    Washburn, L R; Voelker, L L; Ehle, L J; Hirsch, S; Dutenhofer, C; Olson, K; Beck, B

    1995-01-01

    Twenty Mycoplasma arthritidis strains or isolates were compared by a combination of enzyme-linked immunosorbent assay by an antiserum adsorption technique, Western immunoblotting, and restriction analysis of chromosomal DNA. Antigenic markers that defined strains related to strains 158p10p9, PG6, and H606 were identified. In addition, restriction analysis allowed all 20 strains to be divided into six groups. Results of restriction analysis corresponded generally with antigenic similarities, although the former did not allow grouping with as fine a precision as the latter. However, intrastrain antigenic variability, which is common among many Mycoplasma species, including M. arthritidis, introduced a complicating factor into our attempts at antigenic analysis. While serologic and antigenic analyses remain useful, we recommend that they be used with caution and in combination with other techniques for identifying and characterizing new isolates and newly acquired strains. Combinations of these techniques have proven to be useful in our laboratory for quality control and for uncovering interesting relationships among strains subjected to animal passage and their less virulent antecedents and among strains originally classified as the same but obtained from different sources and maintained, sometimes for decades, in different laboratories. PMID:7494014

  7. Comparison of frozen versus desiccated reference human red blood cells for hemagglutination assays.

    PubMed

    Ho, David; Schierts, Jennifer; Zimmerman, Zea; Gadsden, Isaac; Bruttig, Stephen

    2009-10-01

    Red blood cells (RBCs) are commonly used fresh or stored in frozen format for identification of patients' antibodies and serologic specificity of such antibodies at reference laboratories. However, maintaining a large pool of fresh RBCs is impossible in a blood-banking environment and blood in frozen format poses a logistic disadvantage in terms of accessibility, maintenance cost, safety, and sample recovery. This study explores an alternative, desiccation storage method for RBCs to provide a reagent that supports greater utilization and flexibility for reference laboratories. RBCs from five donors were used in the study. RBCs were processed and kept in either frozen or desiccated format. Study variables for either the frozen or the desiccated cells included cell recovery as quantified by cell counts, gross microscopic examination, and hemagglutination assays. The mean percentage of cell recovery for thawed and washed frozen RBCs was 20% versus 50% for rehydrated and washed desiccated RBCs. Microscopic examination of thawed cells from the frozen preparation showed cells with irregular shapes, a sharp contrast when compared with rehydrated cells from the desiccated preparation, where cells are mostly intact, smooth surface, and biconcave in structure. Cells in both preparations performed well in manual agglutination tests. Desiccation preservation of RBCs provides a somewhat better RBC recovery and cell structure stability, while maintaining the necessary antigen-antibody reactions for cell surface markers, which will allow desiccated RBCs to be archived in blood collecting and processing reference laboratories.

  8. Are extraction methods in quantitative assays of pharmacopoeia monographs exhaustive? A comparison with pressurized liquid extraction.

    PubMed

    Basalo, Carlos; Mohn, Tobias; Hamburger, Matthias

    2006-10-01

    The extraction methods in selected monographs of the European and the Swiss Pharmacopoeia were compared to pressurized liquid extraction (PLE) with respect to the yield of constituents to be dosed in the quantitative assay for the respective herbal drugs. The study included five drugs, Belladonnae folium, Colae semen, Boldo folium, Tanaceti herba and Agni casti fructus. They were selected to cover different classes of compounds to be analyzed and different extraction methods to be used according to the monographs. Extraction protocols for PLE were optimized by varying the solvents and number of extraction cycles. In PLE, yields > 97 % of extractable analytes were typically achieved with two extraction cycles. For alkaloid-containing drugs, the addition of ammonia prior to extraction significantly increased the yield and reduced the number of extraction cycles required for exhaustive extraction. PLE was in all cases superior to the extraction protocol of the pharmacopoeia monographs (taken as 100 %), with differences ranging from 108 % in case of parthenolide in Tanaceti herba to 343 % in case of alkaloids in Boldo folium.

  9. Comparison of three typing assays for nicotinamide adenine dinucleotide-independent Actinobacillus pleuropneumoniae.

    PubMed

    Maldonado, Jaime; Blanco, Mónica; Martínez, Eva; Navas, Jesús

    2011-07-01

    Three tests for typing clinical isolates of Actinobacillus pleuropneumoniae biovar 2 were compared: 1) standard coagglutination with type-specific antisera against serovars 1-12 of biovar 1 of A. pleuropneumoniae; 2) a previously described polymerase chain reaction system for detecting the apx genes encoding the ApxI, ApxII, and ApxIII toxins in A. pleuropneumoniae; and 3) a restriction fragment length polymorphism analysis of the transferrin-binding protein B gene. The panel of strains tested included 112 field isolates of biovar 2 recovered from pigs between 1979 and 2007 in Italy and Spain, and reference strains for all described serovars of both biovars. The values of Simpson index of diversity obtained for the 3 methods were 0.68, 0.20, and 0.60, respectively. Coagglutination assays identified the field isolates as belonging to serovars 2 (9 strains), 4 (13 strains), 7 (61 strains), 9 (17 strains), and 11 (1 strain). Eleven strains were not typeable, and cross-reactivity was observed between serovars 2 and 4, 4 and 7, and 9 and 11. Isolates of A. pleuropneumoniae biovar 2 displayed 2 apx patterns: ApxII(+) (94 strains) and ApxI(+)/ApxII(+) (18 strains). The restriction fragment length polymorphism analysis assigned the strains tested to 3 different patterns. This method distinguished between biovar 2 reference strains and field strains that could not be identified by other methods, thus constituting a useful complementary test for the typing of A. pleuropneumoniae biovar 2.

  10. Comparison of molecular assays for detection and typing of human papillomavirus.

    PubMed

    Koidl, Christoph; Bozic, Michael; Hadzisejdic, Ita; Grahovac, Maja; Grahovac, Blazenka; Kranewitter, Wolfgang; Marth, Egon; Kessler, Harald H

    2008-08-01

    The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA). A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

  11. [Analytical quality of assays and comparison of procedures for the sweat test].

    PubMed

    Nguyen-Khoa, Thao; Borgard, Jean-Pierre; Marchand, Martine; Sitruk-Khalfon, Dominique; Feuillet, Marie-Noëlle; Feldmann, Delphine; Vassault, Anne; Rota, Michèle

    2012-01-01

    Sweat test measuring the chloride ion (Cl(-)) concentration in sweat is a tool for the cystic fibrosis (CF) diagnosis. We evaluated analytical criteria of different available methods and compared them into five hospitals and throught a national quality control program. Sweat tests were performed by stimulation using pilocarpine iontophoresis, sweat collection and measurement of sweat Cl(-) (mmol/L) by titration (colorimetric or coulometric end-point) or by in situ direct potentiometry using a chloride-selective electrode. Indirect determination by sweat conductivity measurement was expressed in mmol/L sodium chloride (NaCl) equivalents (Eq). Linearity range was demonstrated for all measurement procedures in the range 10 to 120 mmol/L. Intra-laboratory coefficients of variation (CVs) were <5% for values between 10 and 100 mmol/L. Inter-laboratory CVs were <3% only for conductivity measurement whatever the range. The comparison of results obtained for a same sweat sample, simultaneously by coulometric and conductivity measurements, demonstrated a first degree linear distribution between 30 to 60 mmol/L Cl(-) allowing us to establish an analytical correspondence table for this range. Thus, calculated values for 30, 40 and 60 mmol/L Cl(-) were respectively 57, 66 and 84 mmol/L NaCl Eq. In conclusion, comparison of methods highlighted that the less the sweat test is automatically controlled, the more the operator influence on results quality is important. Our study supports that sweat test result <50 mmol/L NaCl Eq is unlikely with CF diagnosis in absence of clinical arguments.

  12. Multisite Comparison of Anti-Human Immunodeficiency Virus Microbicide Activity in Explant Assays Using a Novel Endpoint Analysis ▿ †

    PubMed Central

    Richardson-Harman, Nicola; Lackman-Smith, Carol; Fletcher, Patricia S.; Anton, Peter A.; Bremer, James W.; Dezzutti, Charlene S.; Elliott, Julie; Grivel, Jean-Charles; Guenthner, Patricia; Gupta, Phalguni; Jones, Maureen; Lurain, Nell S.; Margolis, Leonid B.; Mohan, Swarna; Ratner, Deena; Reichelderfer, Patricia; Roberts, Paula; Shattock, Robin J.; Cummins, James E.

    2009-01-01

    Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development. PMID:19726602

  13. Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

    PubMed

    Richardson-Harman, Nicola; Lackman-Smith, Carol; Fletcher, Patricia S; Anton, Peter A; Bremer, James W; Dezzutti, Charlene S; Elliott, Julie; Grivel, Jean-Charles; Guenthner, Patricia; Gupta, Phalguni; Jones, Maureen; Lurain, Nell S; Margolis, Leonid B; Mohan, Swarna; Ratner, Deena; Reichelderfer, Patricia; Roberts, Paula; Shattock, Robin J; Cummins, James E

    2009-11-01

    Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

  14. Comparison of pepsin-digestion and enzyme-linked immunosorbent assay for the diagnosis of trichinosis in swine.

    PubMed Central

    Smith, H J

    1988-01-01

    Comparison of parasitological and serological diagnosis of trichinosis in swine was carried out on 36 pigs given 15,400 infective larvae each by gavage. Circulating eosinophil levels were determined and sera were examined by enzyme-linked immunosorbent assay for anti-Trichinella antibodies. Two pigs were killed per day from days 15 to 29 postinfection. Muscle was examined by pepsin-digestion and comparable tissue was fed to a rat. Eosinophil counts increased at about day 6 and reached peak levels about day 25 postinfection and returned to approximate preinfection levels about two months postinfection in those pigs still in the study. Infective larvae were recovered from all pigs killed at greater than or equal to 18 days postinfection. Using the criterion of 5 x mean optical density readings of negative sera as positive, seroconversion occurred between days 19 and 26 postinfection. Use of a lower criterion of 3 x mean optical density readings of negative sera resulted in only three of 30 pigs killed greater than or equal to 18 days postinfection seroconverting less than or equal to 18 days postinfection, when infective larvae were first recovered in the musculature. In pigs, even in those heavily infected, there is a lag between the period that trichinae in musculature become infective and development of antibodies as detected by enzyme-linked immunosorbent assay which results in false negative reactions in many animals. This study demonstrated that the enzyme-linked immunosorbent assay using an excretory-secretory antigen should not be used to certify pork or pork products free of infective Trichinella larvae or safe for human consumption. PMID:3127035

  15. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.

    PubMed

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas

    2015-01-07

    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

  16. Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

    PubMed Central

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas

    2015-01-01

    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases. PMID:25566793

  17. Comparison of anti-pertussis toxin ELISA and agglutination assays to assess immune responses to pertussis.

    PubMed

    Khramtsov, Pavel; Bochkova, Maria; Timganova, Valeria; Zamorina, Svetlana; Rayev, Mikhail

    2017-08-01

    The goal of our study was to compare the following two methods of assessment of pertussis post-vaccination immunity: bacterial agglutination test and pertussis toxin enzyme-linked immunosorbent assay (ELISA). The study was carried out in Perm Region, Russia. We measured pertussis immunity using two serological methods: ELISA of IgG to pertussis toxin (PT) and the agglutination test (AT) among 135 children, in the age range from 2 months to 17 years old. The immunization schedule included four doses of DTwP: at 3, 4.5 and 6 months of age and a booster at 18 months. All participants were divided into six age groups. The percentage of samples with IgG level less than the detection limit in vaccinated children was 52.2%. The total seropositivity rate (the percent of children with agglutinin titres ≥1:160) in vaccinated children was 47.8%. Only a weak association was observed between agglutinin and anti-PT IgG titres (R = .3). Neither the primary nor the booster vaccination with DTwP influenced the IgG levels in children. Agglutinin titres significantly increased after vaccination and declined 5 years after the booster dose. Significant growth of IgG concentration was observed in 11-year-olds, indicating the presence of B. pertussis circulation in the childhood population. Based on the obtained results and the results of other authors, we summarize that anti-PT ELISA should be carefully used to assess the population immunity to pertussis. Currently, there is neither a serological test that accurately determines the protection against pertussis nor a distinctive criterion of protection that can be applied in seroepidemiological studies.

  18. Interlaboratory comparison of Taq Nuclease Assays for the quantification of the toxic cyanobacteria Microcystis sp

    PubMed Central

    Schober, Eva; Werndl, Michael; Laakso, Kati; Korschineck, Irina; Sivonen, Kaarina; Kurmayer, Rainer

    2011-01-01

    Summary The application of quantitative real time PCR has been proposed for the quantification of toxic genotypes of cyanobacteria. We have compared the Taq Nuclease Assay (TNA) in quantifying the toxic cyanobacteria Microcystis sp. via the intergenic spacer region of the phycocyanin operon (PC) and mcyB indicative of the production of the toxic heptapeptide microcystin between three research groups employing three instruments (ABI7300, GeneAmp5700, ABI7500). The estimates of mcyB genotypes were compared using (i) DNA of a mcyB containing strain and a non-mcyB containing strain supplied in different mixtures across a low range of variation (0-10% of mcyB) and across a high range of variation (20-100%), and (ii) DNA from field samples containing Microcystis sp. For all three instruments highly significant linear regression curves between the proportion of the mcyB containing strain and the percentage of mcyB genotypes both within the low range and within the high range of mcyB variation were obtained. The regression curves derived from the three instruments differed in slope and within the high range of mcyB variation mcyB proportions were either underestimated (0-50%) or overestimated (0-72%). For field samples cell numbers estimated via both TNAs as well as mcyB proportions showed significant linear relationships between the instruments. For all instruments a linear relationship between the cell numbers estimated as PC genotypes and the cell numbers estimated as mcyB genotypes was observed. The proportions of mcyB varied from 2-28% and did not differ between the instruments. It is concluded that the TNA is able to provide quantitative estimates on mcyB genotype numbers that are reproducible between research groups and is useful to follow variation in mcyB genotype proportion occurring within weeks to months. PMID:17258828

  19. Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis.

    PubMed

    Tanyuksel, Mehmet; Yilmaz, Hasan; Ulukanligil, Mustafa; Araz, Engin; Cicek, Mutalip; Koru, Ozgur; Tas, Zeynep; Petri, William A

    2005-07-01

    Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.

  20. Comparison of peptide mass mapping and electron capture dissociation as assays for histone posttranslational modifications

    NASA Astrophysics Data System (ADS)

    Zhang, Liwen; Freitas, Michael A.

    2004-05-01

    Posttranslational modifications of core histones play a critical role in the structure of chromatin and the regulation of gene activities. Improved techniques for determining these modification sites may lead to a better understanding of histone regulation at the molecular level. LC-MS peptide mass mapping was performed on pepsin, trypsin and Glu-C digests of bovine thymus H4 using a QqTOF instrument. The well established modification sites of H4 (acetylation of K8, 12, 16 and methylation of K20) were observed in addition to several recently discovered modifications including: methylation of K31, 44, 59 and acetylation of K20, 77, 79. For comparison, electron capture dissociation (ECD) was performed on intact H4 along with several peptides from enzymatic digestion. The results from the ECD experiments of histone H4 indicated the acetylation of K5, 12, 16, 31, 91 and the methylation of K20 and 59 in good agreement with the result from peptide mapping. The work is dedicated to Alan G. Marshall on his 60th birthday. His endeavors in the advancement of FT-ICR facilitated experiments reported herein.

  1. Comparison of performance of serum and plasma in panbio dengue and Japanese encephalitis virus enzyme-linked immunosorbent assays.

    PubMed

    Blacksell, Stuart D; Lee, Sue J; Chanthongthip, Anisone; Taojaikong, Thaksinaporn; Thongpaseuth, Soulignasack; Hübscher, Tanja; Newton, Paul N

    2012-09-01

    We examined the comparative performance of serum and plasma (in dipotassium EDTA) in Panbio Dengue enzyme-linked immunosorbent assays (ELISAs) for detection of non-structural protein 1 (NS1), IgM, and IgG, and a dengue/Japanese encephalitis virus (JEV) combination IgM ELISA in a prospective series of 201 patients with suspected dengue in Laos. Paired comparisons of medians from serum and plasma samples were not significantly different for Dengue IgM, and NS1 which had the highest number of discordant pairs (both 2%; P = 0.13 and P = 0.25, respectively). Comparison of qualitative final diagnostic interpretations for serum and plasma samples were not significantly different: only 1.5% (3 of 201 for Dengue/JEV IgM and Dengue IgG) and 2.0% (4 of 201; IgM and NS1) showed discordant pairs. These results demonstrate that plasma containing EDTA is suitable for use in these ELISAs.

  2. Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia

    PubMed Central

    2004-01-01

    Abstract The purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) kits in comparison with a reference AGID kit for the detection of equine infectious anemia (EIA) antibodies in horses for regulatory use in Canada. A total of 285 positive and 315 negative samples by the reference AGID were tested blindly on 2 other AGID and 4 ELISA kits. Commercially available AGID kits for the serodiagnosis of EIA were found equivalent. The 3 ELISAs directed against antibodies to the p26 core protein also performed relatively well in comparison with the reference AGID, with excellent relative accuracy and acceptable precision. The single ELISA directed against antibodies to the gp45 trans-membrane viral protein yielded a lower relative sensitivity. The performance characteristics of the ELISAs directed against antibodies to p26 are, therefore, adequate to support the implementation of ELISA for regulatory purposes in Canada. PMID:15581219

  3. Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia.

    PubMed

    Paré, Julie; Simard, Carole

    2004-10-01

    The purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) kits in comparison with a reference AGID kit for the detection of equine infectious anemia (EIA) antibodies in horses for regulatory use in Canada. A total of 285 positive and 315 negative samples by the reference AGID were tested blindly on 2 other AGID and 4 ELISA kits. Commercially available AGID kits for the serodiagnosis of EIA were found equivalent. The 3 ELISAs directed against antibodies to the p26 core protein also performed relatively well in comparison with the reference AGID, with excellent relative accuracy and acceptable precision. The single ELISA directed against antibodies to the gp45 trans-membrane viral protein yielded a lower relative sensitivity. The performance characteristics of the ELISAs directed against antibodies to p26 are, therefore, adequate to support the implementation of ELISA for regulatory purposes in Canada.

  4. Comparison of optomagnetic and AC susceptibility readouts in a magnetic nanoparticle agglutination assay for detection of C-reactive protein.

    PubMed

    Fock, Jeppe; Parmvi, Mattias; Strömberg, Mattias; Svedlindh, Peter; Donolato, Marco; Hansen, Mikkel Fougt

    2017-02-15

    There is an increasing need to develop biosensor methods that are highly sensitive and that can be combined with low-cost consumables. The use of magnetic nanoparticles (MNPs) is attractive because their detection is compatible with low-cost disposables and because application of a magnetic field can be used to accelerate assay kinetics. We present the first study and comparison of the performance of magnetic susceptibility measurements and a newly proposed optomagnetic method. For the comparison we use the C-reactive protein (CRP) induced agglutination of identical samples of 100nm MNPs conjugated with CRP antibodies. Both methods detect agglutination as a shift to lower frequencies in measurements of the dynamics in response to an applied oscillating magnetic field. The magnetic susceptibility method probes the magnetic response whereas the optomagnetic technique probes the modulation of laser light transmitted through the sample. The two techniques provided highly correlated results upon agglutination when they measure the decrease of the signal from the individual MNPs (turn-off detection strategy), whereas the techniques provided different results, strongly depending on the read-out frequency, when detecting the signal due to MNP agglomerates (turn-on detection strategy). These observations are considered to be caused by differences in the volume-dependence of the magnetic and optical signals from agglomerates. The highest signal from agglomerates was found in the optomagnetic signal at low frequencies. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Comparison of male versus female responses in the Pig-a mutation assay

    PubMed Central

    Labash, Carson; Avlasevich, Svetlana L.; Carlson, Kristine; Torous, Dorothea K.; Berg, Ariel; Bemis, Jeffrey C.; MacGregor, James T.; Dertinger, Stephen D.

    2015-01-01

    Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, 7- or 14-week old male and female Sprague Dawley rats were exposed to N-ethyl-N-nitrosourea (ENU). In the study with the 7-week old rats, exposure was to 0, 1, 5 or 25mg ENU/kg/day for three consecutive days (study Days 1–3). Pig-a mutant phenotype reticulocyte (RETCD59−) and mutant phenotype erythrocyte (RBCCD59−) frequencies were determined on study Days −4, 15, 29 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). Additionally, blood samples collected on Day 4 were analysed for micronucleated reticulocyte (MN-RET) frequency (In Vivo MicroFlow®). The percentage of reticulocytes (%RET) was markedly higher in the 7-week old males compared to females through Day 15 (2.39-fold higher on Day −4). At 25mg/kg/day, ENU reduced Day 4 RET frequencies in both sexes, and the two highest dose levels resulted in elevated MN-RET frequencies, with no sex or treatment × sex interaction. The two highest dose levels significantly elevated the frequencies of mean RETCD59− and RBCCD59− in both sexes from Day 15 onward. RETCD59− and RBCCD59− frequencies were somewhat lower for females compared to males at the highest dose level studied, and differences in RETCD59− resulted in a statistically significant interaction effect of treatment × sex. In the study with 14-week old rats, treatment was for 3 days with 0 or 25mg ENU/kg/day. RET frequencies differed to a lesser degree between the sexes, and in this case there was no evidence of a treatment × sex interaction. These results suggest that the slightly higher response in younger males than in the younger females may be related to differences in erythropoiesis function at that age. In conclusion, while some quantitative differences were noted, there were no

  6. Comparison of four molecular assays for the detection of Tembusu virus.

    PubMed

    Tang, Yi; Yeh, Yin-Ting; Chen, Hao; Yu, Chunmei; Gao, Xuhui; Diao, Youxiang

    2015-10-01

    Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/μL, 20 copies/μL, 2 copies/μL, and 20 copies/μL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.

  7. Comparison of various assays used for detection of beta-lactam antibiotics in poultry meat.

    PubMed

    Popelka, P; Nagy, J; Germuska, R; Marcincák, S; Jevinová, P; De Rijk, A

    2005-06-01

    In this study, microbiological tests for the detection of beta-lactam antibiotics in meat and meat products were evaluated. The traditional FPT (four plate test, containing Bacillus subtilis and Kocuria rhizophila), BsDA (Bacillus stearothermophilus disc assay) and a newly developed microbiological test, Premi Test (containing Bacillus stearothermophilus) were included in the study. The limit of detection (LOD) of the Premi Test was compared with the LOD of the traditional methods. The detection limits of the tests were determined by using beta-lactam antibiotic standards dissolved in meat juice, as well as meat tissue obtained from laying hens after experimental administration of amoxicillin. Positive samples, based on inhibition of growth of the organism in the test, were confirmed by high performance liquid chromatography (HPLC). Growth inhibition in the traditional tests is visible as a clear zone on the plate, whereas for Premi Test, this is based on the absence of a colour change of the test. The LODs of antibiotics tested were as follows: Penicillin G (PENG) 5 microg kg(-1), amoxicillin (AMOX) 10 microg kg(-1), ampicillin (AMP) 25 microg kg(-1), oxacillin (OXA) 30 microg kg(-1), and cloxacillin (CLOX) 30 microg kg(-1) on the plate with Bacillus stearothermophilus. Beta-lactam antibiotics can be detected also on one plate seeded with Kocuria rhizophila, although the LODs are higher: PENG 10 microg kg(-1), AMOX 25 microg kg(-1), AMP 30 microg kg(-1), OXA 50 microg kg(-1), and CLOX 50 microg kg(-1). Premi Test was performed according to the Standard Operating Procedure intended for detection of beta-lactam antibiotics in poultry tissues with following LODs: PENG 4 microg kg(-1), AMOX 5 microg kg(-1), AMP 5 microg kg(-1), OXA 40 microg kg(-1), CLOX 50 microg kg(-1). All tests are able to detect beta-lactam antibiotics such as penicillin G, ampicillin, amoxicillin, oxacillin and cloxacillin below the maximum residue level (MRL). However, the detection limits of

  8. Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis.

    PubMed

    Sachdeva, Poonam; Patel, Achchhe Lal; Sachdev, Divya; Ali, Mashook; Mittal, Aruna; Saluja, Daman

    2009-07-01

    To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

  9. Comparison of Two Commercial Enzyme-Linked Immunosorbent Assays with an Immunofluorescence Assay for Detection of Legionella pneumophila Types 1 to 6

    PubMed Central

    Malan, Annette K.; Martins, Thomas B.; Jaskowski, Troy D.; Hill, Harry R.; Litwin, Christine M.

    2003-01-01

    Members of the genus Legionella are characterized as gram-negative, motile, freshwater-dwelling bacteria that were responsible for a pneumonia outbreak among American Legion members in 1976. Because clinicians routinely order serologic testing for Legionella pneumophila serogroups 1 to 6 as a screen for possible L. pneumophila infections, we evaluated the Wampole Laboratories L. pneumophila type 1 to 6 immunoglobulin G (IgG) and IgM combined enzyme-linked immunosorbent assay (ELISA) and the Zeus Scientific L. pneumophila type 1 to 6 IgG-IgM-IgA multispecific combined ELISA systems and compared them to an IgG-specific immunofluorescence assay (IFA) for L. pneumophila serogroups 1 to 6. The Centers for Disease Control and Prevention recommends that the positive titer cutoff for an IFA be 1:256. Regardless of where the positive IFA cutoff titer is placed, however, the sensitivity of both commercial assays was below what would be acceptable for a screening assay. With a 1:256 IFA titer as the positive cutoff, the agreement, sensitivity, and specificity of the Wampole ELISA were 74.6, 21.4, and 98.4%, respectively. The agreement, sensitivity, and specificity of the Zeus ELISA were 72.6, 10.5, and 100.0%, respectively. We recommend that any laboratories attempting to replace an IFA type 1 to 6 screen with an alternative ELISA carefully investigate the sensitivity of the replacement assay. PMID:12843044

  10. Comparison of immunocytochemical estrogen receptor assay, estrogen receptor enzyme immunoassay, and radioligand-labeled estrogen receptor assay in human breast cancer and uterine tissue

    SciTech Connect

    Heubner, A.; Beck, T.; Grill, H.J.; Pollow, K.

    1986-08-01

    Determination of estrogen receptor content in 82 breast cancer specimens with immunocytochemical estrogen receptor assay (ER-EIA) (Abbott) was compared with our routinely used binding assay using /sup 125/I-estradiol as radioligand with Scatchard plot analysis of the binding data. Although the estrogen receptor content measured with the ER-EIA was approximately 2-fold higher compared with the binding assay, the immunochemical method proved to be a useful alternative for estrogen receptor determination. Furthermore, it is possible to detect estrogen receptors in FPLC Superose 12 (size exclusion column) eluates or in the fractions obtained after sucrose density centrifugation using the ER-EIA. Forty breast cancer samples were analyzed utilizing the immunocytochemical technique (ER-ICA) for visualization of the estrogen receptor content in frozen tumor tissues in relationship to the quantitative results obtained with the ER-EIA assay. Specific staining for estrogen receptor was confined only to the cell nucleus, was distributed irregularly among the tumor cells, and was variable in intensity. The staining intensity and the percentage of positively stained cells increased with increasing level of cytosolic estrogen receptor. In 27 of 40 cases the immunocytochemical results correlated well with the ER-EIA assay. Nine cases were ER-ICA negative with positive ER-EIA, and four were ER-ICA positive with negative ER-EIA.

  11. HIV-1 Viral Load and Phenotypic Antiretroviral Drug Resistance Assays Based on Reverse Transcriptase Activity in Comparison to Amplification Based HIV-1 RNA and Genotypic Assays

    PubMed Central

    Napravnik, Sonia; Cachafeiro, Ada; Stewart, Paul; Eron, Joseph J.; Fiscus, Susan A.

    2009-01-01

    Background Amplification based HIV-1 viral load and genotypic resistance assays are expensive, technologically complex and may be difficult to implement in resource limited settings. Inexpensive, simpler assays are urgently needed. Objectives To determine the suitability of the ExaVir™ Load and ExaVir™ Drug assays for use in patient monitoring. Study Design Specimens from 108 adults were used to compare ExaVir™ Load HIV-1 RT to Amplicor HIV-1 Monitor® HIV-1 RNA, and ExaVir™ Drug phenotype to HIV GenoSure™ genotype. Results HIV-1 RT and HIV-1 RNA levels were comparable (Pearson correlation coefficient 0.83). Most (94%) had detectable results in both assays. The mean difference (HIV-1 RT minus HIV-1 RNA) was -0.21 log10 cps/mL equivalents. Relationship between HIV-1 RT and HIV-1 RNA was not affected by RT mutations, CD4 cell count, or efavirenz (EFV) or nevirapine (NVP) use. Phenotypes were generally consistent with genotype findings for EFV, but not for NVP. Most patients (93.9%) with phenotypic EFV resistance had at least one EFV mutation, while 78.0% of patients with phenotypic NVP resistance had at least one NVP mutation. Eleven of 49 samples tested for EFV susceptibility were found resistant (n=2) or with reduced susceptibility (n=9) despite the absence of genotypic resistance. Eleven of 45 samples tested for NVP susceptibility were found resistant (n=9) or with reduced susceptibility (n=2) with no evidence of genotypic mutations. Conclusions The ExaVir™ Load assay performed well and may be an alternative to amplification based techniques for HIV-1 RNA quantification. The ExaVir™ Drug assay for phenotypic resistance testing requires further evaluation, especially for NVP. PMID:19896416

  12. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

    PubMed

    Pickering, Jerry W; Martins, Thomas B; Schroder, M Carl; Hill, Harry R

    2002-07-01

    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

  13. Electrochemical Measurement of the β-Galactosidase Reporter from Live Cells: A Comparison to the Miller Assay.

    PubMed

    Tschirhart, Tanya; Zhou, Xinyi Y; Ueda, Hana; Tsao, Chen-Yu; Kim, Eunkyoung; Payne, Gregory F; Bentley, William E

    2016-01-15

    In order to match our ability to conceive of and construct cells with enhanced function, we must concomitantly develop facile, real-time methods for elucidating performance. With these, new designs can be tested in silico and steps in construction incrementally validated. Electrochemical monitoring offers the above advantages largely because signal transduction stems from direct electron transfer, allowing for potentially quicker and more integrated measurements. One of the most common genetic reporters, β-galactosidase, can be measured both spectrophotometrically (Miller assay) and electrochemically. However, since the relationship between the two is not well understood, the electrochemical methods have not yet garnered the attention of biologists. With the aim of demonstrating the utility of an electrochemical measurement to the synthetic biology community, we created a genetic construct that interprets and reports (with β-galactosidase) on the concentration of the bacterial quorum sensing molecule autoinducer-2. In this work, we provide a correlation between electrochemical measurements and Miller Units. We show that the electrochemical assay works with both lysed and whole cells, allowing for the prediction of one from the other, and for continuous monitoring of cell response. We further present a conceptually simple and generalized mathematical model for cell-based β-galactosidase reporter systems that could aid in building and predicting a variety of synthetic biology constructs. This first-ever in-depth comparison and analysis aims to facilitate the use of electrochemical real-time monitoring in the field of synthetic biology as well as to facilitate the creation of constructs that can more easily communicate information to electronic systems.

  14. Evaluation of clinical usefulness of second-generation HCV core antigen assay: comparison with COBAS AMPLICOR HCV MONITOR assay version 2.0.

    PubMed

    Yokosuka, Osamu; Kawai, Shigenobu; Suzuki, Yoichi; Fukai, Kenichi; Imazeki, Fumio; Kanda, Tatsuo; Tada, Motohisa; Mikata, Rintarou; Hata, Akira; Saisho, Hiromitsu

    2005-12-01

    Hepatitis C virus (HCV) is an important etiologic agent for chronic liver diseases. The aim of this study was to evaluate the clinical usefulness of second-generation HCV core antigen assay by comparing the results of the assay with those of the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0). HCV core antigen was detectable by this assay in 142/149 (95.3%) of serotype 1 (3821+/-322 fmol/l; mean+/-SD), in 56/58 (96.6%) of serotype 2 (2589+/-449 fmol/l), and in 6/6 (100%) of serotypes 1+2 (1240+/-548 fmol/l). The HCV core antigen levels measured by this assay correlated well with the HCV RNA levels by COBAS v2.0 (r=0.848, P<0.0001). In relation to the outcome of interferon monotherapy, the pretreatment HCV core antigen levels of sustained and non-sustained virological responders were 659+/-189 and 4904+/-376 fmol/l in serotype 1, 1993+/-740 and 3145+/-519 fmol/l in serotype 2. The cutoff values with the best accuracy for HCV core Ag levels to discriminate between sustained and non-sustained virological response were 699 fmol/l for serotype 1 and 292 fmol/l for serotype 2, respectively, by receiver operating characteristic curve analysis. This new assay was considered to be useful in evaluating the HCV levels in patients with chronic hepatitis C.

  15. Comparison of a PCR serotyping assay, Check&Trace assay for Salmonella, and Luminex Salmonella serotyping assay for the characterization of Salmonella enterica identified from fresh and naturally contaminated cilantro.

    PubMed

    Jean-Gilles Beaubrun, J; Ewing, L; Jarvis, K; Dudley, K; Grim, C; Gopinath, G; Flamer, M-L; Auguste, W; Jayaram, A; Elmore, J; Lamont, M; McGrath, T; Hanes, D E

    2014-09-01

    Salmonella enterica isolated from fresh cilantro samples collected through the USDA/AMS Microbiological Data Program (MDP) were used to compare a PCR serotyping assay against the Check&Trace assay and the Luminex (BioPlex) Salmonella serotyping assay. The study was conducted to evaluate the effectiveness of the three methods for serotyping Salmonella from both enrichment broth cultures and pure Salmonella cultures. In this investigation, Salmonella spp. serotyping was conducted using 24 h enrichment broth cultures and pure Salmonella cultures from cilantro samples, with the PCR serotyping assay. Conversely, the Check&Trace and Luminex for Salmonella assays required pure cultures for Salmonella serotyping. The cilantro samples contained S. enterica serovar Montevideo, Newport, Saintpaul, and Tennessee, identified by the PCR serotyping assay and Check&Trace for Salmonella, but the Luminex assay only identified two of the four serotypes of the cilantro samples. The anticipated impact from this study is that the PCR serotyping assay provides a time- and cost-effective means for screening, identifying and serotyping Salmonella using DNA extracted from 24 h enrichment cilantro samples.

  16. 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.

    PubMed

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

    2017-07-26

    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) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  17. A comparison of the human buccal cell assay and the pollen abortion assay in assessing genotoxicity in an urban-rural gradient.

    PubMed

    Fleck, Alan da Silveira; Vieira, Mariana; Amantéa, Sergio Luís; Rhoden, Claudia Ramos

    2014-08-27

    Air pollution is exacerbated near heavy traffic roads in cities. Air pollution concentration and composition vary by region and depend on urban-rural gradients. The aim of this study was to evaluate the distribution of air pollution in areas of varying population densities and to compare plant biomonitoring with an established biomarker of human exposure to traffic-related air pollution in children. The areas of study were selected near a major street in 3 different regions. Areas A, B and C represent high, intermediate and low population densities, respectively. Micronucleus assay, an established biomarker of human exposure, was performed in children from these areas. For a plant biomonitoring assay, the pollen abortion assay was performed on Bauhinia variegata in these areas. NO2 and O3 concentrations were determined by passive sampling. We report here that the pollen abortion frequency in Bauhinia variegata is correlated with NO2 concentration (P = 0.004) and is strongly associated with vehicular flow and population density in the studied areas. Micronuclei frequency in buccal cells of children was higher in the regions with more degree of urbanization (P < 0.001) following the same pattern of O3 concentrations (P = 0.030). In conclusion, our results demonstrate that high concentrations of air pollutants in Porto Alegre are related to both human and plant genotoxicity. Areas with different concentration of pollutants demonstrated to have an urbanization gradient dependent pattern which also reflected on genotoxic damage among these areas.

  18. A Comparison of the Human Buccal Cell Assay and the Pollen Abortion Assay in Assessing Genotoxicity in an Urban-Rural Gradient

    PubMed Central

    Fleck, Alan da Silveira; Vieira, Mariana; Amantéa, Sergio Luís; Rhoden, Claudia Ramos

    2014-01-01

    Air pollution is exacerbated near heavy traffic roads in cities. Air pollution concentration and composition vary by region and depend on urban-rural gradients. The aim of this study was to evaluate the distribution of air pollution in areas of varying population densities and to compare plant biomonitoring with an established biomarker of human exposure to traffic-related air pollution in children. The areas of study were selected near a major street in 3 different regions. Areas A, B and C represent high, intermediate and low population densities, respectively. Micronucleus assay, an established biomarker of human exposure, was performed in children from these areas. For a plant biomonitoring assay, the pollen abortion assay was performed on Bauhinia variegata in these areas. NO2 and O3 concentrations were determined by passive sampling. We report here that the pollen abortion frequency in Bauhinia variegata is correlated with NO2 concentration (P = 0.004) and is strongly associated with vehicular flow and population density in the studied areas. Micronuclei frequency in buccal cells of children was higher in the regions with more degree of urbanization (P < 0.001) following the same pattern of O3 concentrations (P = 0.030). In conclusion, our results demonstrate that high concentrations of air pollutants in Porto Alegre are related to both human and plant genotoxicity. Areas with different concentration of pollutants demonstrated to have an urbanization gradient dependent pattern which also reflected on genotoxic damage among these areas. PMID:25166920

  19. Measuring permeability with a whole cell-based biosensor as an alternate assay for angiogenesis: comparison with common in vitro assays.

    PubMed

    Ghosh, Gargi; Mehta, Ishan; Cornette, Abagail L; Anderson, Kimberly W

    2008-02-28

    Angiogenesis plays cardinal role in normal developmental processes as well as in numerous pathologies. Multiple cytokines are released and act simultaneously to activate endothelial cells in vivo. The present study investigated the relative ability of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), tumor necrosis factor-alpha (TNF-alpha) in modulating cell monolayer permeability, migration, proliferation and tube formation individually and in combination. While the common methods for assaying angiogenesis were conducted for studying cell migration, proliferation and differentiation, endothelial cell monolayer permeability studies were carried out using a whole cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell monolayer permeability dysfunction to detect the presence of small quantities of cytokines. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. The response obtained after exposing this sensor to different cytokines for 1 and 3h, can be attributed to the modulation of monolayer permeability by these cytokines. The present study demonstrated that at the concentrations experimented with, the relative change in permeability assay in the presence of cytokines compared to the control was much higher than that observed in other assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis.

  20. Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm

    SciTech Connect

    Haas, G.G. Jr.; D'Cruz, O.J.; DeBault, L.E. )

    1991-02-01

    The relative sensitivities of the indirect immunobead test, the indirect flo cytometric immunofluorescence assay, and an indirect radiolabeled antiglobulin assay were compared. Eighteen immunobead test positive sera and 18 negative sera were used as the standard for the other two assays. Of the 18 positive sera, 14 (77%) and 5 (27%) were positive in the immunofluorescence assay and the radiolabeled antiglobulin assay, respectively. Four (22%) of the low titer immunobead test positive sera were negative by both the immunofluorescence assay and the radiolabeled antiglobulin assay. However, there was a significant positive correlation between the results of the immunofluorescence assay and the radiolabeled antiglobulin assay (r = 0.73) and between the results of the radiolabeled antiglobulin assay and the titer of the immunobead test (r = 0.82). The use of an unselected sperm population in the radiolabeled antiglobulin assay and the classical indirect immunofluorescence method using methanol-fixed sperm gave false-positive results in the radiolabeled antiglobulin assay and the immunofluorescence assay. These results suggested that immunoglobulin G antisperm antibody positive sera may be reactive both to sperm surface and internalized sperm antigens.

  1. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

    PubMed Central

    Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael

    2016-01-01

    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569

  2. Performance comparison of the versant HCV genotype 2.0 assay (LiPA) and the abbott realtime HCV genotype II assay for detecting hepatitis C virus genotype 6.

    PubMed

    Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying; Wei, Lai

    2014-10-01

    The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

    PubMed Central

    Yang, Ruifeng; Cong, Xu; Du, Shaocai; Fei, Ran; Rao, Huiying

    2014-01-01

    The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1. PMID:25100817

  4. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    EPA Science Inventory

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  5. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    EPA Science Inventory

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  6. Assay design affects the interpretation of T-cell receptor gamma gene rearrangements: comparison of the performance of a one-tube assay with the BIOMED-2-based TCRG gene clonality assay.

    PubMed

    Cushman-Vokoun, Allison M; Connealy, Solomon; Greiner, Timothy C

    2010-11-01

    Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay.

  7. Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes.

    PubMed

    Nasci, Roger S; Gottfried, Kristy L; Burkhalter, Kristen L; Kulasekera, Varuni L; Lambert, Amy J; Lanciotti, Robert S; Hunt, Ann R; Ryan, Jeffrey R

    2002-12-01

    Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed.

  8. Comparison of indirect immunofluorescent-antibody assay, enzyme-linked immunosorbent assay, and Western immunoblot for the diagnosis of Lyme disease in dogs.

    PubMed Central

    Lindenmayer, J; Weber, M; Bryant, J; Marquez, E; Onderdonk, A

    1990-01-01

    Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody assay (IFA), and Western immunoblot were used to test serum samples from 128 dogs for the presence of antibody to Borrelia burgdorferi. Sera included 72 samples from dogs suspected of having Lyme disease, 32 samples from dogs residing in areas in which Lyme disease was not considered endemic, and 24 samples from dogs with clinical and serologic evidence of immune-mediated disease (n = 10), Rocky Mountain spotted fever (n = 5), or leptospirosis (n = 9). Results of Western immunoblotting were used as the standard against which performances of ELISA and IFA were measured. ELISA was significantly more sensitive than IFA (84.8 versus 66.7%), although both tests were equally specific (93.5%). Eight samples that were positive by Western immunoblot were simultaneously negative by ELISA and IFA. Of these eight, four were from dogs suspected of having immune-mediated disease, two were from dogs suspected of having leptospirosis, and two were from dogs suspected of having Lyme disease. These results may indicate that sera from dogs with immune-mediated disease, and to a lesser extent sera from those with leptospirosis, cross-react with B. burgdorferi antigens. Alternatively, Western immunoblot results may not truly reflect Lyme disease status, particularly in the case of dogs with immune-mediated diseases. At present, however, the use of Western immunoblotting as a diagnostic standard for dogs offers the best alternative to a clinical definition of disease. PMID:2405018

  9. Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay.

    PubMed

    Lumor, Stephen E; Fredrickson, Neal R; Ronningen, Ian; Deen, Bronwyn D; Smith, Kenneth; Diez-Gonzalez, Francisco; Labuza, Theodore P

    2012-06-01

    This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

  10. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  11. Anti-cyclic citrullinated peptide antibodies: a comparison of different assays for the diagnosis of rheumatoid arthritis.

    PubMed

    Debaugnies, F; Servais, G; Badot, V; Noubouossie, D; Willems, D; Corazza, F

    2013-01-01

    Anti-cyclic citrullinated peptide (anti-CCP) antibodies are highly specific markers of rheumatoid arthritis (RA). Considering the heterogeneity of the target antigens involved, and the test platforms and conjugates proposed in commercial anti-CCP assays, we assessed the diagnostic performances of four fully automated anti-CCP assays in a cohort of patients with RA compared to patients with other autoimmune and inflammatory disorders. We also evaluated the agreement between the qualitative results of these immunoassays. We evaluated three anti-CCP2 assays [Eurodiagnostica enzyme-linked immunosorbent assay (ELISA), Elecsys electrochemiluminescence immunoassay (ECLIA) on the Modular E170 Analyzer, and Zenit chemiluminescence immunoassay (CLIA) on the Zenit RA Analyzer] and one anti-CCP3 assay (Inova ELISA). ELISAs were performed on an automated workstation. Samples from 112 patients with RA and a disease control group of 136 patients (53 with autoimmune diseases, 65 non-autoimmune disorders, and 18 infectious diseases) were studied (included 161 samples submitted consecutively to the laboratory). At a fixed specificity of 92%, the anti-CCP3 assay presented the highest sensitivity (75%) compared to the anti-CCP2 assays evaluated (63-72%). The Zenit anti-CCP2 assay gave the most false-positive results (especially in patients with viral infections and connective tissue diseases). The agreement between assays ranged from 86.3% to 95.2% and Kappa coefficients ranged from 0.724 to 0.899. Recently released automated workstations provide a valuable alternative to ELISA to diagnose RA. However, differences in diagnostic performances are highlighted in our experience, especially for the Zenit assay. In our cohort, the anti-CCP3 assay gave slightly better performances than the anti-CCP2 assays (with the exception of the Zenit assay).

  12. Comparison of amino acid digestibility of feedstuffs determined with the precision-fed cecectomized rooster assay and the standardized ileal amino acid digestibility assay.

    PubMed

    Kim, E J; Utterback, P L; Applegate, T J; Parsons, C M

    2011-11-01

    The objective of this study was to evaluate and compare amino acid digestibility of several feedstuffs using 2 commonly accepted methods: the precision-fed cecectomized rooster assay (PFR) and the standardized ileal amino acid assay (SIAAD). Six corn, 6 corn distillers dried grains with or without solubles (DDGS/DDG), one wet distillers grains, one condensed solubles, 2 meat and bone meal (MBM) and a poultry byproduct meal were evaluated. Due to insufficient amounts, the wet distillers grains and condensed solubles were only evaluated in roosters. Standardized amino acid digestibility varied among the feed ingredients and among samples of the same ingredient for both methods. For corn, there were generally no differences in amino acid digestibility between the 2 methods. When differences did occur, there was no consistent pattern among the individual amino acids and methods. Standardized amino acid digestibility was not different between the 2 methods for 4 of the DDG samples; however, the PFR yielded higher digestibility values for a high protein DDG and a conventionally processed DDGS. The PFR yielded higher amino acid digestibility values than the SIAAD for several amino acids in 1 MBM and the poultry byproduct meal, but it yielded lower digestibility values for the other MBM. Overall, there were no consistent differences between methods for amino acid digestibility values. In conclusion, the PFR and SIAAD methods are acceptable for determining amino acid digestibility. However, these procedures do not always yield similar results for all feedstuffs evaluated. Thus, further studies are needed to understand the underlying causes in this variability.

  13. Serologic testing for avian influenza viruses in wild birds: comparison of two commercial competition enzyme-linked immunosorbent assays.

    PubMed

    Pérez-Ramírez, Elisa; Rodríguez, Vanessa; Sommer, Dagmar; Blanco, Juan Manuel; Acevedo, Pelayo; Heffels-Redmann, Ursula; Höfle, Ursula

    2010-03-01

    Serologic testing of wild birds for avian influenza virus (AIV) surveillance poses problems due to species differences and nonspecific inhibitors that may be present in sera of wild birds. Recently available competitive enzyme-linked immunosorbent assay (cELISA) kits offer a new species-independent approach. In this study we compare two commercial competitive cELISAs, using a total of 184 serum and plasma samples from 23 species of wild birds belonging to 10 orders. Thirteen samples were from experimentally high pathogenicity AI and low pathogenicity AI infected red-legged partridges (Alectoris rufa), 77 samples were from a flock of sentinel hybrid ducks confirmed infected by AI by real-time PCR, and 94 samples were from wild birds admitted to a rehabilitation center. Both ELISAs detected AI antibodies in the experimentally infected partridges, whereas hemagglutination inhibition (HI) was negative. Concordance in results between the two ELISAs was 51.5%. When specific subtype-H5/H7 HI-positive samples were considered for comparison, ELISA 1 appeared to perform better on ducks, whereas ELISA 2 appeared to perform better in other wild bird species. Overall, 68.2% of H5/H7 positive samples tested positive by ELISA 1 and 36% by ELISA 2. Both ELISAs detected AIV-antibody-positive samples negative by specific HI against 9 of the 16 existing hemagglutinin (HA) subtypes. Presumably this reflects either higher sensitivity of cELISA when compared to HI, presence of antibodies against HA subtypes not tested, or unspecific reactions. Performance of ELISA 1 on ducks appears to be comparable to in-house cELISA previously used by other authors in wild birds, but requires a relatively large sample volume. Alternatively, although ELISA 2 required a smaller sample volume, it was less effective at identifying HI-positive samples. The results reflect the necessity of validation of cELISA tests for individual species or at least families, as required by the OIE.

  14. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

    PubMed

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Multiplex fluorescent immunoassay of 139 patients' blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy. The results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract. Multiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

  15. Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay.

    PubMed

    Gould, Matthew K; Vu, Xuan Lan; Seebeck, Thomas; de Koning, Harry P

    2008-11-15

    The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC(50) values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC(50) values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.

  16. Determination of glutamic acid decarboxylase antibodies (GADA) IgG subclasses – comparison of three immunoprecipitation assays (IPAs)

    PubMed Central

    Hillman, M; Törn, C; Landin-Olsson, M

    2007-01-01

    IgG subclasses of glutamic acid decarboxylase (GAD65) antibodies (GADA) may reflect the immunological state in the pancreas of GADA-positive patients with autoimmune diabetes. The use of biotin-conjugated antibodies and streptavidin Sepharose are used commonly in immunoprecipitation assays (IPA) based on 125I- or 35S-labelled antigens to capture IgG subclasses directed against IA-2 or GAD65. We have compared three different immunoprecipitation assays for the determination of GADA IgG subclasses. Two of the assays were based on the biotin and streptavidin systems provided in a solid (immobilized) or liquid (mobilized) phase binding environment. The third assay was based on N-hydroxysuccinimide (immobilized) interaction with primary amines (i.e. lysine residues) on the antibody. We found the liquid phase binding assay (LPBA) to be the most stable assay, with a comparatively low coefficient of variation and background. PMID:17666094

  17. Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

    PubMed

    Kandel, Yuba R; Haudenshield, James S; Srour, Ali Y; Islam, Kazi Tariqul; Fakhoury, Ahmad M; Santos, Patricia; Wang, Jie; Chilvers, Martin I; Hartman, Glen L; Malvick, Dean K; Floyd, Crystal M; Mueller, Daren S; Leandro, Leonor F S

    2015-12-01

    The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

  18. Performance evaluation of Siemens ADVIA Centaur enhanced estradiol assay and a split sample comparison with liquid chromatography-tandem mass spectrometry.

    PubMed

    Chen, Yu; Kinney, Lois; Soldin, Steven J

    2012-07-01

    The objective of this study was to evaluate the newly developed Siemens ADVIA Centaur enhanced Estradiol (eE2) assay and compare it with a well-established estradiol liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The Siemens eE2 assay was evaluated using the Clinical and Laboratory Standards Institute evaluation protocols. Split patient samples were compared with the eE2 assay, the current ADVIA Centaur E2-6 Ill assay; and LC-MS/MS method by API5000 mass spectrometer. Within-run and total imprecision of the eE2 assay demonstrated coefficient of variations of 5.7%, 3.2%, 1.5%, and 10.4%, 7.3%, and 6.8%, at levels of 380, 752, and 2051 pmol/L, respectively. The method comparisons showed: eE2=0.903(E2-6 III) -16.2, R(2)=0.938, average bias=-12.3%; and eE2=0.946(LC-MS/MS)+19.5, R(2)=0.925, average bias: 0%. The Siemens eE2 assay correlates well with LC-MS/MS. This method is reliable, and appropriate for routine clinical laboratory use. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  19. Comparison of enzyme-linked immunosorbent assay, DNA hybridization, hemagglutination, and electron microscopy for detection of canine parvovirus infections.

    PubMed Central

    Teramoto, Y A; Mildbrand, M M; Carlson, J; Collins, J K; Winston, S

    1984-01-01

    Canine fecal samples were analyzed by enzyme-linked immunosorbent assays (ELISA) by using monoclonal antibodies to the canine parvovirus hemagglutinating protein. These data were compared with results obtained with DNA hybridization assays, hemagglutination assays, and electron microscopy. The highest correlation was observed between the ELISA and the hemagglutination tests, with 94.4% of samples showing agreement. Lower correlation was obtained between ELISA and DNA hybridization tests (73.3%). Correlation between ELISA and electron microscopy was 60.9%. The studies indicated that the ELISA can be used as a sensitive and specific diagnostic assay for canine parvovirus infections. Images PMID:6092425

  20. Comparison of multiple assays for detecting human antibodies directed against surface antigens on normal and malignant human tissue culture cells.

    PubMed

    Rosenberg, S A; Schwarz, S; Anding, H; Hyatt, C; Williams, G M

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cell lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual and end-point cytolysis assay and the 51Chromium release assay were equally sensitive in measuring complement mediated antibody cytoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody.

  1. Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods

    PubMed Central

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q.; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. PMID:25789875

  2. Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells.

    PubMed

    Karlsson, Annika C; Martin, Jeffrey N; Younger, Sophie R; Bredt, Barry M; Epling, Lorrie; Ronquillo, Rollie; Varma, Arjun; Deeks, Steven G; McCune, Joseph M; Nixon, Douglas F; Sinclair, Elizabeth

    2003-12-01

    The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.

  3. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    PubMed

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  4. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  5. Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars.

    PubMed

    Widén, F; Everett, H; Blome, S; Fernandez Pinero, J; Uttenthal, A; Cortey, M; von Rosen, T; Tignon, M; Liu, L

    2014-10-01

    Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    USGS Publications Warehouse

    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.

  7. Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

    PubMed Central

    Phillips, Richard Odame; Badziklou, Kossi; Piten, Ebekalisai; Maman, Issaka; Sarfo, Fred Stephen; Huber, Kristina Lydia; Rhomberg, Agata; Symank, Dominik; Wagner, Magdalena; Wiedemann, Franz; Nitschke, Jörg; Banla Kere, Abiba; Herbinger, Karl-Heinz; Adjei, Ohene; Löscher, Thomas; Bretzel, Gisela

    2014-01-01

    This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence. PMID:24478404

  8. Comparison of three immunoglobulin G assays for the diagnosis of failure of passive transfer of immunity in neonatal alpacas.

    PubMed

    Pinn, Toby L; Gagliardo, Lucille F; Purdy, Steve R; Appleton, Judith A; Stokol, Tracy

    2013-01-01

    Measurement of serum immunoglobulin G (IgG) is used for the assessment of passive transfer of immunity in neonatal crias, with an IgG concentration <10 g/l being suggestive of failure of passive transfer (FPT). The purpose of the current study was to determine whether 3 commercially available immunologic assays yielded comparable results for IgG in alpacas. Serum samples from 91 alpacas were used and were stored frozen until batch analysis on the same day with the 3 assays. Immunoglobulin G was measured by radial immunodiffusion (RID) and 2 immunoturbidimetric (IT) assays (IT1, configured for automated chemistry analyzers; IT2, a point-of-care test). Median IgG concentrations were significantly different between the 3 assays, with the RID (median: 15 g/l) and IT1 (median: 16 g/l) assays, which used the same standard, yielding significantly higher IgG values than IT2 (median: 11 g/l). Results indicated a diagnostic discordance in 1-17% of samples at an IgG threshold of 10 g/l. Protein electrophoresis revealed that the RID and IT1 standard contained mostly albumin (>60%), whereas the IT2 standard consisted of beta and gamma globulins. The discrepant results between assays IT1 and IT2 were eliminated when the same standard was used (IT1: median 11 g/l; IT2: 10 g/l; n = 19 and 17, respectively). The IT1 assay had the highest precision, while the RID assay had the lowest. The results indicate that camelid IgG measurement is highly dependent on the assay standard and is not directly comparable between assays, potentially resulting in underdiagnosis of FPT in some crias.

  9. Diagnosis of Coxiella burnetii infection: comparison of a whole blood interferon-gamma production assay and a Coxiella ELISPOT.

    PubMed

    Schoffelen, Teske; Limonard, Gijs J M; Bleeker-Rovers, Chantal P; Bouwman, John J M; van der Meer, Jos W M; Nabuurs-Franssen, Marrigje; Sprong, Tom; van Deuren, Marcel

    2014-01-01

    Diagnosis of ongoing or past infection with Coxiella burnetii, the causative agent of Q fever, relies heavily on serology: the measurement of C. burnetii-specific antibodies, reflecting the host's humoral immune response. However, cell-mediated immune responses play an important, probably even more relevant, role in infections caused by the intracellular C. burnetii bacterium. Recent studies have investigated interferon-gamma (IFN-γ) based assays, including a whole-blood IFN-γ production assay and a Coxiella enzyme-linked immunospot (Coxiella ELISPOT), as potential diagnostic tools for Q fever diagnosis. Both are in-house developed assays using stimulating antigens of different origin. The main objective of this study was to compare the test performance of the IFN-γ production assay and the Coxiella ELISPOT for detecting a cellular immune response to C. burnetii in Q fever patients, and to assess the correlation between both assays. To that end, both tests were performed in a well-defined patient group of chronic Q fever patients (n = 16) and a group of healthy seronegative individuals (n = 17). Among patients, both the Coxiella ELISPOT and the IFN-γ production assay detected positive response in 14/16. Among controls, none were positive in the Coxiella ELISPOT, whereas the IFN-γ production assay detected positive results in 1/17 and 3/17, when using Henzerling and Nine Mile as stimulating antigens, respectively. These results suggest the Coxiella ELISPOT has a somewhat higher specificity than the IFN-γ production assay when Nine Mile is used as antigen stimulus. The assays showed moderate correlation: the Spearman correlation coefficient r ranged between 0.37-0.60, depending on the antigens used. Further investigation of the diagnostic potential for C. burnetii infection of both assays is warranted.

  10. Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples.

    PubMed

    Kuczynska, Ewa; Boyer, Douglas G; Shelton, Daniel R

    2003-04-01

    Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to

  11. Antiproliferative effects of 5-fluorouracil and oxaliplatin in colon cancer cell lines: comparison of three different cytotoxicity assays.

    PubMed

    Failli, A; Legitimo, A; Orsini, G; Castagna, M; Spisni, R; Miccoli, P; Consolini, R

    2013-01-01

    Adjuvant therapy in colorectal cancer has evolved to become the standard of care, whereas the tumor capability of activating effective mechanisms of defence against both chemical and physical cytotoxic agents represents a serious obstacle to the successful therapy of human tumors. Therefore, the possibility to have an assay useful to measure the drug sensitivity of tumor cells has a great importance. A number of cytotoxicity assays are currently available, each of them using a specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. The purpose of this study is to compare, under identical experimental conditions, three common cytotoxicity assays (ATP-lite, MTT and CCK-8 assays) in the assessment of the anti-proliferative effects of 5-fluorouracil (5-FU) and oxaliplatin (OHP) on three colon cancer cell lines (WiDr, SW620 and HT-29). Regarding 5-FU, the three assays were found to be significantly correlated with a moderate or high correlation coefficient, whereas in the case of OHP we found different outcomes among the assays. Our study demonstrates that the CCK-8 is the most sensitive assay for detecting changes of cell viability, suggesting that the viability measured in cells after drug exposure depends on several parameters like the drug used, the biological characteristics of the target cell and the specific approach employed by the method to detect distinct cell growth and metabolic functions.

  12. Use of limited protocols to evaluate the genotoxicity of hazardous wastes in mammalian cell assays: comparison to Salmonella

    SciTech Connect

    DeMarini, D.M.; Brusick, D.J.; Lewtas, J.

    1987-01-01

    Dichloromethane extracts of four diverse hazardous wastes (coke plant, herbicide manufacturing, pulp and paper, and oil refining) were evaluated for mutagenicity in strains TA98 and TA100 of Salmonella. These extracts also were tested for biological activity in short-term mammalian cell assays, including mutagenicity in L5178Y/TK +/- mouse lymphoma cells, chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells, morphological transformation in BALB/c-3T3 cells, and teratogenic potential in mouse limb bud cells. The mammalian cell assays were performed using limited protocols that consisted of a preliminary testing of the extracts for cytotoxicity in CHO cells in order to estimate the appropriate dose range for the other assays. These assays were then performed once with only a few doses of extract; all but the mouse limb bud assay were performed in the presence of metabolic activation. Although all four of the wastes were presumptively positive for either mutation or cytogenetic effects, none of the wastes transformed BALB/c-3T3 cells. Further studies are needed to establish which mammalian cell assays, if any, might be useful complements to the Salmonella assay for the purpose of screening hazardous wastes.

  13. Comparison of two real-time PCR assays for the detection of malaria parasites from hemolytic blood samples - Short communication.

    PubMed

    Hagen, Ralf Matthias; Hinz, Rebecca; Tannich, Egbert; Frickmann, Hagen

    2015-06-01

    We compared the performance of an in-house and a commercial malaria polymerase chain reaction (PCR) assay using freeze-thawed hemolytic blood samples. A total of 116 freeze-thawed ethylenediamine tetraacetic acid (EDTA) blood samples of patients with suspicion of malaria were analyzed by an in-house as well as by a commercially available real-time PCR. Concordant malaria negative PCR results were reported for 39 samples and malaria-positive PCR results for 67 samples. The in-house assay further detected one case of Plasmodium falciparum infection, which was negative in the commercial assay as well as five cases of P. falciparum malaria and three cases of Plasmodium vivax malaria, which showed sample inhibition in the commercial assay. The commercial malaria assay was positive in spite of a negative in-house PCR result in one case. In all concordant results, cycle threshold values of P. falciparum-positive samples were lower in the commercial PCR than in the in-house assay. Although Ct values of the commercial PCR kit suggest higher sensitivity in case of concordant results, it is prone to inhibition if it is applied to hemolytic freeze-thawed blood samples. The number of misidentifications was, however, identical for both real-time PCR assays.

  14. Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle.

    PubMed

    Tombácz, Kata; Mwangi, Duncan; Werling, Dirk; Gibson, Amanda J

    2017-05-01

    PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

  15. HPV16 Seropositivity and Subsequent HPV16 Infection Risk in a Naturally Infected Population: Comparison of Serological Assays

    PubMed Central

    Lin, Shih-Wen; Ghosh, Arpita; Porras, Carolina; Markt, Sarah C.; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Kemp, Troy J.; Pinto, Ligia A.; Gonzalez, Paula; Wentzensen, Nicolas; Esser, Mark T.; Matys, Katie; Meuree, Ariane; Quint, Wim; van Doorn, Leen-Jan; Herrero, Rolando; Hildesheim, Allan; Safaeian, Mahboobeh

    2013-01-01

    Background Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays. Methodology/Principal Findings Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27–0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28–0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection. Conclusions/Significance Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity. PMID:23301022

  16. Comparison of Quantitation of Cytomegalovirus DNA by Real-Time PCR in Whole Blood with the Cytomegalovirus Antigenemia Assay

    PubMed Central

    Kwon, Seonhee; Jung, Bo Kyeung; Ko, Sun-Young; Lee, Chang Kyu

    2015-01-01

    Background Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. Method 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. Results The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. Conclusions Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients. PMID:25553288

  17. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    PubMed

    Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

    2015-08-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

  18. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  19. Comparison of in vitro eye irritation potential by bovine corneal opacity and permeability (BCOP) assay to erythema scores in human eye sting test of surfactant-based formulations.

    PubMed

    Cater, Kathleen C; Harbell, John W

    2008-01-01

    The bovine corneal opacity and permeability (BCOP) assay can be used to predict relative eye irritation potential of surfactant-based personal care formulations relative to a corporate benchmark. The human eye sting test is typically used to evaluate product claims of no tears/no stinging for children's bath products. A preliminary investigation was conducted to test a hypothesis that the BCOP assay could be used as a prediction model for relative ranking of human eye irritation responses under conditions of a standard human eye sting test to surfactant-based formulations. BCOP assays and human eye sting tests were conducted on 4 commercial and 1 prototype body wash (BW) developed specifically for children or as mild bath products. In the human eye sting test, 10 mul of a 10% dosing solution is instilled into one eye of each panelist (n = 20), and the contralateral eye is dosed with sterile water as a control. Bulbar conjunctival erythema responses of each eye are graded at 30 seconds by an ophthalmologist. The BCOP assay permeability values (optical density at 490 nm [OD(490)]) for the 5 BWs ranged from 0.438 to 1.252 (i.e., least to most irritating). By comparison, the number of panelists exhibiting erythema responses (mild to moderately pink) ranged from 3 of 20 panelists for the least irritating BW to 10 of 20 panelists for the most irritating BW tested. The relative ranking of eye irritation potential of the 5 BWs in the BCOP assay compares favorably with the relative ranking of the BWs in the human eye sting test. Based on these findings, the permeability endpoint of the BCOP assay, as described for surfactant-based formulations, showed promise as a prediction model for relative ranking of conjunctival erythema responses in the human eye. Consequently, screening of prototype formulations in the BCOP assay would allow for formula optimization of mild bath products prior to investment in a human eye sting test.

  20. Development of candidate reference reagent for HIV-1 RNA and comparison analysis for different HIV-1 RNA quantitative assay.

    PubMed

    Park, Borae G; Park, Ae Ja; Choi, Jee-Hye; Park, Jina; Kim, Sung Soon; Wang, Jin-Sook; Kee, Mee Kyung; Choi, Ju-yeon

    2011-09-01

    Human immunodeficiency virus type-1 (HIV-1) RNA viral load is a surrogate marker that is routinely used to determine indications for, and monitor the effectiveness of HIV-1 treatment. We developed three reagents for potential use in routine quality control of HIV-1 RNA quantitative assays. In this report, we compare the stability of these re-agents in storage and compare their performance in three different HIV-1 RNA quantitative assays. The candidate reagents were derived from readily available pre-existing reagents and examined for stability at different storage temperatures. They were compared in three commercially available HIV-1 RNA quantitative assays: the Cobas TaqMan HIV-1 Test (Cobas TaqMan), the RealTime HIV-1 Assay (Abbott RealTime), and the NucliSens EasyQ HIV-1 Assay v1.1 (NucliSens EasyQ). The candidate reagent derived from an HIV culture supernatant (candidate CS) was the most stable of the three candidates and showed good reproducibility. Candidate CS yielded the highest HIV-1 titer of the three candidates in the Cobas TaqMan assay and the lowest HIV-1 titer and stability of the three candidates in the NucliSens EasyQ system. The candidate CS is the most appropriate of the three candidate reagents for quantitative testing of HIV-1 RNA. This working reagent should be useful for use in routine calibration for quality control in centers with limited financial resources. The Cobas TaqMan assay tended to yield higher viral load results than the other assays when used with our three candidate reagents.

  1. Comparison of three different commercial PCR assays for the detection of pathogens in critically ill sepsis patients.

    PubMed

    Schreiber, J; Nierhaus, A; Braune, S A; de Heer, G; Kluge, S

    2013-05-01

    The high mortality rate associated with sepsis necessitates a timely identification of the causative organism in order to optimize antimicrobial therapy. PCR assays are increasingly being used for this purpose. The aim of this study was to compare three commercially available PCR systems for the diagnosis of systemic infections. In a prospective observational study, a broad-range (SepsiTest®; Molzym, Bremen, Germany) and two multiplex PCR assays (VYOO®; SIRS-Lab, Jena, Germany and LightCycler® SeptiFast; Roche, Mannheim, Germany) were compared to blood cultures with respect to the clinical course of 50 critically ill patients with sepsis, severe sepsis or septic shock. Pathogens were detected by PCR in 12 % (SepsiTest®), 10 % (VYOO®) and 14 % (LightCycler® SeptiFast) of samples and in 26 % by blood culture. Negative results were obtained using all four methods in 32 samples (64 %) and 3 (6 %) samples were positive in all tests. Upon consideration of additional diagnostic findings and the clinical course, eight (16 %) of the positive blood culture results were deemed clinically relevant. All three PCR assays could also identify the causative organism (or a specific gene thereof) in three of these eight positive blood cultures, whereas for five of the eight, all three PCR assays were negative. In one patient with a negative blood culture, the SepsiTest®, VYOO® and LightCycler® SeptiFast assays were positive for Streptococcus species. The PCR assays appeared to be less susceptible than blood cultures to false-positive results arising from contamination with coagulase-negative staphylococcal organisms. There was some variability between the three PCR assays tested and the corresponding blood cultures with regards to the type of pathogen detected. The three PCR assays appeared to be less susceptible to false-positive results than blood cultures.

  2. Comparison of various assays for detection of AFP in differentiating patients with primary hepatocellular carcinoma from controls.

    PubMed Central

    Chan, S H; Heng, S H; Yo, S L; Oon, C J

    1980-01-01

    Four assays for the detection of alphafetoprotein (AFP) in the diagnosis of primary hepatocellular carcinoma (HCC) were evaluated. The frequency of AFP detection was compared in 89 HCC patients and in 71 patients with chronic active hepatitis/cirrhosis. Of the four assays, immunodiffusion, counterimmunoelectrophoresis (CIE), supplement CIE, and radioimmunoassay, the best for differentiating patients with HCC from those with chronic active hepatitis and cirrhosis was CIE. PMID:6159370

  3. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity.

    PubMed

    Legler, Juliette; Zeinstra, Laura M; Schuitemaker, Femke; Lanser, Peter H; Bogerd, Jan; Brouwer, Abraham; Vethaak, A Dick; De Voogt, Pim; Murk, Albertinka J; Van der Burg, Bart

    2002-10-15

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.

  4. Comparison of in vitro assays of cellular toxicity in the human hepatic cell line HepG2.

    PubMed

    Miret, Silvia; De Groene, Els M; Klaffke, Werner

    2006-03-01

    Cytotoxicity testing allows determining whether a compound or extract contains significant quantities of biologically harmful chemicals. Cytotoxicity test methods are useful for screening because they serve to separate toxic from nontoxic materials, providing predictive evidence of compound safety. However, a wide range of assays measuring different aspects of cell death is available in the market, but it is difficult to determine which one(s) to use when evaluating a selection of compounds. The objective of this study was to compare different commercially available in vitro assays for cytotoxicity in HepG2 cells according to its sensitivity, reproducibility, simplicity, cost, and speed. The assays evaluated included Alamar Blue for the measurement of mitochondrial activity, ATPlite and ViaLight for the determination of cellular adenosine triphosphate (ATP), ToxiLight as an indicator of cellular necrosis, and Caspase-3 Fluorometric Assay, Apo-ONE Caspase-3/7 Homogeneous Assay, and Caspase-Glo for the determination of caspase-3/7 activity. All assays were performed using 4 compounds of previously reported cytotoxic activity: DMSO, butyric acid, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and camptothecine. Overall, it was concluded that the best way to evaluate the potential cytotoxicity of a compound is to employ a battery of assays that focus on different aspects of cell death. In this case, the focus has been on ATP levels, cell necrosis, and capsase-3/7 activation. Many other kits are commercially available in the market for these and other aspects of necrosis and/or apoptosis. However, the use of ViaLight Plus, ToxiLight, and Caspase-3 Fluorometric Assay resulted in the most useful combination when working with HepG2 cells.

  5. Comparison of phenotypically indistinguishable but geographically distinct Neisseria meningitidis Group B isolates in a serum bactericidal antibody assay.

    PubMed

    Findlow, Jamie; Holland, Ann; Andrews, Nick; Weynants, Vincent; Sotolongo, Franklin; Balmer, Paul; Poolman, Jan; Borrow, Ray

    2007-11-01

    The "gold standard" assay for measuring serologic protection against Neisseria meningitidis group B (MenB) is the serum bactericidal antibody (SBA) assay. Of vital importance to the outcome of the SBA assay is the choice of the target strain(s), which is often chosen on the basis of phenotype or genotype. We therefore investigated the effect on the results produced by the SBA assay of using phenotypically indistinguishable but geographically distinct MenB isolates. Nine PorA P1.19,15 and 11 PorA P1.7-2,4 MenB isolates were incorporated into the SBA assay using human complement and were assayed against sera obtained either before or after outer membrane vesicle vaccination. Large differences in the results produced by the isolates in the SBA assay were demonstrated. These included differences as great as 5.8-fold in SBA geometric mean titers and in the proportions of subjects with SBA titers of >/=4. Ranges of as many as 9 SBA titers were achieved by individual sera across the panels of isolates. To determine the reasons for the differences observed, investigations into the expression of capsular polysaccharide, PorA, PorB, Opc, and lipooligosaccharide (LOS) and into LOS sialylation were completed. However, minor differences were found between strains, indicating similar expression and no antigen masking. These results have implications for the choice of MenB target strains for inclusion in future studies of MenB vaccines and highlight the requirement for standardization of target strains between laboratories.

  6. Comparison of rosetting, phagocytosis, and IgG binding assays for detection of IgG on old red cells

    SciTech Connect

    Bassel, P.; Bosman, G.; Kay, M.

    1986-03-01

    Various methods have been used for detecting or inferring the presence of IgG on senescent red cells. In the authors studies, they have used a method for directly measuring IgG on senescent red cells. In our studies, the authors have used a method for directly measuring IgG on cells (e.g. scanning immunoelectron microscopy) along with determining phagocytosis. Thus, phagocytosis is used as a biological assay for determining the biological significance of the IgG on cells. However, the phagocytosis assay as performed in the authors laboratory is tedious, time-consuming, and requires meticulous technique. In contrast, rosetting is a quick, simple assay that does not require special techniques or supplies. Therefore, the authors compared the phagocytosis assay employed by us to rosetting, and correlated each of these with the amount of IgG present on red cells as determined with an /sup 125/I protein A binding assay. Although senescent red cells were phagocytized, they did not form rosettes with K562 cells even at 25 RBC:K562. Further experiments indicated that the rosette assay depended on the RBC:K561 cell ratio and not on the amount of IgG/red cell. Rosette formation (%) at varying RBC:K562 ratios was as follows: 100:1, 81 +/- 12; 50:1, 65 +/- 18; 25:1, 34 +/- 30, 10:1, 20 +/- 33; 5:1, 15 +/- 29; 1: 1, 3 +/- 7 (n = 14). In contrast, phagocytosis of old RBC correlated well with the amount of IgG present on red cells (r = 0.96, 0.94, 0.92 and 0.94 in each of 4 different experiments with n = 16, 19, 14, and 19 respectively). Thus, the phagocytosis assay the authors have used correlates with IgG on red cells; whereas rosette formation does not.

  7. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients

    PubMed Central

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer

    2017-01-01

    Background The third-generation bio-intact parathyroid hormone (PTH) (1–84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Methods Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1–84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Results Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=−0.435, r=−0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=−0.319, r=−0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=−0.717, r=−0.688; P<0.0001, respectively). Conclusions Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. PMID:28643486

  8. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

    PubMed

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

    2017-09-01

    The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=-0.435, r=-0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=-0.319, r=-0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=-0.717, r=-0.688; P<0.0001, respectively). Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD.

  9. Comparison of Microtox and Xenoassay Light as a Near Real Time River Monitoring Assay for Heavy Metals

    PubMed Central

    Halmi, M. I. E.; Jirangon, Hussain; Johari, W. L. W.; Abdul Rachman, A. R.; Shukor, M. Y.; Syed, M. A.

    2014-01-01

    Luminescence-based assays for toxicants such as Microtox, ToxAlert, and Biotox have been used extensively worldwide. However, the use of these assays in near real time conditions is limited due to nonoptimal assay temperature for the tropical climate. An isolate that exhibits a high luminescence activity in a broad range of temperatures was successfully isolated from the mackerel, Rastrelliger kanagurta. This isolate was tentatively identified as Photobacterium sp. strain MIE, based on partial 16S rDNA molecular phylogeny. Optimum conditions that support high bioluminescence activity occurred between 24 and 30°C, with pH 5.5 to 7.5, 10 to 20 g/L of sodium chloride, 30 to 50 g/L of tryptone, and 4 g/L of glycerol as the carbon source. Assessment of near real time capability of this bacterial system, Xenoassay light to monitor heavy metals from a contaminated river running through the Juru River Basin shows near real time capability with assaying time of less than 30 minutes per samples. Samples returned to the lab were tested with a standard Microtox assay using Vibrio fishceri. Similar results were obtained to Xenoassay light that show temporal variation of copper concentration. Thus, this strain is suitable for near real time river monitoring of toxicants especially in the tropics. PMID:24977231

  10. Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q fever by quality assessment.

    PubMed

    Herremans, Tineke; Hogema, Boris M; Nabuurs, Marrigje; Peeters, Marcel; Wegdam-Blans, Marjolijn; Schneeberger, Peter; Nijhuis, Carla; Notermans, Daan W; Galama, Joep; Horrevorts, Anton; van Loo, Inge H M; Vlaminckx, Bart; Zaaijer, Hans L; Koopmans, Marion P; Berkhout, Hanneke; Socolovschi, Cristina; Raoult, Didier; Stenos, John; Nicholson, William; Bijlmer, Henk

    2013-01-01

    The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    PubMed Central

    Maleki-Ravasan, N; Oshaghi, MA; Javadian, E; Rassi, Y; Sadraei, J; Mohtarami, F

    2009-01-01

    Background We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method. Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006–2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the PCR products by Hae III enzyme. Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%). Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed. PMID:22808367

  12. Comparison of Y1 mouse adrenal cell and coagglutination assays for detection of Escherichia coli heat labile enterotoxin.

    PubMed Central

    Chapman, P A; Daly, C M

    1989-01-01

    A commercial coagglutination assay (COA; Phadebact LT-ETEC) was compared with a Y1 mouse adrenal cell assay for detecting the heat labile enterotoxin of Escherichia coli. Of four different media evaluated for use with the COA, only one (modified blood agar) gave a positive result with all strains known to produce heat labile enterotoxin. With modified blood agar, the COA detected 74 (85%) of 87 such strains. Eighty six strains negative by cell culture assay were also negative by COA, and one strain positive by COA could not be confirmed by cell culture. The Phadebact LT-ETEC kit provides a simple, sensitive, and economical method for detecting E coli heat labile enterotoxin. PMID:2668342

  13. Osteocalcin production in vivo and in vitro after 1,25-dihydroxycholecalciferol stimulation comparison of different assays.

    PubMed

    Villa, I; Banfi, G; Daverio, R; Resmini, G; Rubinacci, A

    1996-09-01

    The study was designed to assess the sensitivity of three commercial assays (which differ in methodology, standard and antibodies) for osteocalcin, used for detecting changes in osteocalcin secretion induced by calcitriol (1,25-dihydroxycholecalciferol) in vivo and in vitro. Osteocalcin levels were determined in serum samples of 10 osteoporotic women after short term calcitriol treatment, and in the culture medium of human osteoblast-like cells (n = 22) after 48 h calcitriol exposure. All assays displayed similar sensitivity in detecting osteocalcin production in vivo after a 1 microgram daily dose of calcitriol. A novel IRMA (CIS), claimed to detect intact osteocalcin, showed higher osteocalcin values than the other assays, and in vitro showed the best sensitivity; it provides an appropriate index of the osteocalcin synthetic activity of cultured human osteoblasts.

  14. Colorimetric and electrochemical phosphodiesterase inhibition assays for yessotoxin detection: development and comparison with LC-MS/MS.

    PubMed

    Campàs, Mònica; de la Iglesia, Pablo; Fernández-Tejedor, Margarita; Diogène, Jorge

    2010-03-01

    This work describes the development and applicability of two functional assays for the detection of yessotoxin (YTX), a polycyclic ether marine toxin produced by dinoflagellates. The assays are based on the interaction between this toxin and the phosphodiesterase (PDE) enzyme and the subsequent measurement of the enzyme activity by colorimetric and electrochemical methods. Firstly, several enzyme substrates were tested in order to select those able to be detected by colorimetry or electrochemistry after enzymatic hydrolysis. The substrates that provided the highest absorbance values and density currents were p-nitrophenyl phenylphosphonate and alpha-naphthyl phosphate, respectively. After optimisation of the experimental parameters, limits of detection of 0.8 and 0.6 microM were attained by colorimetry and electrochemistry, respectively. An inhibitory effect of YTX on the PDE activity was observed. The assays have been applied to the analysis of YTX production by Protoceratium reticulatum cultures, and results were compared with liquid chromatography-tandem mass spectrometry analysis.

  15. Comparison of food antioxidants and iron chelators in two cellular free radical assays: strong protection by luteolin.

    PubMed

    Hofer, Tim; Jørgensen, Trond Ø; Olsen, Ragnar L

    2014-08-20

    Liver (HepG2) cells were incubated with 21 edible flavonoids, carotenoids, polyunsaturated fatty acid (PUFA) chromones, and metal chelators for 1 h, washed in PBS, and challenged in the cellular antioxidant activity (CAA) and the cellular lipid peroxidation antioxidant activity (CLPAA) assays. These microplate format assays assess the compounds' ability to protect against cytosolic peroxyl radicals (CAA) and induced membrane lipid peroxidation (CLPAA), respectively. Incubation encompassing a broad compound concentration range determined half-maximal inhibitory concentrations (IC(50)) by using sigmoidal curve fits. Overall, considering both assays, luteolin offered the greatest protection. The carotenoid astaxanthin offered only modest protection, whereas β-carotene was ineffective. Subtle structural differences between flavonoids were found to have amplified effects on protective abilities, and mechanisms of flavonoid antioxidant action are discussed. Membrane-permeable iron chelators (deferasirox and SIH) offered strong protective effects in CLPAA, but not in CAA, suggesting that CLPAA is dependent on membrane-associated free iron ions.

  16. Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

    PubMed Central

    Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

    2012-01-01

    Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

  17. Comparison of on-treatment HCV RNA during direct antiviral therapy using two different COBAS TaqMan HCV assays.

    PubMed

    Vermehren, Johannes; Bourlière, Marc; Pol, Stanislas; Marcellin, Patrick; Hyland, Robert H; Jiang, Deyuan; Brainard, Diana M; Zeuzem, Stefan; Welzel, Tania M

    2017-04-01

    Repeated measurements of hepatitis C virus (HCV) RNA levels during antiviral therapy are recommended to monitor treatment efficacy and adherence. Throughout most direct antiviral agent (DAA) approval studies, HCV RNA cutoffs and endpoints were established with the COBAS TaqMan assay for use with the High Pure System (HPS/CTM). Different assays used in clinical practice may yield different quantitative results and possibly impact treatment decisions. The concordance of the fully-automated COBAS AmpliPrep/COBAS TaqMan assay (CAP/CTM) with HPS/CTM and its ability to predict response to DAA-treatment with ledipasvir/sofosbuvir was assessed in cirrhotic patients with HCV genotype-1-infection who had failed prior treatment with protease inhibitor-based regimens. Serum samples from patients (n=154) treated in the phase-2 SIRIUS-study were collected at baseline and during antiviral therapy (weeks 1-8), and were tested in parallel by both assays. The mean difference between HPS/CTM and CAP/CTM at baseline (n=153) was 0.32 log10 IU/mL HCV RNA. Discordant results were observed in 12% of samples collected at treatment weeks 1-8, with the greatest differences observed at weeks 2 and 4 (14% and 29%, respectively, for undetectable HCV RNA). SVR rates were 96%-97% in the study and were not significantly different between patients with detectable vs. undetectable HCV RNA according to both assays at weeks 1-4 of antiviral therapy. CAP/CTM and HPS/CTM showed significantly different response rates during the early stages of ledipasvir/sofosbuvir treatment. However, on-treatment response was not predictive of SVR with either assay, indicating that determination of on-treatment HCV RNA levels may not be useful to guide treatment decisions. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens.

    PubMed

    Rose, Alexandra; Funk, Deborah; Neiger, Reto

    2016-04-01

    To compare total protein (TP) concentrations in canine pleural and abdominal fluid specimens as measured by refractometry and biuret assay. Diagnostic test evaluation. Data regarding 92 pleural and 148 abdominal fluid specimens from dogs with various diseases. TP concentrations in fluid specimens as measured by refractometry and biuret assay were recorded. Strength of association between sets of measurements was assessed by Spearman rank correlations and Bland-Altman plots. Optimal concentration cutoff for diagnostic discrimination between exudate and nonexudate was identified by construction of receiver operating characteristic curves. Median TP concentration in pleural fluid specimens was 2.7 g/dL (range, 0.3 to 4.8 g/dL) for refractometry and 2.9 g/dL (range, 0.7 to 5.8 g/dL) for biuret assay. Median TP concentration in abdominal fluid specimens was 3.5 g/dL (range, 0.1 to 6.0 g/dL) for refractometry and 3.5 g/dL (range, 0.6 to 5.7 g/dL) for biuret assay. Correlation was significant between refractometric and biuret results for pleural (ρ = 0.921) and abdominal (ρ = 0.908) fluid. Bland-Altman plots revealed bias of -0.18 g/dL for pleural fluid and -0.03 g/dL for abdominal fluid for refractometry versus biuret assay. With a TP concentration of ≥ 3 g/dL used to distinguish exudate from nonexudate, sensitivity of refractometry was 77% for pleural fluid and 80% for abdominal fluid. Specificity was 100% and 94%, respectively. Refractometry yielded acceptable results for measurement of TP concentration in canine pleural and abdominal fluid specimens, providing a more rapid and convenient method than biuret assay.

  19. Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus.

    PubMed

    Drigo, Michele; Franzo, Giovanni; Belfanti, Ilaria; Martini, Marco; Mondin, Alessandra; Ceglie, Letizia

    2014-06-01

    The accuracy and rapid diagnosis of PRRSV infection is a major prerequisite for every control and/or eradication strategy. In this study two real time RT-PCR based on different chemistry analysis (TaqMan Probes and SYBR Green) have been developed and validated before comparison to an end point two-step RT-PCR validated previously. All assays were aimed at discrimination between PRRSV genotypes. Furthermore, an exogenous internal control (IC) system had also been implemented in qRT-PCR. A rigorous analytical validation, executed on infected cell cultures and serum, demonstrated good sensitivity, specificity and repeatability. In particular RT-PCR was exceptionally sensitive and could detect a viral titre in the order of a magnitude of 1 copies/μL, 10-fold lower than other qRT-PCR described in this study. Optimal diagnostic performances have been demonstrated analyzing samples retrieved from an experimental infection, with RT-PCR again outperforming real time RT-PCR assays. All tests, showing substantial agreement between them, were able to detect early stages of viraemia (1 DPI) and some animals were classified as positive until the end of the study (76 DPI). Therefore, this supports the assays usefulness in animals with different clinical conditions and in a broad range of epidemiological scenarios. The benefits and disadvantages of different assays were also considered and discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Comparison of aerobic and anaerobic [3H]leucine incorporation assays for determining pollution-induced bacterial community tolerance in copper-polluted, irrigated soils.

    PubMed

    Aaen, Karoline Nolsø; Holm, Peter E; Priemé, Anders; Hung, Ngoc Ngo; Brandt, Kristian Koefoed

    2011-03-01

    Pollution-induced community tolerance (PICT) constitutes a sensitive and ecologically relevant impact parameter in ecotoxicology. We report the development and application of a novel anaerobic [(3) H]leucine incorporation assay and its comparison with the conventional aerobic [(3) H]leucine incorporation assay for PICT detection in soil bacterial communities. Selection of bacterial communities was performed over 42 d in bulk soil microcosms (no plants) and in rice (Oryza sativa) rhizosphere soil mesocosms. The following experimental treatments were imposed using a full factorial design: two soil types, two soil water regimes, and four Cu application rates (0, 30, 120, or 280 µg g(-1)). Bacterial communities in bulk soil microcosms exhibited similar Cu tolerance patterns when assessed by aerobic and anaerobic PICT assays, whereas aerobic microorganisms tended to be more strongly selected for Cu tolerance than anaerobic microorganisms in rhizosphere soil. Despite similar levels of water-extractable Cu, bacterial Cu tolerance was significantly higher in acid sulfate soil than in alluvial soil. Copper amendment selected for significant PICT development in soils subjected to alternate wetting and drying, but not in continuously flooded soils. Our results demonstrate that soil bacterial communities subjected to alternate wetting and drying may be more affected by Cu than bacterial communities subjected to continuous flooding. We conclude that the parallel use of anaerobic and aerobic [(3) H]leucine PICT assays constitutes a valuable improvement over existing procedures for PICT detection in irrigated soils and other redox gradient environments such as sediments and wetlands. Copyright © 2010 SETAC.

  1. Concentrations of plasma immunoglobulins in the dog as determined by laser nephelometry. Comparison with radial immunodiffusion and enzyme-linked immunosorbent assay.

    PubMed

    Ginel, P J; Margarito, J M; Lucena, R; Molleda, J M

    1997-03-01

    Concentrations of immunoglobulins were determined by laser nephelometry and, for comparison, by single radial immunodiffusion assay and a sandwich enzyme-linked immunosorbent assay (ELISA) in plasma samples of normal dogs. Correlation between methods was high, although the mean IgM concentrations determined by laser nephelometry were significantly higher than those determined by the single radial immunodiffusion assay or ELISA. In contrast, the mean IgA concentrations were significantly lower than those determined by ELISA. Laser nephelometry was the most reproducible method. The between-assay coefficient of variation was 4.12% for IgG, 6.98% for IgM and 6.35% for IgA. Laser nephelometry and ELISA showed similar accuracies, and both were more accurate than single radial immunodiffusion. Finally, laser nephelometry was much more sensitive than single radial immunodiffusion, but less sensitive than the ELISA method. In conclusion, results of the three methods were in general comparable. The higher range of linearity precision and accuracy of laser nephelometry, and the availability of automated nephelometers make this the method of choice for quantifying canine plasma immunoglobulins.

  2. Comparison of Cobas® HPV and Anyplex™ II HPV28 assays for detecting and genotyping human papillomavirus.

    PubMed

    Pasquier, Christophe; Sauné, Karine; Raymond, Stéphanie; Boisneau, Jérôme; Courtade, Monique; Izopet, Jacques

    2017-01-01

    Persistent infection with high-risk human papillomavirus (HPV-HR) is a recognized cause of cervical cancer. The aim of this study was to compare analytical and clinical performances of the Cobas® HPV and Anyplex™ II HPV28 assays for HPV detection and genotyping. A total of 94 cervical samples were tested. For HPV-HR, the results agreed very well (94.68%), with 100% agreement when detecting CIN2+. The Anyplex™ II HPV28 assay detected more genotypes than the Cobas® HPV Test, but their clinical performances were similar. Copyright © 2016. Published by Elsevier Inc.

  3. Comparison of Limulus assay, standard plate count, and total coliform count for microbiological assessment of renovated wastewater.

    PubMed Central

    Jorgensen, J H; Lee, J C; Alexander, G A; Wolf, H W

    1979-01-01

    The Limulus endotoxin assay was compared to the standard plate count and total coliform count for assessment of the bacteriological quality of reclaimed wastewater. A total of 48 water samples from an advanced waste treatment plant in Dallas, Tex. were examined by the three techniques. Limulus assays were technically simpler to perform and provided results much sooner than conventional culture methods. However, the endotoxin values did not correlate extremely well with determinations of viable bacterial numbers. This lack of correlation may have been due to alterations in the normal ratio of viable gram-negative cells to endotoxin caused by water reclamation procedures. PMID:384901

  4. A novel method for the determination of chemical purity and assay of menaquinone-7. Comparison with the methods from the official USP monograph.

    PubMed

    Jedynak, Łukasz; Jedynak, Maria; Kossykowska, Magdalena; Zagrodzka, Joanna

    2017-02-20

    An HPLC method with UV detection and separation with the use of a C30 reversed phase analytical column for the determination of chemical purity and assay of menaquinone-7 (MK7) in one chromatographic run was developed. The method is superior to the methods published in the USP Monograph in terms of selectivity, sensitivity and accuracy, as well as time, solvent and sample consumption. The developed methodology was applied to MK7 samples of active pharmaceutical ingredient (API) purity, MK7 samples of lower quality and crude MK7 samples before purification. The comparison of the results revealed that the use of USP methodology could lead to serious overestimation (up to a few percent) of both purity and MK7 assay in menaquinone-7 samples.

  5. Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations.

    PubMed

    Maki, Katsuyuki; Holmes, Ann R; Watabe, Etsuko; Iguchi, Yumi; Matsumoto, Satoru; Ikeda, Fumiaki; Tawara, Shuichi; Mutoh, Seitaro

    2007-01-01

    The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.

  6. ASSAYS FOR ENDOGENOUS COMPONENTS OF HUMAN MILK: COMPARISON OF FRESH AND FROZEN SAMPLES AND CORRESPONDING ANALYTES IN SERUM

    EPA Science Inventory

    Breast milk is a primary source of nutrition that contains many endogenous compounds that may affect infant development. The goals of this study were to develop reliable assays for selected endogenous breast milk components and to compare levels of those in milk and serum collect...

  7. CONFIRMATIONAL IDENTIFICATION OF ESCHERICHIA COLI, A COMPARISON OF GENOTYPIC AND PHENOTYPIC ASSAYS FOR GLUTAMATE DECARBOXYLASE AND B-D-GLUCURONIDASE

    EPA Science Inventory

    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  8. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction.

    PubMed

    Gregory, Carl A; Gunn, W Grady; Peister, Alexandra; Prockop, Darwin J

    2004-06-01

    Alizarin red S (ARS) staining has been used for decades to evaluate calcium-rich deposits by cells in culture. It is particularly versatile in that the dye can be extracted from the stained monolayer and assayed. This study describes a sensitive method for the recovery and semiquantification of ARS in a stained monolayer by acetic acid extraction and neutralization with ammonium hydroxide followed by colorimetric detection at 405 nm. This method was three times more sensitive than an older method involving cetylpyridinium chloride (CPC) extraction and resulted in a better signal to noise ratio, especially for weakly stained monolayers. The assay facilitates detailed inspection of mineralization by phase microscopy and semiquantification of the entire monolayer by extraction and quantification. The sensitivity of the assay is improved by the extraction of the calcified mineral at low pH and, since the mineral is already stained in a quantitative manner, there is no requirement for an additional colorimetric quantification step. Furthermore, the linear range is much wider than those of conventional assays for calcium, making dilutions of mineral extracts prior to measurement unnecessary. It has a wide range of potential uses including tumor characterization, mesenchymal stem cell evaluation, and osteogenic compound screening. Although more labor intensive than CPC extraction, the protocol is more sensitive and yields more reliable results for weakly mineralizing samples.

  9. A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods

    PubMed Central

    Yarrow, Justin C; Perlman, Zachary E; Westwood, Nicholas J; Mitchison, Timothy J

    2004-01-01

    Background Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. Results We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. Conclusions The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail. PMID:15357872

  10. CONFIRMATIONAL IDENTIFICATION OF ESCHERICHIA COLI, A COMPARISON OF GENOTYPIC AND PHENOTYPIC ASSAYS FOR GLUTAMATE DECARBOXYLASE AND B-D-GLUCURONIDASE

    EPA Science Inventory

    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  11. ASSAYS FOR ENDOGENOUS COMPONENTS OF HUMAN MILK: COMPARISON OF FRESH AND FROZEN SAMPLES AND CORRESPONDING ANALYTES IN SERUM

    EPA Science Inventory

    Breast milk is a primary source of nutrition that contains many endogenous compounds that may affect infant development. The goals of this study were to develop reliable assays for selected endogenous breast milk components and to compare levels of those in milk and serum collect...

  12. Evaluation and comparison of three enzyme-linked immunosorbent assay formats for the detection of ricin in milk and serum

    USDA-ARS?s Scientific Manuscript database

    When applied to the detection of a specific protein toxin in food or biological fluids in the incidence of a potential contamination, it is crucial that the assay be both sensitive and specific. In order to identify an immunoassay which is sensitive, simple, and accurate for the detection of ricin i...

  13. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method ...

  14. Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster

    EPA Science Inventory

    There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method p...

  15. High performance liquid chromatography with two simultaneous on-line antioxidant assays: Evaluation and comparison of espresso coffees

    SciTech Connect

    Barnett, Neil W; Gritti, Fabrice; Guiochon, Georges A; Shalliker, R. Andrew

    2010-01-01

    The antioxidant profiles of various espresso coffees were established using HPLC with UV-absorbance detection and two rapid, simultaneous, on-line chemical assays that enabled the relative reactivity of sample components to be screened. The assays were based on (i) the colour change associated with reduction of the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH{sm_bullet}); and (ii) the emission of light (chemiluminescence) upon reaction with acidic potassium permanganate. Results from the two approaches were similar and reflected the complex array of antioxidant species present in the samples. However, some differences in selectivity were observed. Chromatograms generated with the chemiluminescence assay contained more peaks, which was ascribed to the greater sensitivity of the reagent towards minor, readily oxidisable sample components. The three coffee samples produced closely related profiles, signifying their fundamentally similar chemical compositions and origin. Nevertheless, the overall intensity and complexity of the samples in both UV absorption and antioxidant assay chromatograms were aligned with the manufacturers description of flavour intensity and character.

  16. Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

    USDA-ARS?s Scientific Manuscript database

    Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific...

  17. Comparison of a new immunoturbidometric assay of human serum lipoprotein (a) to the ELISA and the IRMA methods.

    PubMed

    Sundvall, J; Sulonen, G B; Hiltunen, O; Kiuru, J; Pursiainen, M; Jauhiainen, M

    1995-04-01

    In the present work we have tested a new immunoturbidometric (IT) lipoprotein(a) (Lp(a)) assay and compared it with two other Lp(a) assay systems (IRMA and ELISA) commonly used in clinical chemistry laboratories. In addition, we have examined the effects of long-term storage and plasminogen on the results of Lp(a). Intra-assay and inter-assay coefficients of variation of the IT method were from 1.6 to 5.5% and from 2.8 to 10.7% depending on the concentration of Lp(a). The correlations between methods were 0.957 (IRMA vs. IT), 0.969 (IRMA vs. ELISA) and 0.956 (IT vs. ELISA) and the regression curves were IRMA = 1.07*IT + 11, IRMA = 1.84*ELISA + 2 and IT = 1.62*ELISA + 17, respectively. Storage of the samples for 5 years at -70 degrees C did not affect serum Lp(a) levels. There was a slight increasing effect of high concentrations of plasminogen on the Lp(a) results, but on physiological serum levels of plasminogen the effect was not significant. We conclude that the IT method provides a simple way to screen serum Lp(a) levels.

  18. Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

    PubMed

    Whiley, Phillip J; de la Hoya, Miguel; Thomassen, Mads; Becker, Alexandra; Brandão, Rita; Pedersen, Inge Sokilde; Montagna, Marco; Menéndez, Mireia; Quiles, Francisco; Gutiérrez-Enríquez, Sara; De Leeneer, Kim; Tenés, Anna; Montalban, Gemma; Tserpelis, Demis; Yoshimatsu, Toshio; Tirapo, Carole; Raponi, Michela; Caldes, Trinidad; Blanco, Ana; Santamariña, Marta; Guidugli, Lucia; de Garibay, Gorka Ruiz; Wong, Ming; Tancredi, Mariella; Fachal, Laura; Ding, Yuan Chun; Kruse, Torben; Lattimore, Vanessa; Kwong, Ava; Chan, Tsun Leung; Colombo, Mara; De Vecchi, Giovanni; Caligo, Maria; Baralle, Diana; Lázaro, Conxi; Couch, Fergus; Radice, Paolo; Southey, Melissa C; Neuhausen, Susan; Houdayer, Claude; Fackenthal, Jim; Hansen, Thomas Van Overeem; Vega, Ana; Diez, Orland; Blok, Rien; Claes, Kathleen; Wappenschmidt, Barbara; Walker, Logan; Spurdle, Amanda B; Brown, Melissa A

    2014-02-01

    Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

  19. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

    PubMed

    Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

    2013-12-01

    Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

  20. Comparison of manual and automated nucleic acid isolation methods for HBV-DNA and HCV-RNA assays.

    PubMed

    Yagmur, Gulhan; Altun, Hatice Uludag; Gökahmetoglu, Selma; Basok, Ela

    2015-09-01

    In the diagnosis and monitoring of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, it is important to use methods that can provide rapid and reliable results. The present study aimed to compare the automated and manual extraction methods during the nucleic acid isolation phase for HBV-DNA and HCV-RNA assays. The study included 93 serum samples, 49 of which were for the HBV-DNA assay and 44 for the HCV-RNA assay. DNA and RNA isolation from the samples was performed manually with a "QIAmpMin Elute Kit" (Qiagen, Germany) and the automated isolation system, NucliSens easyMAG (BioMérieux, France). All the extraction products were amplified using the iCycler device (Bio-Rad, USA). With both methods, compliance was found in 21 (42.8%) samples in the HBV-DNA assay; nine (18.3%) samples had a higher amount of viral nucleic acid with the manual method, whereas 19 samples (38.7%) were found to have a higher amount of nucleic acid with the automated system. For the HCV-RNA assay, total compliance was found in 31 (70.4%) samples; 12 (27.2%) samples had a higher amount of viral nucleic acid with the manual method whereas one sample (2.2%) was found to have a higher amount of nucleic acid with the automated system. It was concluded that the NucliSens easyMAG automated isolation system can be used with confidence for nucleic acid extraction due to its higher sensitivity, providing results in a shorter time, and assured standardization.

  1. Comparison of nucleic acid amplification assays with BD affirm VPIII for diagnosis of vaginitis in symptomatic women.

    PubMed

    Cartwright, Charles P; Lembke, Bryndon D; Ramachandran, Kalpana; Body, Barbara A; Nye, Melinda B; Rivers, Charles A; Schwebke, Jane R

    2013-11-01

    A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerellavaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

  2. Comparison of Nucleic Acid Amplification Assays with BD Affirm VPIII for Diagnosis of Vaginitis in Symptomatic Women

    PubMed Central

    Lembke, Bryndon D.; Ramachandran, Kalpana; Body, Barbara A.; Nye, Melinda B.; Rivers, Charles A.; Schwebke, Jane R.

    2013-01-01

    A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerellavaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis. PMID:23985917

  3. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts

    PubMed Central

    Redmile-Gordon, M.A.; Armenise, E.; White, R.P.; Hirsch, P.R.; Goulding, K.W.T.

    2013-01-01

    Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts. PMID:24302786

  4. Comparison of in vivo (Draize method) and in vitro (Corrositex assay) dermal corrosion values for selected industrial chemicals.

    PubMed

    Stobbe, Jody L; Drake, Kevin D; Maier, Kurt J

    2003-01-01

    Skin irritation is a common occupational hazard for employees engaged in the manufacture, transport, and use of industrial chemicals. The most common method used to evaluate dermal irritation and/or corrosion has typically been in vivo tests using rabbits (Draize method). Several in vitro test methods have been developed, with Corrositex being the first to gain approval by a regulatory agency (U.S. Department of Transportation). The purpose of this study was to compare the results of in vitro (Corrositex) assays of dermal irritation/corrosion to in vivo test data for several industrial chemical formulations and to determine the predictability and usefulness of the Corrositex assay for these types of products. Twenty-four (24) formulations were qualified, categorized, and evaluated using the Corrositex method and the results compared to available animal data for each of the formulations. The Corrositex assay accurately predicted a corrosive end point in 8 (57.1%) of the 14 formulations identified as corrosive by the in vivo evaluations. Corrositex accurately predicted a noncorrosive end point for 1 (10%) of 10 formulations determined to be noncorrosive in animal studies. The Corrositex assay overpredicted the packing group for 12 (50%) of the 24 formulations, and underpredicted the packing group for 7 (29.2%) of the 24 formulations. Compared to the in vivo results, Corrositex correctly classified as corrosive or noncorrosive 37.5% of the formulations tested. A concordance of 20.8% for the packing group assignments of the evaluated formulations was calculated. The Corrositex assay did not accurately predict a corrosive end point or packing group assignment for all of the formulations used in this study. Manufacturers should assess the relevance of this method to their products prior to relying on it for compliance with hazardous material and worker safety regulations.

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

    PubMed

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

    2017-10-01

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

  6. Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

    DTIC Science & Technology

    2013-07-12

    assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference...Southeast Asia. Keywords: Immediate ex vivo Plasmodium falciparum drug susceptibility testing, HRP-2 ELISA, Malaria SYBR green fluorescence assay, Cambodia... malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates. Malar J 2012, 11:325. 22. Le

  7. Performance characteristics of the VIDAS® 25-OH Vitamin D Total assay - comparison with four immunoassays and two liquid chromatography-tandem mass spectrometry methods in a multicentric study.

    PubMed

    Moreau, Emmanuel; Bächer, Silvia; Mery, Sophie; Le Goff, Caroline; Piga, Nadia; Vogeser, Michael; Hausmann, Michael; Cavalier, Etienne

    2016-01-01

    The study was conducted to evaluate the analytical and clinical performance of the VIDAS® 25-OH Vitamin D Total assay. The clinical performance of the assay was compared with four other immunoassays against the results of two different liquid chromatography/mass spectrometry methods (LC-MS/MS) standardized to NIST reference materials. VIDAS® 25-OH Vitamin D Total assay precision, linearity, detection limits and sample matrix comparison were assessed following CLSI guidelines. For method comparison, a total of 150 serum samples ranging from 7 to 92 ng/mL were analyzed by all the methods. Correlation was studied using Passing-Bablok regression and Bland-Altman analysis. The concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and the reference LC-MS/MS method. In addition, samples containing endogenous 25(OH)D2 were used to assess each immunoassay's ability to detect this analyte. Pregnancy and hemodialysis samples were used to the study the effect of vitamin D binding protein (DBP) concentration over VIDAS® assay performance. The VIDAS® 25-OH Vitamin D Total assay showed excellent correlation to the LC-MS/MS results (y=1.01x+0.22 ng/mL, r=0.93), as obtained from two different sites and distinct LC-MS/MS methods. The limit of quantification was determined at 8.1 ng/mL. Cross-reactivity for 25(OH)D2 was over 80%. At concentrations of 10.5, 26 and 65.1 ng/mL, within-run CVs were 7.9%, 3.6% and 1.7%, while total CVs (between runs, calibrations, lots and instruments) were 16.0%, 4.5% and 2.8%. The VIDAS® performance was not influenced by altered DBP levels, though under-recovery of 25(OH)D as compared to LC-MS/MS was observed for hemodialysis samples. The VIDAS® 25-OH Vitamin D Total assay is therefore considered suitable for assessment of vitamin D status in clinical routine.

  8. Comparison of solid-phase and eluate assays to gauge the ecotoxicological risk of organic wastes on soil organisms.

    PubMed

    Domene, Xavier; Alcañiz, Josep M; Andrés, Pilar

    2008-02-01

    Development of methodologies to assess the safety of reusing polluted organic wastes in soil is a priority in Europe. In this study, and coupled with chemical analysis, seven organic wastes were subjected to different aquatic and soil bioassays. Tests were carried out with solid-phase waste and three different waste eluates (water, methanol, and dichloromethane). Solid-phase assays were indicated as the most suitable for waste testing not only in terms of relevance for real situations, but also because toxicity in eluates was generally not representative of the chronic effects in solid-phase. No general correlations were found between toxicity and waste pollutant burden, neither in solid-phase nor in eluate assays, showing the inability of chemical methods to predict the ecotoxicological risks of wastes. On the contrary, several physicochemical parameters reflecting the degree of low organic matter stability in wastes were the main contributors to the acute toxicity seen in collembolans and daphnids.

  9. Pulmonary toxicity of nanomaterials: a critical comparison of published in vitro assays and in vivo inhalation or instillation studies.

    PubMed

    Landsiedel, Robert; Sauer, Ursula G; Ma-Hock, Lan; Schnekenburger, Jürgen; Wiemann, Martin

    2014-11-01

    To date, guidance on how to incorporate in vitro assays into integrated approaches for testing and assessment of nanomaterials is unavailable. In addressing this shortage, this review compares data from in vitro studies to results from in vivo inhalation or intratracheal instillation studies. Globular nanomaterials (ion-shedding silver and zinc oxide, poorly soluble titanium dioxide and cerium dioxide, and partly soluble amorphous silicon dioxide) and nanomaterials with higher aspect ratios (multiwalled carbon nanotubes) were assessed focusing on the Organisation for Economic Co-Operation and Development (OECD) reference nanomaterials for these substances. If in vitro assays are performed with dosages that reflect effective in vivo dosages, the mechanisms of nanomaterial toxicity can be assessed. In early tiers of integrated approaches for testing and assessment, knowledge on mechanisms of toxicity serves to group nanomaterials thereby reducing the need for animal testing.

  10. Comparison of EMIT II, CEDIA, and DPC RIA assays for the detection of lysergic acid diethylamide in forensic urine samples.

    PubMed

    Wiegand, Russell F; Klette, Kevin L; Stout, Peter R; Gehlhausen, Jay M

    2002-10-01

    In an effort to determine a practical, efficient, and economical alternative for the use of a radioimmunoassay (RIA) for the detection of lysergic acid diethylamide (LSD) in human urine, the performance of two photometric immunoassays (Dade Behring EMIT II and Microgenics CEDIA) and the Diagnostics Products Corp. (DPC) RIA were compared. Precision, accuracy, and linearity of the 3 assays were determined by testing 60 replicates (10 for RIA) at 5 different concentrations below and above the 500-pg/mL LSD cut-off. The CEDIA and RIA exhibited better accuracy and precision than the EMIT II immunoassay. In contrast, the EMIT II and CEDIA demonstrated superior linearity r2 = 0.9809 and 0.9540, respectively, as compared with the RIA (r2 = 0.9062). The specificity of the three assays was assessed using compounds that have structural and chemical properties similar to LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the 144 compounds studied, the EMIT II cross-reacted with twice as many compounds as did the CEDIA and RIA. Specificity was also assessed in 221 forensic human urine specimens that previously screened positive for LSD by the EMIT II assay. Of these, only 11 tested positive by CEDIA, and 3 were positive by RIA. This indicated a comparable specificity performance between CEDIA and RIA. This also was consistent with a previously reported high false-positive rate of EMIT II (low specificity). Each of the immunoassays correctly identified LSD in 23 out of 24 human urine specimens that had previously been found to contain LSD by gas chromatography-mass spectrometry at a cut-off concentration of 200 pg/mL. The CEDIA exhibited superior precision, accuracy, and decreased cross-reactivity to compounds other than LSD as compared with the EMIT II assay and does not necessitate the handling of radioactive materials.

  11. Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

    PubMed Central

    Aldridge, Andrew; Kouroupis, Dimitrios; Churchman, Sarah; English, Anne; Ingham, Eileen; Jones, Elena

    2013-01-01

    Background aims Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. Methods Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4′,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. Results Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources. Conclusions Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low

  12. Analytic Validation of Immunohistochemical Assays: A Comparison of Laboratory Practices Before and After Introduction of an Evidence-Based Guideline.

    PubMed

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

    2017-09-01

    - Laboratories must demonstrate analytic validity before any test can be used clinically, but studies have shown inconsistent practices in immunohistochemical assay validation. - To assess changes in immunohistochemistry analytic validation practices after publication of an evidence-based laboratory practice guideline. - A survey on current immunohistochemistry assay validation practices and on the awareness and adoption of a recently published guideline was sent to subscribers enrolled in one of 3 relevant College of American Pathologists proficiency testing programs and to additional nonsubscribing laboratories that perform immunohistochemical testing. The results were compared with an earlier survey of validation practices. - Analysis was based on responses from 1085 laboratories that perform immunohistochemical staining. Of 1057 responses, 65.4% (691) were aware of the guideline recommendations before this survey was sent and 79.9% (550 of 688) of those have already adopted some or all of the recommendations. Compared with the 2010 survey, a significant number of laboratories now have written validation procedures for both predictive and nonpredictive marker assays and specifications for the minimum numbers of cases needed for validation. There was also significant improvement in compliance with validation requirements, with 99% (100 of 102) having validated their most recently introduced predictive marker assay, compared with 74.9% (326 of 435) in 2010. The difficulty in finding validation cases for rare antigens and resource limitations were cited as the biggest challenges in implementing the guideline. - Dissemination of the 2014 evidence-based guideline validation practices had a positive impact on laboratory performance; some or all of the recommendations have been adopted by nearly 80% of respondents.

  13. Genomewide linkage analysis of bipolar disorder by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay: a comparison with microsatellite marker assays and finding of significant linkage to chromosome 6q22.

    PubMed

    Middleton, F A; Pato, M T; Gentile, K L; Morley, C P; Zhao, X; Eisener, A F; Brown, A; Petryshen, T L; Kirby, A N; Medeiros, H; Carvalho, C; Macedo, A; Dourado, A; Coelho, I; Valente, J; Soares, M J; Ferreira, C P; Lei, M; Azevedo, M H; Kennedy, J L; Daly, M J; Sklar, P; Pato, C N

    2004-05-01

    We performed a linkage analysis on 25 extended multiplex Portuguese families segregating for bipolar disorder, by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay, the GeneChip Human Mapping 10K Array (HMA10K). Of these families, 12 were used for a direct comparison of the HMA10K with the traditional 10-cM microsatellite marker set and the more dense 4-cM marker set. This comparative analysis indicated the presence of significant linkage peaks in the SNP assay in chromosomal regions characterized by poor coverage and low information content on the microsatellite assays. The HMA10K provided consistently high information and enhanced coverage throughout these regions. Across the entire genome, the HMA10K had an average information content of 0.842 with 0.21-Mb intermarker spacing. In the 12-family set, the HMA10K-based analysis detected two chromosomal regions with genomewide significant linkage on chromosomes 6q22 and 11p11; both regions had failed to meet this strict threshold with the microsatellite assays. The full 25-family collection further strengthened the findings on chromosome 6q22, achieving genomewide significance with a maximum nonparametric linkage (NPL) score of 4.20 and a maximum LOD score of 3.56 at position 125.8 Mb. In addition to this highly significant finding, several other regions of suggestive linkage have also been identified in the 25-family data set, including two regions on chromosome 2 (57 Mb, NPL = 2.98; 145 Mb, NPL = 3.09), as well as regions on chromosomes 4 (91 Mb, NPL = 2.97), 16 (20 Mb, NPL = 2.89), and 20 (60 Mb, NPL = 2.99). We conclude that at least some of the linkage peaks we have identified may have been largely undetected in previous whole-genome scans for bipolar disorder because of insufficient coverage or information content, particularly on chromosomes 6q22 and 11p11.

  14. Genomewide Linkage Analysis of Bipolar Disorder by Use of a High-Density Single-Nucleotide–Polymorphism (SNP) Genotyping Assay: A Comparison with Microsatellite Marker Assays and Finding of Significant Linkage to Chromosome 6q22

    PubMed Central

    Middleton, F. A.; Pato, M. T.; Gentile, K. L.; Morley, C. P.; Zhao, X.; Eisener, A. F.; Brown, A.; Petryshen, T. L.; Kirby, A. N.; Medeiros, H.; Carvalho, C.; Macedo, A.; Dourado, A.; Coelho, I.; Valente, J.; Soares, M. J.; Ferreira, C. P.; Lei, M.; Azevedo, M. H.; Kennedy, J. L.; Daly, M. J.; Sklar, P.; Pato, C. N.

    2004-01-01

    We performed a linkage analysis on 25 extended multiplex Portuguese families segregating for bipolar disorder, by use of a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the GeneChip Human Mapping 10K Array (HMA10K). Of these families, 12 were used for a direct comparison of the HMA10K with the traditional 10-cM microsatellite marker set and the more dense 4-cM marker set. This comparative analysis indicated the presence of significant linkage peaks in the SNP assay in chromosomal regions characterized by poor coverage and low information content on the microsatellite assays. The HMA10K provided consistently high information and enhanced coverage throughout these regions. Across the entire genome, the HMA10K had an average information content of 0.842 with 0.21-Mb intermarker spacing. In the 12-family set, the HMA10K-based analysis detected two chromosomal regions with genomewide significant linkage on chromosomes 6q22 and 11p11; both regions had failed to meet this strict threshold with the microsatellite assays. The full 25-family collection further strengthened the findings on chromosome 6q22, achieving genomewide significance with a maximum nonparametric linkage (NPL) score of 4.20 and a maximum LOD score of 3.56 at position 125.8 Mb. In addition to this highly significant finding, several other regions of suggestive linkage have also been identified in the 25-family data set, including two regions on chromosome 2 (57 Mb, NPL = 2.98; 145 Mb, NPL = 3.09), as well as regions on chromosomes 4 (91 Mb, NPL = 2.97), 16 (20 Mb, NPL = 2.89), and 20 (60 Mb, NPL = 2.99). We conclude that at least some of the linkage peaks we have identified may have been largely undetected in previous whole-genome scans for bipolar disorder because of insufficient coverage or information content, particularly on chromosomes 6q22 and 11p11. PMID:15060841

  15. Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

    PubMed

    Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, M Jesús

    2016-12-01

    Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

  16. Comparison of MTT assay, flow cytometry, and RT-PCR in the evaluation of cytotoxicity of five prosthodontic materials.

    PubMed

    Wang, Xue; Xia, Yang; Liu, Laikui; Liu, Mei; Gu, Ning; Guang, Hanbing; Zhang, Feimin

    2010-11-01

    In the present study, the cytotoxic effects of five prosthodontic materials on the L929 cell line were assessed by flow cytometry (FCM), reverse transcription PCR (RT-PCR), and MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoli-umbromide) assay. The cells were treated with eluates resin (RE), pressable ceramics (PC), Co-Cr alloy-porcelain (CC), Ni-Cr alloy-porcelain (NC), and diatomite ceramics (DC). The cytotoxicity of all the materials tested by the MTT assay was grade 1. By FCM analysis, apoptosis rates of DC and PC were low, with no significant difference from the control (p > 0.05). The rest of the groups induced much higher apoptosis rates (p < 0.05), with the highest in the RE group. The necrotic cell levels of RE was also significantly increased (p < 0.05). Bcl-2 and Bax mRNA expression were determined by RT-PCR, and the Bax/Bcl-2 ratio in the DC and PC groups were not significantly different from the control (p > 0.05), whereas CC, NC, and RE groups showed significant differences (p < 0.05). Taken together, the results suggest that FCM and RT-PCR analyses can supplement the traditional MTT assay in evaluating the cytotoxicity of prosthodontic materials for selecting highly biocompatible materials. © 2010 Wiley Periodicals, Inc.

  17. Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM).

    PubMed

    Schiffmann, Christian; Hansen, Rasmus; Baumann, Sven; Kublik, Anja; Nielsen, Per Halkjær; Adrian, Lorenz; von Bergen, Martin; Jehmlich, Nico; Seifert, Jana

    2014-01-01

    Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

  18. Diagnosis of preeclampsia with soluble Fms-like tyrosine kinase 1/placental growth factor ratio: an inter-assay comparison.

    PubMed

    Andersen, Louise Bjørkholt; Frederiksen-Møller, Britta; Work Havelund, Kathrine; Dechend, Ralf; Jørgensen, Jan Stener; Jensen, Boye L; Nielsen, Jan; Lykkedegn, Sine; Barington, Torben; Christesen, Henrik Thybo

    2015-02-01

    The angiogenic factor ratio soluble Fms-kinase 1 (sFlt-1)/placental growth factor (PlGF) is a novel diagnostic tool for preeclampsia. We compared the efficacy of the KRYPTOR (BRAHMS) automated assays for sFlt-1 and PlGF with the Elecsys (Roche) assays in a routine clinical setting. Preeclamptic women (n = 39) were included shortly after the time of diagnosis. Normotensive control pregnancies were matched by gestational age (n = 76). The KRYPTOR assays performed comparably or superior to Elecsys (sFlt-1/PlGF area under the curve 0.746 versus 0.735; P = .09; for non-obese 0.820 versus 0.805, P = .047). For early-onset preeclampsia, KRYPTOR area under the curve increased to 0.929 with a 100% specificity for preeclampsia at cut-off 85 and an 88.9% sensitivity for preeclampsia at cut-off 33. For women with preeclampsia and preterm delivery or Hemolysis, Elevated Liver enzymes, Low Platelet count (HELLP) syndrome, the KRYPTOR sFlt-1/PlGF ratio was manifold increased (P < .01). The sFlt-1/PlGF ratio proved especially useful in early-onset preeclampsia, preeclampsia with preterm delivery or HELLP, and among non-obese women. Copyright © 2015 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.

  19. A comparison of FVII:C and FVIIa assays for the monitoring of recombinant factor VIIa treatment.

    PubMed

    Cid, A R; Lorenzo, J I; Haya, S; Montoro, J M; Casaña, P; Aznar, J A

    2001-01-01

    The use of recombinant factor VIIa (rFVIIa) is on the increase, not only to treat haemophilic patients with inhibitors, but also patients with other clotting disorders. However, the most appropriate method of monitoring this treatment remains a question that has yet to be resolved. We studied 24 plasma samples from patients receiving rFVIIa treatment (three had haemophilia A with inhibitors, and three a congenital FVII deficiency) and compared the results obtained from the FVII:C and FVIIa assays. Although a good correlation between the two methods was obtained (r = 0.91), the values of the FVII:C method were 1.63 higher than those of the FVIIa method, with a relatively wide margin in the interval of the FVII:C/FVIIa ratios obtained [95% confidence interval (CI) 1.38--1.88, range 0.68--3.68]. This interval became wider when we compared values of over 6 IU mL(-1), which led us to conclude that the two methods cannot be considered equivalent. As the FVIIa method specifically measures FVIIa, and FVII:C assay is known to have a wide interlaboratory variability, we believe that the FVIIa assay would be more suitable for the monitoring of rFVIIa treatment.

  20. Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

    PubMed

    Costa, Cristina; Sidoti, Francesca; Mantovani, Samantha; Gregori, Gabriella; Proietti, Alex; Ghisetti, Valeria; Cavallo, Rossana

    2016-07-01

    In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection.

  1. Latent tuberculosis infection amongst new recruits to the Chinese army: comparison of ELISPOT assay and tuberculin skin test.

    PubMed

    Wu, Xueqiong; Li, Qiaoke; Yang, Yourong; Zhang, Chuiying; Li, Juan; Zhang, Junxian; Liang, Yan; Cheng, Hongbing; Zhang, Jingzhuan; Zhu, Lin; Zhang, Guangyu; Wang, Lan

    2009-07-01

    In China, latent tuberculosis infection (LTI) is frequent. To protect the health of soldiers and monitor TB infection, new recruits were routinely examined for LTI by tuberculin skin testing (TST). This is the first report on the extent of LTI in the Chinese army comparing TST and enzyme-linked immunospot (ELISPOT) assay. New recruits to the army were interviewed, routinely examined and injected intradermally with purified protein derivative (PPD) in March 2007. At the same time, 100 male soldier volunteers were detected with ELISPOT assay using recombinant CFP-10/ESAT-6 fusion protein (rCFP-10/ESAT-6) as a stimulus. The prevalence of LTI, as estimated by TST and ELISPOT assay, was 41% and 21% of new recruits, respectively. Vaccination scars on the arms could be found in 83% of volunteers with positive TST and 19% of volunteers with negative TST. Five individuals with strongly positive TST of whom 2 had negative ELISPOT were not given chemotherapy, and were observed for 20 months. None developed active TB. The prevalence of LTI in new recruits to the Chinese army is not as high as previously reported. ELISPOT technique may be the most accurate screening method for the TB infection in China, which was not interfered by BCG vaccination.

  2. Anti-glomerular basement membrane antibodies in the diagnosis of Goodpasture syndrome: a comparison of different assays.

    PubMed

    Sinico, Renato Alberto; Radice, Antonella; Corace, Caterina; Sabadini, Ettore; Bollini, Bruna

    2006-02-01

    The role of anti-glomerular basement membrane (GBM) antibodies in the pathogenesis of Goodpasture syndrome (GPS) is firmly established. Untreated, the disease may follow a fulminating course. Early identification of patients has important implications in terms of management and prognosis. Therefore, a diagnostic test for the determination of circulating anti-GBM antibodies, of very high sensitivity and specificity, is necessary. A number of assays, using different antigenic substrates, are available, but studies comparing the 'performances' of the different tests are scarce. The aim of our work was to evaluate the sensitivity and specificity of four immunoassay-based anti-GBM antibodies kits. Thirty-four serum samples from 19 GPS patients, 41 pathological and 28 normal controls were studied retrospectively (the follow-up samples were not included in the analysis of performance data). Cut-off limits were derived from receiver operating characteristics curve analysis. All the assays showed a comparable good sensitivity (between 94.7 and 100.0%), whereas specificity varied considerably (from 90.9 to 100.0%). The better performance in terms of sensitivity/specificity was achieved by a fluorescence immunoassay which utilizes a recombinant antigen. All the assays have a good performance, with high sensitivity; however, the specificity may vary considerably.

  3. Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

    PubMed

    Wise, Stephen A; Phinney, Karen W; Tai, Susan S-C; Camara, Johanna E; Myers, Gary L; Durazo-Arvizu, Ramon; Tian, Lu; Hoofnagle, Andrew N; Bachmann, Lorin M; Young, Ian S; Pettit, Juanita; Caldwell, Grahame; Liu, Andrew; Brooks, Stephen P J; Sarafin, Kurtis; Thamm, Michael; Mensink, Gert B M; Busch, Markus; Rabenberg, Martina; Cashman, Kevin D; Kiely, Mairead; Kinsella, Michael; Galvin, Karen; Zhang, Joy Y; Oh, Kyungwon; Lee, Sun-Wha; Jung, Chae L; Cox, Lorna; Goldberg, Gail; Guberg, Kate; Prentice, Ann; Carter, Graham D; Jones, Julia; Brannon, Patsy M; Lucas, Robyn M; Crump, Peter M; Cavalier, Etienne; Merkel, Joyce; Betz, Joseph M; Sempos, Christopher T

    2017-09-01

    The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

  4. Comparison of nested and ELISA based polymerase chain reaction assays for detecting Chlamydia trachomatis in pregnant women with preterm complications.

    PubMed

    Sulaiman, S; Chong, P P; Mokhtarudin, R; Lye, M S; Wan Hassan, W H

    2014-03-01

    Identification of pregnant women infected with Chlamydia trachomatis is essential to allow early antibiotic treatment in order to prevent adverse pregnancy outcomes. In this study, two nucleic acid amplification tests (NAAT) namely nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA) were evaluated in terms of sensitivity and specificity for the detection of C. trachomatis DNA in pregnant women with preterm complications. A cross-sectional study was carried out in two public hospitals in Southern Selangor, Malaysia. Endocervical swabs obtained were subjected to DNA amplification using nested PCR (BioSewoom, Korea) and Amplicor CT/NG (Roche Diagnostic, USA). A total of 83 endocervical swabs obtained from pregnant women of less than 37 weeks gestation and presented with preterm complications were subjected to chlamydial DNA detection using both assays. The study shows that Amplicor CT/NG assay is more effective in the detection of C. trachomatis DNA from endocervical swabs compared to Biosewoom nested PCR kit. Agreement between the two assays were poor (kappa=0.094) with nested PCR showing a low sensitivity of 10.81% and a 97.83% specificity when compared to Amplicor CT/NG. The results obtained indicated that BioSewoom nested PCR was less sensitive than Amplicor CT/ NG for detecting C. trachomatis in endocervical specimens and that another more reliable test is required for confirmatory result.

  5. Comparison of ante-mortem assays to assess progression/regression of paratuberculosis in individual dairy animals.

    PubMed

    Click, Robert E; Van Kampen, Craig L

    2010-01-01

    Johne's disease, caused by Mycobacterium avium, subspecies paratuberculosis (MAP) is becoming increasingly widespread on dairy farms worldwide, due in part, to the absence of vaccine/drug or curative modalities.  This spread is of concern since MAP is at the center of a controversy as to its role in Crohn's disease.  None of the methods presently available to define paratuberculosis in cattle have been examined for their ability to assess progression/regression of any treatment or intervention of this disease   The research presented herein, therefore was designed to assess the reliability and accuracy of available ante-mortem assays to predict disease change of individual animals undergoing a probiotic, potentially therapeutic, treatment.  Paratuberculosis positive (n = 75) and negative (n = 10) animals were longitudinally monitored over their natural lifetimes with specific serum antibody and fecal shedding assays, and for development of end-stage clinical disease.  Longitudinal, increasing/decreasing serum ELISA values were associated with, and predictive of, progression/regression of disease.  Changes in fecal shedding and serum AGID were of value at only specific stages.  Documentation that ELISA-positive animals were positive for paratuberculosis was done by a compilation of ELISA-independent assays--succumbing with end-stage clinical disease, autopsy, AGID, and MAP fecal shedding.

  6. Method for detecting classes of microcystins by combination of protein phosphatase inhibition assay and ELISA: comparison with LC-MS.

    PubMed

    Mountfort, Douglas O; Holland, Patrick; Sprosen, Jan

    2005-02-01

    Depending on the class of microcystin the protein phosphatase inhibition assay shows different sensitivities to different classes of toxin. We have determined that the IC50 values obtained from dose-response curves for the inhibition of the enzyme by micro-cystin LR, nodularin, YR, and RR were 2.2, 1.8, 9 and 175 nM, respectively. When equimolar amounts of these toxins were determined by the ELISA assay with microcystin LR as the standard, the assay showed equivalence in toxin responses. However, when the toxins were determined by the protein phosphatase inhibition assay using microcystin LR as the standard, the ratios of the values determined by PP-2A to ELISA decreased in the order: nodularin (2.23) microcystin LR (1.1)> microcystin YR (0.63)> microcystin RR (0.06). When the ratios for each standard were plotted against the IC50 values, the log-log plot was negative linear, and the lowest value for the IC50 corresponded with the lowest ratio. The differential sensitivity of the PP-2A assay to the various standards was used to establish an indicative toxicity ranking (ITR) where a ranking of 1 (the highest) was assigned to ratios of > or = 0.8 or greater, and 3 (the lowest) to values < or = 0.2. The three ranking classes corresponded to toxin equivalence represented by the four standards. The new method allows not only the determination of microcystin toxins in terms of stoichiometry (ELISA) but also in terms of indicative toxicity. The method can be performed using the same instrument (e.g. multiwell fluorimeter with absorbance capability) and offers an advantage to methods presently used to determine microcystins (e.g. ELISA or LC-MS). The former has the propensity to overestimate toxicity because it measures equivalence to microcystin LR and is a stoichiometric measurement and the latter has the disadvantage in that relatively few of the microcystins that occur naturally are available as standards. The new method was applied to the analysis of sample from lakes

  7. Baseline morning cortisol level as a predictor of pituitary-adrenal reserve: a comparison across three assays.

    PubMed

    Sbardella, Emilia; Isidori, Andrea M; Woods, Conor P; Argese, Nicola; Tomlinson, Jeremy W; Shine, Brian; Jafar-Mohammadi, Bahram; Grossman, Ashley B

    2017-02-01

    The short ACTH stimulation test (250 μg) is the dynamic test most frequently used to assess adrenal function. It is possible that a single basal cortisol could be used to predict the dynamic response, but research has been hampered by the use of different assays and thresholds. To propose a morning baseline cortisol criterion of three of the most commonly used modern cortisol immunoassays - Advia Centaur (Siemens), Architect (Abbott) and the Roche Modular System (Roche) - that could predict adrenal sufficiency. Observational, retrospective cross-sectional study at two centres. Retrospective analysis of the results of 1019 Short Synacthen tests (SSTs) with the Advia Centaur, 449 SSTs with the Architect and 2050 SSTs with the Roche Modular System assay. Serum cortisol levels were measured prior to injection of 250 μg Synacthen and after 30 min. Overall, we were able to collate data from a total of 3518 SSTs in 3571 patients. Using receiver-operator curve analysis, baseline cortisol levels for predicting passing the SST with 100% specificity were 358 nmol/l for Siemens, 336 nmol/l for Abbott and 506 nmol/l for Roche. Utilizing these criteria, 589, 158 and 578 SSTs, respectively, for Siemens, Abbott and Roche immunoassays could have been avoided. We have defined assay-specific morning cortisol levels that are able to predict the integrity of the hypothalamo-pituitary-adrenal axis. We propose that this represents a valid tool for the initial assessment of adrenal function and has the potential to obviate the need for dynamic testing in a significant number of patients. © 2016 John Wiley & Sons Ltd.

  8. Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease.

    PubMed

    Flood, V H; Gill, J C; Christopherson, P A; Wren, J S; Friedman, K D; Haberichter, S L; Hoffmann, R G; Montgomery, R R

    2012-07-01

    von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Collagen binding assays (VWF:CB), however, are not part of the routine work-up for von Willebrand disease (VWD). This study presents data on collagen binding for healthy controls and VWD subjects to compare three different collagens. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen were examined for samples obtained from the Zimmerman Program. Mean VWF:CB in healthy controls was similar and highly correlated for types I, III and VI collagen. The mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen were 1.31, 1.19 and 1.21, respectively. In type 1 VWD subjects, VWF:CB was similar to VWF:Ag with mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen of 1.32, 1.08 and 1.1, respectively. For type 2A and 2B subjects, VWF:CB was uniformly low, with mean ratios of 0.62 and 0.7 for type I collagen, 0.38 and 0.4 for type III collagen, and 0.5 and 0.47 for type VI collagen. Normal ranges for type I, III and VI collagen are correlated, but higher values were obtained with type I collagen as compared with types III and VI. The low VWF:CB in type 2A and 2B subjects suggests that VWF:CB may also supplement analysis of multimer distribution. However, these results reflect only one set of assay conditions per collagen type and therefore may not be generalizable to all collagen assays. © 2012 International Society on Thrombosis and Haemostasis.

  9. Comparison of different assays to assess human papillomavirus (HPV) type 16- and 18-specific antibodies after HPV infection and vaccination.

    PubMed

    Scherpenisse, Mirte; Schepp, Rutger M; Mollers, Madelief; Mooij, Sofie H; Meijer, Chris J L M; Berbers, Guy A M; van der Klis, Fiona R M

    2013-08-01

    We compared the measurement of human papillomavirus (HPV)-specific serum antibody levels with the virus-like-particle multiplex immunoassay (VLP-MIA), competitive Luminex immunoassay (cLIA), and glutathione S-transferase (GST) L1-based MIA. Using a large panel of serum samples, these assays showed mutually good correlations for both naturally induced and vaccine-derived HPV-specific antibody levels. However, an adaptation of the GST L1-based MIA resulted in an improved correlation with both cLIA and VLP-MIA.

  10. Comparison of Different Assays To Assess Human Papillomavirus (HPV) Type 16- and 18-Specific Antibodies after HPV Infection and Vaccination

    PubMed Central

    Schepp, Rutger M.; Mollers, Madelief; Mooij, Sofie H.; Meijer, Chris J. L. M.; Berbers, Guy A. M.; van der Klis, Fiona R. M.

    2013-01-01

    We compared the measurement of human papillomavirus (HPV)-specific serum antibody levels with the virus-like-particle multiplex immunoassay (VLP-MIA), competitive Luminex immunoassay (cLIA), and glutathione S-transferase (GST) L1-based MIA. Using a large panel of serum samples, these assays showed mutually good correlations for both naturally induced and vaccine-derived HPV-specific antibody levels. However, an adaptation of the GST L1-based MIA resulted in an improved correlation with both cLIA and VLP-MIA. PMID:23740920

  11. Comparison of two highly discriminatory molecular fingerprinting assays for analysis of multiple Aspergillus fumigatus isolates from patients with invasive aspergillosis.

    PubMed

    de Valk, Hanneke A; Meis, Jacques F G M; de Pauw, Ben E; Donnelly, Peter J; Klaassen, Corné H W

    2007-05-01

    Two highly discriminatory fingerprinting assays, short tandem repeat typing and amplified fragment length polymorphism (AFLP), were compared to determine the genetic relatedness between 55 isolates of Aspergillus fumigatus obtained from 15 different patients suffering from proven invasive aspergillosis. Both techniques showed that interpatient isolates belonged to different genotypes and that intrapatient isolates from deep sites were all of the same genotype. By contrast, multiple genotypes were found among isolates originating from respiratory samples. Both techniques have specific advantages and disadvantages. AFLP is more universally applicable, but short tandem repeat analysis offers better discriminatory power and should be the preferred method for standardizing typing of clinical isolates of Aspergillus fumigatus.

  12. Comparison of real-time PCR and antigen assays for detection of hepatitis E virus in blood donors.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2014-06-01

    Hepatitis E virus (HEV) infection is recognized as an emerging and often undiagnosed disease in industrialized countries, with asymptomatic infections actually occurring in blood donors. Sensitive detection of HEV-RNA is crucial for diagnosis and monitoring of disease progression. We evaluated the analytical sensitivity and performance of three HEV RT-PCR assays (RealStar HEV reverse transcription-PCR [RT-PCR], hepatitis@ceeramTools, and ampliCube HEV RT-PCR) for screening of individuals for HEV infections (ID-nucleic acid amplification technology [ID-NAT]) and for blood donor pool screening (minipool-NAT [MP-NAT]). RNA was extracted using NucliSens easyMAG (ID-NAT) and a high-volume extraction protocol (4.8 ml, chemagic Viral 5K, MP-NAT). Three NAT assays were evaluated for ID-NAT but only two assays for MP-NAT due to inhibition of the ampliCube HEV RT-PCR kit using the corresponding RNA extract. Assays provided good analytical sensitivity, ranging from 37.8 to 180.1 IU/ml (ID-NAT) and from 4.7 to 91.2 IU/ml (MP-NAT). The applicability of HEV antigen (HEV-Ag) screening was compared to that of RT-PCR screening and detection of HEV-IgM antibodies using seroconversion panels of 10 HEV genotype 3-infected individuals. Four individuals revealed a positive HEV-Ag detection result, with corresponding viremias ranging from 1.92 E + 03 to 2.19 E + 05 IU/ml, while the progression of HEV-Ag followed that of HEV viremia. The other six individuals showed no presence of HEV-Ag although the corresponding viremias were also in the range of >1.0 E + 03. Anti-HEV-IgM antibodies were detectable in seven donors; one donor presented parallel positivities of HEV-Ag and anti-HEV IgM. The evaluated NAT methods present powerful tools providing sensitive HEV detection. Application of HEV-Ag or anti-HEV IgM screening is currently inferior for the early detection of HEV infection due to the decreased sensitivity compared to NAT methods.

  13. Comparison of Real-Time PCR and Antigen Assays for Detection of Hepatitis E Virus in Blood Donors

    PubMed Central

    Knabbe, C.; Dreier, J.

    2014-01-01

    Hepatitis E virus (HEV) infection is recognized as an emerging and often undiagnosed disease in industrialized countries, with asymptomatic infections actually occurring in blood donors. Sensitive detection of HEV-RNA is crucial for diagnosis and monitoring of disease progression. We evaluated the analytical sensitivity and performance of three HEV RT-PCR assays (RealStar HEV reverse transcription-PCR [RT-PCR], hepatitis@ceeramTools, and ampliCube HEV RT-PCR) for screening of individuals for HEV infections (ID-nucleic acid amplification technology [ID-NAT]) and for blood donor pool screening (minipool-NAT [MP-NAT]). RNA was extracted using NucliSens easyMAG (ID-NAT) and a high-volume extraction protocol (4.8 ml, chemagic Viral 5K, MP-NAT). Three NAT assays were evaluated for ID-NAT but only two assays for MP-NAT due to inhibition of the ampliCube HEV RT-PCR kit using the corresponding RNA extract. Assays provided good analytical sensitivity, ranging from 37.8 to 180.1 IU/ml (ID-NAT) and from 4.7 to 91.2 IU/ml (MP-NAT). The applicability of HEV antigen (HEV-Ag) screening was compared to that of RT-PCR screening and detection of HEV-IgM antibodies using seroconversion panels of 10 HEV genotype 3-infected individuals. Four individuals revealed a positive HEV-Ag detection result, with corresponding viremias ranging from 1.92E + 03 to 2.19E + 05 IU/ml, while the progression of HEV-Ag followed that of HEV viremia. The other six individuals showed no presence of HEV-Ag although the corresponding viremias were also in the range of >1.0E + 03. Anti-HEV-IgM antibodies were detectable in seven donors; one donor presented parallel positivities of HEV-Ag and anti-HEV IgM. The evaluated NAT methods present powerful tools providing sensitive HEV detection. Application of HEV-Ag or anti-HEV IgM screening is currently inferior for the early detection of HEV infection due to the decreased sensitivity compared to NAT methods. PMID:24740079

  14. Determination of immunoglobulin A in saliva by immunobead enzyme-linked immunosorbent assay: comparison with single radial immunodiffusion.

    PubMed Central

    Bratthall, D; Ellen, R P

    1982-01-01

    For the quantitative measurement of immunoglobulin A in human saliva, an immunobead enzyme-linked immunosorbent assay was investigated in detail. The method proved to be fast and sensitive, and it gave reproducible results. There was a strong correlation with results obtained by a single radial immunodiffusion method. The immunobead method gave 1.8 times higher values than the single radial immunodiffusion method with a lower correction factor for low-immunoglobulin A saliva and a higher factor for immunoglobulin A-rich saliva. PMID:6818248

  15. Standardization of a bottle assay--an indigenous method for laboratory and field monitoring of insecticide resistance and comparison with WHO adult susceptibility test.

    PubMed

    Elamathi, N; Barik, Tapan Kumar; Verma, Vaishali; Velamuri, Poonam Sharma; Bhatt, R M; Sharma, S K; Raghavendra, Kamaraju

    2014-10-01

    The WHO adult susceptibility test is in use for insecticide resistance monitoring. Presently, materials are being imported from the Universiti Sains Malaysia, Malaysia and sometimes it is cost prohibitive. As an alternative, we present here a method of bottle bioassay using indigenous material. Different aspects related to the assay were studied and validated in the field. Bottle assay was standardized in the laboratory by using locally sourced material and laboratory-maintained insecticide-susceptible Anopheles stephensi and Aedes aegypti strains against technical grade deltamethrin and cyfluthrin insecticides dissolved in ethanol in a range of different concentrations. The frequency of use of the deltamethrin-coated bottles and shelf-life were determined. Discriminating dose for deltamethrin and cyfluthrin was 10 μg against An. stephensi and 2 μg against Ae. aegypti females. Insecticide-coated bottles stored at 25 to 35 °C can be used for three exposures within 7 days of coating. The study carried out in the laboratory was validated on wild caught An. culicifacies in the states of Odisha and Chhattisgarh against deltamethrin-coated bottles in comparison to WHO adult susceptibility test. Results of the study indicated that deltamethrin-coated bottles were effective up to three exposures within 7 days of coating for field population and 100% mortality was recorded within 35 min as observed in laboratory studies for field collected susceptible population. Also in the WHO adult susceptibility test, 100% knock-down within 35 min and 100% mortality after 24 h holding period were observed in susceptible population, while in it was 50% knock-down in 1 h and 64% mortality after 24 h holding period for resistant population (50% mortality in bottle assay in 60 min). The bottle assay can be used as an alternative to the WHO adult susceptibility test both in the laboratory and field for monitoring insecticide resistance in mosquito vectors using locally sourced material.

  16. Comparison of the Diagnostic Value Between Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Histopathologic Examination in Sentinel Lymph Nodes for Patients With Gastric Carcinoma.

    PubMed

    Kwak, Yoonjin; Nam, Soo Kyung; Shin, Eun; Ahn, Sang-Hoon; Lee, Hee Eun; Park, Do Joong; Kim, Woo Ho; Kim, Hyung-Ho; Lee, Hye Seung

    2016-05-01

    Sentinel lymph node (SLN)-based diagnosis in gastric cancers has shown varied sensitivities and false-negative rates in several studies. Application of the reverse transcription-polymerase chain reaction (RT-PCR) in SLN diagnosis has recently been proposed. A total of 155 SLNs from 65 patients with cT1-2, N0 gastric cancer were examined. The histopathologic results were compared with results obtained by real-time RT-PCR for detecting molecular RNA (mRNA) of cytokeratin (CK)19, carcinoembryonic antigen (CEA), and CK20. The sensitivity and specificity of the multiple marker RT-PCR assay standardized against the results of the postoperative histological examination were 0.778 (95% confidence interval [CI], 0.577-0.914) and 0.781 (95% CI, 0.700-0.850), respectively. In comparison, the sensitivity and specificity of intraoperative diagnosis were 0.819 (95% CI, 0.619-0.937) and 1.000 (95% CI, 0.972-1.000), respectively. The positive predictive value of the multiple-marker RT-PCR assay was 0.355 (95% CI, 0.192-0.546) for predicting non-SLN metastasis, which was lower than that of intraoperative diagnosis (0.813, 95% CI, 0.544-0.960). The real-time RT-PCR assay could detect SLN metastasis in gastric cancer. However, the predictive value of the real-time RT-PCR assay was lower than that of precise histopathologic examination and did not outweigh that of our intraoperative SLN diagnosis. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Comparison of ante-mortem assays to assess progression/regression of paratuberculosis in individual dairy animals

    PubMed Central

    Van Kampen, Craig L

    2010-01-01

    Johne disease, caused by Mycobacterium avium, subspecies paratuberculosis (MAP) is becoming increasingly widespread on dairy farms worldwide, due in part, to the absence of vaccine/drug or curative modalities. This spread is of concern since MAP is at the center of a controversy as to its role in Crohn disease. None of the methods presently available to define paratuberculosis in cattle have been examined for their ability to assess progression/regression of any treatment or intervention of this disease. The research presented herein, therefore was designed to assess the reliability and accuracy of available ante-mortem assays to predict disease change of individual animals undergoing a probiotic, potentially therapeutic, treatment. Paratuberculosis positive (n = 75) and negative (n = 10) animals were longitudinally monitored over their natural lifetimes with specific serum antibody and fecal shedding assays, and for development of end-stage clinical disease. Longitudinal, increasing/decreasing serum ELISA values were associated with, and predictive of, progression/regression of disease. Changes in fecal shedding and serum AGID were of value at only specific stages. Documentation that ELISA-positive animals were positive for paratuberculosis was done by a compilation of ELISA-independent assays—succumbing with end-stage clinical disease, autopsy, AGID and MAP fecal shedding. PMID:21178432

  18. Prevalence and comparison of serologic assays, necropsy, and tongue examination for the diagnosis of porcine cysticercosis in Peru.

    PubMed

    Gonzalez, A E; Cama, V; Gilman, R H; Tsang, V C; Pilcher, J B; Chavera, A; Castro, M; Montenegro, T; Verastegui, M; Miranda, E

    1990-08-01

    Swine cysticercosis, a severe zoonotic disease which is part of the Taenia solium life cycle, causes major economic losses in pig husbandry. Throughout South America, farmers diagnose cysticercosis by examining the tongues of their pigs for cysticercus nodules. Farmers do not bring pigs believed to be infected to the slaughterhouse for fear of confiscation. Therefore, reliable statistics on porcine cysticercosis can only be acquired at the household level. We examined the utility of the tongue test as a diagnostic tool for porcine cysticercosis. The results of the tongue test was compared with 2 serologic methods for the detection of cysticercosis, the enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunoelectrotransfer blot assay (EITB), and with necropsy results. We examined 11 animals from an endemic area (Huancayo) and 42 animals from an area free of cysticercosis (Lima). The tongue test has a sensitivity of 70% and a specificity of 100%, the EITB a sensitivity and specificity of 100%, and the ELISA a sensitivity of 79% and a specificity of 75%. Thus, the tongue examination, being a test essentially without cost and having fair sensitivity and high specificity, can be useful in epidemiological surveys. Prevalence for porcine cysticercosis in Huancayo is 23.4% by tongue examination, 31.2% by necropsy, 37.7% by ELISA, and 51.9% by EITB.

  19. Evaluation of DNA recombinant methodologies for the diagnosis of Plasmodium falciparum and their comparison with the microscopy assay.

    PubMed

    Urdaneta, L; Guevara, P; Ramirez, J L

    1998-01-01

    Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolivar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r = 1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies.

  20. Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

    PubMed Central

    Moore, Caroline E.; Juan, Jeanette; Lin, Yanping; Gaskill, Cynthia L.; Puschner, Birgit

    2016-01-01

    Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1). A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS) detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS. PMID:27005635

  1. Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens.

    PubMed Central

    Germani, Y; Guesdon, J L; Phalente, L; Begaud, E; Moreau, J P

    1988-01-01

    Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia. False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride. Images PMID:3290242

  2. Comparison of high-pressure liquid chromatography and microbiological assay for the determination of biliary elimination of ciprofloxacin in humans.

    PubMed Central

    Brogard, J M; Jehl, F; Monteil, H; Adloff, M; Blickle, J F; Levy, P

    1985-01-01

    Serum kinetics and biliary, urinary, and fecal elimination of ciprofloxacin, a new quinolone derivative, were studied in 12 recently cholecystectomized patients provided with T-tube drainage during 24 h after oral administration of a single 500-mg dose of this substance. Drug concentrations were measured by both high-pressure liquid chromatography (HPLC) and microbiological assay. The results were comparable for the concentrations in serum (average of peaks, 2.0 +/- 0.2 micrograms/ml by HPLC and 2.3 +/- 0.3 micrograms/ml by the microbiological method) and urine (0 to 6 h, 267 +/- 74 and 241 +/- 58 micrograms/ml, respectively). This was not the case for biliary values, for which the microbiological assay yielded significantly higher concentrations than did HPLC (average of peak concentrations, 21.2 +/- 2.6 and 16.0 +/- 2.5 micrograms/ml, respectively [P less than 0.02]), nor for total 24-h biliary output (2,167 +/- 288 and 1,587 +/- 222 micrograms, respectively [P less than 0.01]). This suggests hepatic biotransformation of ciprofloxacin into microbiologically active metabolites. The apparent broad antibacterial spectrum of ciprofloxacin and its higher biliary levels than simultaneously determined serum concentrations suggest that this derivative is suitable for the treatment of biliary tract infections. PMID:2939796

  3. Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases.

    PubMed

    Moore, Caroline E; Juan, Jeanette; Lin, Yanping; Gaskill, Cynthia L; Puschner, Birgit

    2016-03-09

    Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1). A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS) detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.

  4. Evaluation and comparison of radical scavenging properties of solvent extracts from Justicia adhatoda leaf using DPPH assay.

    PubMed

    Jha, Deepak Kumar; Panda, Likun; Ramaiah, Sudha; Anbarasu, Anand

    2014-12-01

    2,2-Diphenyl-1-picrylhydrazyl (DPPH) method is routinely practiced for the assessment of antioxidant activity of compounds and their mixtures. The method is based on the spectrophotometric measurement of DPPH(·) concentration that changes resulting from the DPPH radical reaction with an antioxidant. The amount of remaining DPPH(·) in the examined system is a measure of the antioxidant activity of compounds. Our study aims at exploring the antioxidant properties of Justicia adhatoda leaf extract and comparing the results in terms of effective concentration which scavenges 50 % radical (EC50). The correlation of the activities for both cold and Soxhlet methanolic extracts is reported with DPPH assay. The antioxidant capacity of the methanolic extract derived by two different methods is positively correlated. Correlation between antioxidant capacity and phenolic content of methanolic extract in both the cases indicates the efficiency of the extraction procedure. Positive correlation and p value <0.05 validate the efficiency of the procedures and results.

  5. Comparison of Mismatch Amplification Mutation Assay with DNA Sequencing for Characterization of Fluoroquinolone Resistance in Neisseria gonorrhoeae

    PubMed Central

    Sultan, Zafar; Nahar, Shamsun; Wretlind, Bengt; Lindback, Emma; Rahman, Motiur

    2004-01-01

    A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of gyrA at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of gyrA in 117 Neisseria gonorrhoeae isolates (with ciprofloxacin MICs of 0.004 to >32 μg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the gyrA genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in N. gonorrhoeae. PMID:14766821

  6. Comparison of the antiinflammatory effects of Drosera rotundifolia and Drosera madagascariensis in the HET-CAM assay.

    PubMed

    Paper, Dietrich H; Karall, Elisabeth; Kremser, Michaela; Krenn, Liselotte

    2005-04-01

    The antiinflammatory effects of ethanol and aqueous extracts from Drosera rotundifolia and from Drosera madagascariensis were compared in vivo in the HET-CAM assay. Both extracts from D. rotundifolia and the ethanol extract from D. madagascariensis showed remarkable efficacy at doses of 500 microg/pellet. The inhibition of the inflammation by the extracts was stronger than that by 50 microg hydrocortisone/pellet. In contrast, there was only a very weak effect observed at a dose of 500 microg/pellet of the water extract from D. madagascariensis. The chemical analyses of the extracts showed that the effect cannot be attributed to naphthoquinones, but might be due to flavonoids. Ellagic acid obviously plays an important role in the antiangiogenic effect of the Drosera extracts. (c) 2005 John Wiley & Sons, Ltd.

  7. A rapid and simple assay for lamotrigine in serum/plasma by HPLC, and comparison with an immunoassay.

    PubMed

    Morgan, Phillip E; Fisher, Danielle S; Evers, Richard; Flanagan, Robert J

    2011-07-01

    Monitoring serum/plasma concentrations of lamotrigine may be useful under certain circumstances. An HPLC column packed with strong cation-exchange (SCX)-modified microparticulate silica together with a 100% methanol eluent containing an ionic modifier permits direct injection of sample extracts. An HPLC-UV method developed using this principle for the measurement of serum/plasma lamotrigine is simple, sensitive and selective. The analysis time is less than 5 min. Intra- and inter-assay precision and accuracy meet acceptance criteria, and sample stability, and potential interferences from other compounds have been evaluated. There was good agreement with consensus mean results from external quality assessment samples (n = 32). Analysis of patient samples (n = 115) using the HPLC method and the Seradyn QMS® Lamotrigine immunoassay showed that the immunoassay over-estimated lamotrigine by 21% on average.

  8. Analytic validation of a clinical-grade PTEN immunohistochemistry assay in prostate cancer by comparison with PTEN FISH.

    PubMed

    Lotan, Tamara L; Wei, Wei; Ludkovski, Olga; Morais, Carlos L; Guedes, Liana B; Jamaspishvili, Tamara; Lopez, Karen; Hawley, Sarah T; Feng, Ziding; Fazli, Ladan; Hurtado-Coll, Antonio; McKenney, Jesse K; Simko, Jeffrey; Carroll, Peter R; Gleave, Martin; Lin, Daniel W; Nelson, Peter S; Thompson, Ian M; True, Lawrence D; Brooks, James D; Lance, Raymond; Troyer, Dean; Squire, Jeremy A

    2016-08-01

    PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated, and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to four-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for the absence of PTEN gene deletion (549/602 tumors with two copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for the presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for the presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed two intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had two copies of the PTEN gene detected by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions, or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.

  9. Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

    PubMed Central

    Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F

    2017-01-01

    The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased. PMID:28243563

  10. Comparison of Xpert MTB/RIF with Line Probe Assay for Detection of Rifampin-Monoresistant Mycobacterium tuberculosis

    PubMed Central

    Rufai, Syed Beenish; Kumar, Parveen; Singh, Amit; Prajapati, Suneel; Balooni, Veena

    2014-01-01

    The MTBDRplus line probe assay (LPA) and Xpert MTB/RIF have been endorsed by the World Health Organization for the rapid diagnosis of drug-resistant tuberculosis. However, there is no clarity regarding the superiority of one over the other. In a double-blinded prospective study, we evaluated the efficacy of the Xpert MTB/RIF on samples that were first tested by LPA under the revised national tuberculosis control program of India. A total of 405 sputum samples from suspected drug-resistant tuberculosis patients were included. Of these, 285 smear-positive samples were subjected to LPA. Seventy-two (25.8%) samples showed multidrug resistance, 62 (22.2%) showed rifampin monoresistance, 29 (10.3%) showed isoniazid monoresistance, and 116 (41.5%) were pan-susceptible. Six (2.1%) of the samples gave invalid results. Of the 62 rifampin-monoresistant samples by LPA, 38 (61.4%) showed rifampin resistance, while 21 (33.8%) were found susceptible to rifampin by Xpert MTB/RIF using cartridge version G4. Three (4.8%) samples gave an error. Of the 116 pan-susceptible samples, only 83 were available for Xpert MTB/RIF testing; 4 (5.1%) were rifampin resistant, 74 (94.8%) were susceptible, and 5 (6.0%) showed an error. The 25 discrepant samples were further subjected to MGIT960 drug susceptibility testing. The MGIT960 results showed 100% agreement with LPA results but only 64.4% agreement with Xpert MTB/RIF results. Sequencing analysis of discrepant samples showed 91.3% concordance with LPA but only 8.7% concordance with the Xpert MTB/RIF assay. These findings indicate that by using Xpert MTB/RIF testing we might be underestimating the burden of drug-resistant tuberculosis and indicate that country-specific probes need to be designed to increase the sensitivity of the Xpert MTB/RIF. PMID:24648554

  11. Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

    PubMed

    Santos, Paula Fernanda Gonçalves Dos; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M

    2017-06-01

    To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

  12. Use of RNA amplification and electrophoresis for studying virus aerosol collection efficiency and their comparison with plaque assays.

    PubMed

    Jiang, Xiao; Pan, Maohua; Hering, Susanne V; Lednicky, John A; Wu, Chang-Yu; Fan, Z Hugh

    2016-10-01

    The spread of virus-induced infectious diseases through airborne routes of transmission is a global concern for economic and medical reasons. To study virus transmission, it is essential to have an effective aerosol collector such as the growth tube collector (GTC) system that utilizes water-based condensation for collecting virus-containing aerosols. In this work, we characterized the GTC system using bacteriophage MS2 as a surrogate for a small RNA virus. We investigated using RNA extraction and reverse transcription- polymerase chain reaction (RT-PCR) to study the total virus collection efficiency of the GTC system. Plaque assays were also used to enumerate viable viruses collected by the GTC system compared to that by a commercially available apparatus, the SKC® Biosampler. The plaque assay counts were used to enumerate viable viruses whereas RT-PCR provides a total virus count, including those viruses inactivated during collection. The effects of relative humidity (RH) and other conditions on collection efficiency were also investigated. Our results suggest that the GTC has a collection efficiency for viable viruses between 0.24 and 1.8% and a total virus collection efficiency between 18.3 and 79.0%, which is 1-2 orders of magnitude higher than that of the SKC® Biosampler. Moreover, higher RH significantly increases both the viable and total collection efficiency of the GTC, while its effect on the collection efficiency of the SKC® Biosampler is not significant. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Pharmacokinetics of cefuroxime in normal and impaired renal function: comparison of high-pressure liquid chromatography and microbiological assays.

    PubMed Central

    Bundtzen, R W; Toothaker, R D; Nielson, O S; Madsen, P O; Welling, P G; Craig, W A

    1981-01-01

    The pharmacokinetics of cefuroxime were studied after a single dose of 750 mg was given intravenously to each of 21 male volunteers grouped according to their creatinine clearances; these clearances were 60 to 120, 20 to 59, and less than 20 ml/min per 1.73 m,2 respectively, for groups 1 (12 subjects), 2 (4 subjects), and 3 (5 subjects). Cefuroxime obeyed two-compartment model kinetics in all three groups. Initial serum levels of cefuroxime were approximately 130 microgram/ml in group 1 and 2 and 80 microgram/ml in group 3. the levels then declined rapidly for 0.5 to 1 h after injection. After that time, cefuroxime levels declined more slowly, and the elimination rate became monoexponential. The mean serum half-lives for cefuroxime in groups 2, 2, and 3 were 1.7, 2.4, and 17.6 h, respectively. Mean cefuroxime levels in serum were greater than 8 microgram/ml for 3 h in group 1, for 6 h in group 2, and for 30 h in group 3. Cumulative 24-h urinary excretion accounted for essentially 100% of the dose in group 1 and 2, and for 40% in group 3. Urine levels exceeded the minimal inhibitory concentration for susceptible organisms for more than 12 h in all groups. Cefuroxime distribution characteristics were independent of renal function. In patients with creatinine clearances less than 20 ml/min per 1.73 m2, doses of cefuroxime needs to be reduced. A microbiological disk diffusion assay and a high-pressure liquid chromatography assay for cefuroxime yielded statistically identical results, except for serum levels in uremic patients (group 3). PMID:7247369

  14. Comparison of thrombin generation assay with conventional coagulation tests in evaluation of bleeding risk in patients with rare bleeding disorders.

    PubMed

    Zekavat, Omid R; Haghpanah, Sezaneh; Dehghani, Javad; Afrasiabi, Abdolreza; Peyvandi, Flora; Karimi, Mehran

    2014-09-01

    Based on the premise that the capacity of plasma to generate thrombin in vitro is a comprehensive and precise functional test of the clotting system, we designed a cross-sectional, single-center study involving 83 patients with rare bleeding disorders (RBDs) to compare the usefulness of the thrombin generation (TG) assay versus conventional tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT) in predicting bleeding risk in patients with RBD in southern Iran. The TG parameters consisted of endogenous thrombin potential, lag time, peak, time to peak (ttPeak), and start tail. The area under the receiver-operating characteristic (ROC) curve showed statistically significant associations between bleeding risk and lag time, ttPeak, and start tail. We determined cutoff values for these 3 TG parameters and obtained a negative predictive value of 86% to 90% in patients with RBD who had a bleeding score (BS) ≤13. The ROC curves for the association of PT and aPTT with BS did not indicate any significant association. Correlation analysis supported the results of ROC curve analysis, only lag time, ttPeak, and start tail showed significant positive correlations with BS (P < .05). Disease severity based on plasma factor activity was significantly associated with prolonged lag time and ttPeak and with prolonged PT (P <.05). We suggest that TG assay is a potentially more useful tool for predicting the bleeding risk in patients with RBD. However, the small sample size in different RBD subgroups precluded subgroup analysis. Prospective multicenter studies with larger numbers of patients are therefore advisable.

  15. Liquid Chromatography-Tandem Mass Spectrometry Assay to Detect Ethyl Glucuronide in Human Fingernail: Comparison to Hair and Gender Differences.

    PubMed

    Jones, Joseph; Jones, Mary; Plate, Charles; Lewis, Douglas; Fendrich, Michael; Berger, Lisa; Fuhrmann, Daniel

    2012-01-01

    Over the past decade, the use of hair specimens for the long-term detection of the alcohol biomarker ethyl glucuronide has been increasing in popularity and usage. We evaluated the usefulness of fingernail clippings as a suitable alternative to hair for ethyl glucuronide detection. A liquid chromatography-tandem mass spectrometry method for the detection of ethyl glucuronide in fingernail clippings was fully validated and used to analyze the hair and/or fingernail specimens of 606 college-aged study participants. The limit of detection was 2 pg/mg, the limit of quantitation was 8 pg/mg and the method was linear from 8 to 2000 pg/mg. Intra- and inter-assay imprecision studies at three different concentrations (20, 40, 200 pg/mg) were all within 7.8% and all intra- and inter-assay bias studies at these levels were within 115.1% of target concentration. Ethyl glucuronide levels in fingernail (mean = 29.1 ± 55.6 pg/mg) were higher than ethyl glucuronide levels in hair (mean = 9.48 ± 22.3 pg/mg) and a correlation of the matched pairs was observed (r = 0.552, P < 0.01, n = 529). Evaluating each gender separately revealed that the correlation of male fingernail to male hair was large and significant (r = 0.782, P < 0.01, n = 195) while female hair to female fingernail was small yet significant (r = 0.249, P < 0.01, n = 334). The study results demonstrated that fingernail may be a suitable alternative to hair for ethyl glucuronide detection and may be the preferred sample type due to the lack of a gender bias.

  16. Analytic Validation of a Clinical-Grade PTEN Immunohistochemistry Assay in Prostate Cancer by Comparison to PTEN FISH

    PubMed Central

    Lotan, Tamara L.; Wei, Wei; Ludkovski, Olga; Morais, Carlos L.; Guedes, Liana B.; Jamaspishvili, Tamara; Lopez, Karen; Hawley, Sarah T.; Feng, Ziding; Fazli, Ladan; Hurtado-Coll, Antonio; McKenney, Jesse K.; Simko, Jeffrey; Carroll, Peter R.; Gleave, Martin; Lin, Daniel W.; Nelson, Peter S.; Thompson, Ian M.; True, Lawrence D.; Brooks, James D.; Lance, Raymond; Troyer, Dean; Squire, Jeremy A.

    2016-01-01

    PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to 4-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for absence of PTEN gene deletion, (549/602 tumors with 2 copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed 2 intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had 2 copies of the PTEN gene by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number. PMID:27174589

  17. Liquid Chromatography-Tandem Mass Spectrometry Assay to Detect Ethyl Glucuronide in Human Fingernail: Comparison to Hair and Gender Differences

    PubMed Central

    Jones, Joseph; Jones, Mary; Plate, Charles; Lewis, Douglas; Fendrich, Michael; Berger, Lisa; Fuhrmann, Daniel

    2015-01-01

    Over the past decade, the use of hair specimens for the long-term detection of the alcohol biomarker ethyl glucuronide has been increasing in popularity and usage. We evaluated the usefulness of fingernail clippings as a suitable alternative to hair for ethyl glucuronide detection. A liquid chromatography-tandem mass spectrometry method for the detection of ethyl glucuronide in fingernail clippings was fully validated and used to analyze the hair and/or fingernail specimens of 606 college-aged study participants. The limit of detection was 2 pg/mg, the limit of quantitation was 8 pg/mg and the method was linear from 8 to 2000 pg/mg. Intra- and inter-assay imprecision studies at three different concentrations (20, 40, 200 pg/mg) were all within 7.8% and all intra- and inter-assay bias studies at these levels were within 115.1% of target concentration. Ethyl glucuronide levels in fingernail (mean = 29.1 ± 55.6 pg/mg) were higher than ethyl glucuronide levels in hair (mean = 9.48 ± 22.3 pg/mg) and a correlation of the matched pairs was observed (r = 0.552, P < 0.01, n = 529). Evaluating each gender separately revealed that the correlation of male fingernail to male hair was large and significant (r = 0.782, P < 0.01, n = 195) while female hair to female fingernail was small yet significant (r = 0.249, P < 0.01, n = 334). The study results demonstrated that fingernail may be a suitable alternative to hair for ethyl glucuronide detection and may be the preferred sample type due to the lack of a gender bias. PMID:27134762

  18. Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

    PubMed Central

    dos Santos, Paula Fernanda Gonçalves; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M

    2017-01-01

    BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages. PMID:28591399

  19. Comparison of murine fibroblast cell response to fluor-hydroxyapatite composite, fluorapatite and hydroxyapatite by eluate assay.

    PubMed

    Jantová, Sona; Letasiová, Silvia; Theiszová, Marica; Palou, M

    2009-03-01

    Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetic composite that contains the same molecular concentration of OH(-) groups and F(-) ions. The aim of this experiment was to evaluate the cellular responses of murine fibroblast NIH-3T3 cells in vitro to solid solutions of FHA and FA and to compare them with the effect of hydroxyapatite (HA). We studied 24, 48 and 72 h effects of biomaterials on cell morphology, proliferation and cell cycle of NIH-3T3 cells by eluate assay. Furthermore, we examined the ability of FHA, FA and HA to induce cell death and DNA damage. Our cytotoxic/antiproliferative studies indicated that any of tested biomaterials did not cause the total inhibition of cell division. Biomaterials induced different antiproliferative effects increasing in the order HA < FHA < FA which were time- and concentration-dependent. None of the tested biomaterials induced necrotic/apoptotic death of NIH-3T3 cells. On the other hand, after 72 h we found that FHA and FA induced G0/G1 arrest of NIH-3T3 cells, while HA did not affect any cell cycle phases. Comet assay showed that while HA demonstrated weaker genotoxicity, DNA damage induced by FHA and FA caused G0/G1 arrest of NIH-3T3 cells. Fluoridation of hydroxyapatite and different FHA and FA structure caused different cell response of NIH-3T3 cells to biomaterials.

  20. Comparison of Xpert MTB/RIF with line probe assay for detection of rifampin-monoresistant Mycobacterium tuberculosis.

    PubMed

    Rufai, Syed Beenish; Kumar, Parveen; Singh, Amit; Prajapati, Suneel; Balooni, Veena; Singh, Sarman

    2014-06-01

    The MTBDRplus line probe assay (LPA) and Xpert MTB/RIF have been endorsed by the World Health Organization for the rapid diagnosis of drug-resistant tuberculosis. However, there is no clarity regarding the superiority of one over the other. In a double-blinded prospective study, we evaluated the efficacy of the Xpert MTB/RIF on samples that were first tested by LPA under the revised national tuberculosis control program of India. A total of 405 sputum samples from suspected drug-resistant tuberculosis patients were included. Of these, 285 smear-positive samples were subjected to LPA. Seventy-two (25.8%) samples showed multidrug resistance, 62 (22.2%) showed rifampin monoresistance, 29 (10.3%) showed isoniazid monoresistance, and 116 (41.5%) were pan-susceptible. Six (2.1%) of the samples gave invalid results. Of the 62 rifampin-monoresistant samples by LPA, 38 (61.4%) showed rifampin resistance, while 21 (33.8%) were found susceptible to rifampin by Xpert MTB/RIF using cartridge version G4. Three (4.8%) samples gave an error. Of the 116 pan-susceptible samples, only 83 were available for Xpert MTB/RIF testing; 4 (5.1%) were rifampin resistant, 74 (94.8%) were susceptible, and 5 (6.0%) showed an error. The 25 discrepant samples were further subjected to MGIT960 drug susceptibility testing. The MGIT960 results showed 100% agreement with LPA results but only 64.4% agreement with Xpert MTB/RIF results. Sequencing analysis of discrepant samples showed 91.3% concordance with LPA but only 8.7% concordance with the Xpert MTB/RIF assay. These findings indicate that by using Xpert MTB/RIF testing we might be underestimating the burden of drug-resistant tuberculosis and indicate that country-specific probes need to be designed to increase the sensitivity of the Xpert MTB/RIF.

  1. Comparison of two subtelomeric assays for the screening of chromosomal rearrangements: analysis of 383 patients, literature review and further recommendations.

    PubMed

    Santa María, Lorena; Faundes, Víctor; Curotto, Bianca; Morales, Paulina; Morales, Karla; Aliaga, Solange; Pugin, Ángela; Alliende, María Angélica

    2016-02-01

    Intellectual disability (ID) and global development delay (GDD) are caused by genetic factors such as subtelomeric rearrangements (SR) in 25 % of patients. There are several assays currently available to detect SR, but subtelomeric fluorescence in situ hybridisation (Subt-FISH) and subtelomeric multiplex ligation-dependent probe amplification (Subt-MLPA) have been the most frequently used. However, the diagnostic yield of each technique has not been compared. We reviewed the results of SR screening over a ten-year period in Chilean patients with ID/GDD using Subt-FISH and/or Subt-MLPA, compared the diagnostic yield of both tools and reviewed the corresponding literature. A total of 383 cases were included in this study, of which 53.8 % were males. The overall diagnostic yield was 8.9 % between both methods, but Subt-MLPA showed a higher performance than Subt-FISH (p = 0.002). A total of 4,181 patients with ID/GDD have been studied worldwide with Subt-MLPA and other subtelomeric assays, and 244 (5.84 %) had a pathogenic SR. It is estimated that Subt-MLPA may detect 92.6 % of the total cases with SR. The capacity of detecting tandem duplication and other critical regions, as well as the use of two MLPA kits, may explain the higher performance of this tool over Subt-FISH. Therefore, we recommend the use of this subtelomeric method as a cost-effective way to study ID/GDD patients.

  2. A comparison of cyst age and assay method of the efficacy of contact lens disinfectants against Acanthamoeba

    PubMed Central

    Kilvington, S.; Anger, C.

    2001-01-01

    AIMS—(i) To determine effect of Acanthamoeba cyst age, method of production, and (ii) to assay technique on the efficacy of multipurpose solutions (MPS) and hydrogen peroxide based contact lens disinfectants. (iii) To establish if MPS can remove mature cysts from contact lenses according to the ISO/DIS 14729 regimen test for microbe removal.
METHODS—Immature and mature cysts of A polyphaga were tested against the MPS Opti-Free express and the hydrogen peroxide based solutions Oxysept 1Step and Oxysept 1 using two assay methods. Simulated patient regimen testing was performed with the Opti-Free express and Complete using mature cysts inoculated on to group I or group IV lenses.
RESULTS—Immature cysts were sensitive to disinfection by all solutions. No killing was observed with mature cysts with Opti-Free express, while immature cysts yielded a 1-2 log reduction in viability. Oxysept 1Step gave a 1.1 (SD 0.3) log reduction in mature cysts after 6 hours. Oxysept 1 gave a 2.4 (0.3) log reduction in mature cysts after 4 hours and a 3.8 (0.5) log reduction after 6 hours. Patient regimen testing using Opti-Free express and Complete resulted in no recovery of viable mature cysts from the contact lenses or from the soaking solutions.
CONCLUSION—Cyst age but not method of production used in this study influences the efficacy of contact lens disinfectants against Acanthamoeba. MPS are effective in removing cysts from contact lens surfaces and may have a role in the prevention of acanthamoeba keratitis.

 PMID:11222342

  3. Comparison of loop-mediated isothermal amplification assay and smear microscopy with culture for the diagnostic accuracy of tuberculosis.

    PubMed

    Gelaw, Baye; Shiferaw, Yitayal; Alemayehu, Marta; Bashaw, Abate Assefa

    2017-01-17

    Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of death from infectious diseases worldwide. Sputum smear microscopy remains the most widely available pulmonary TB diagnostic tool particularly in resource limited settings. A highly sensitive diagnostic with minimal infrastructure, cost and training is required. Hence, we assessed the diagnostic performance of Loop-mediated isothermal amplification (LAMP) assay in detecting M.tuberculosis infection in sputum sample compared to LED fluorescent smear microscopy and culture. A cross-sectional study was conducted at the University of Gondar Hospital from June 01, 2015 to August 30, 2015. Pulmonary TB diagnosis using sputum LED fluorescence smear microscopy, TB-LAMP assay and culture were done. A descriptive analysis was used to determine demographic characteristics of the study participants. Analysis of sensitivity and specificity for smear microscopy and TB-LAMP compared with culture as a reference test was performed. Cohen's kappa was calculated as a measure of agreement between the tests. A total of 78 pulmonary presumptive TB patients sputum sample were analyzed. The overall sensitivity and specificity of LAMP were 75 and 98%, respectively. Among smear negative sputum samples, 33.3% sensitivity and 100% specificity of LAMP were observed. Smear microscopy showed 78.6% sensitivity and 98% specificity. LAMP and smear in series had sensitivity of 67.8% and specificity of 100%. LAMP and smear in parallel had sensitivity of 85.7% and specificity of 96%. The agreement between LAMP and fluorescent smear microscopy tests was very good (κ = 0.83, P-value ≤0.0001). TB-LAMP showed similar specificity but a slightly lower sensitivity with LED fluorescence microscopy. The specificity of LAMP and smear microscopy in series was high. The sensitivity of LAMP was insufficient for smear negative sputum samples.

  4. Validation of fluorescence polarization assay (FPA) and comparison with other tests used for diagnosis of B. melitensis infection in sheep.

    PubMed

    Minas, A; Stournara, A; Minas, M; Papaioannou, A; Krikelis, V; Tselepidis, S

    2005-12-20

    Fluorescence polarization assay (FPA) is a new test for the serological diagnosis of Brucella spp. infection in animals. The FPA is validated for the diagnosis of B. melitensis infection in sheep. For this purpose, 166 sera originated from natural infected sheep (verified by culture) and 851 sera originated from healthy animals (reared in areas where B. melitensis was never been isolated) were tested. The optimum cut-off value that offers the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 87mP with the use of ROC analysis. The DSn and DSp of FPA using this cut-off value are calculated at 97.6 and 98.9% with a 95% confidence interval (CI) of 93.9-99.3% and 98.0-99.5%, respectively. The DSn and DSp of FPA have been assessed also using as positive reference (n=587), sera that gave positive results at least in two tests used for diagnosis of B. melitensis in sheep as Rose Bengal Test (RBT), modified Rose Bengal Test (m-RBT), complement fixation test (CFT), indirect Elisa (i-Elisa) and competition Elisa (c-Elisa) originated from animals reared in flocks infected by B. melitensis. The optimum cut-off value using the above panel of positive reference sera was the same offering a DSn of 95.9% with a 95% CI, 94.0-97.4%, since the DSp remains the same. The DSn and DSp as well as performance, accuracy and agreement of FPA's result were compared with those of other tests used. The accuracy of FPA is very high, similar with that of i-Elisa. FPA is a promising assay, which offers a DSn and accuracy better that of those of the tests currently approved for the diagnosis of B. melitensis in sheep and goats. Due to its simplicity, the sort time that results can be obtained and its accuracy it can be used and improve the laboratory testing capacity as well as the efficacy of the eradication program based on test-and-slaughter policy.

  5. [Comparison of direct immune-fluorescent assay and real-time quantitative PCR in detecting the Hantavirus].

    PubMed

    Yu, Peng-bo; Li, Shen; Wei, Jing; Ma, Chang-an; Lu, Xiao-ling; DU, Shui-quan; Guan, Lu-yuan; Zheng, Yuan; Dong, Jian-hua; Ma, Chao-feng; Wang, Jing-jun

    2013-04-01

    To compare the differences between the direct immuno-fluorescent assay (DFA) and real-time quantitative PCR in detecting the Hantavirus (HV) in rat lungs. From April to October in 2012, a total of 479 rats were caught by mouse-trap in residential or wild areas in Huxian, Jingyang, and Meixian of Shaanxi province, where haemorrhagic fever with renal syndrome (HFRS) was highly prevalent. The rats were dissected to take the two lungs, one was frozen and applied immuno-fluorescent assay to detect HV antigen while the other one was extracted its RNA and detected HV nucleic acid by real-time quantitative PCR. Then we compared the positive rate of the two methods. Out of the 479 rats, 105 were caught from residential areas and the other 374 were caught from wild areas. Among the 105 rats caught from residential areas, no HV were detected out neither by DFA nor by real-time quantitative PCR. Among the 374 wild rats, 13.1% (49/374) were detected HV positive by DFA and 14.7% (55/374) were detected HV positive by real-time quantitative PCR. The difference showed no statistical significance (χ(2) = 0.402, P = 0.526). When detecting each lung sample, the HV positive rate was 10.2% (49/479) under the detection by DFA while the HV positive rate was 11.5% (55/479) under the detection by real-time quantitative PCR. The difference had no statistical significance (χ(2) = 1.286, P = 0.257) and the consistency coefficient was 68.2% under the paired chi-square test analysis, which showed high consistency (u = 11.759, P < 0.05). The sensitivity of real-time quantitative PCR to detect HV was 77.6% (38/49) comparing with DFA as standard, and the specificity was 96.1% (413/430). Out of the 9 suspected HV positive sample detected by DFA, 6 were confirmed positive by real-time quantitative PCR and 3 were denied. Compared with the DFA, real-time quantitative PCR could also be used to detect the infection of HV in rats, and the result might be more stable.

  6. Comparison and correlation of neisseria meningitidis serogroup B immunologic assay results and human antibody responses following three doses of the Norwegian meningococcal outer membrane vesicle vaccine MenBvac.

    PubMed

    Findlow, Jamie; Taylor, Stephen; Aase, Audun; Horton, Rachel; Heyderman, Robert; Southern, Jo; Andrews, Nick; Barchha, Rita; Harrison, Ewan; Lowe, Ann; Boxer, Emma; Heaton, Charlotte; Balmer, Paul; Kaczmarski, Ed; Oster, Philipp; Gorringe, Andrew; Borrow, Ray; Miller, Elizabeth

    2006-08-01

    The prediction of efficacy of Neisseria meningitidis serogroup B (MenB) vaccines is currently hindered due to the lack of an appropriate correlate of protection. For outer membrane vesicle (OMV) vaccines, immunogenicity has primarily been determined by the serum bactericidal antibody (SBA) assay and OMV enzyme-linked immunosorbent assay (ELISA). However, the opsonophagocytic assay (OPA), surface labeling assay, whole blood assay (WBA), and salivary antibody ELISA have been developed although correlation with protection is presently undetermined. Therefore, the aim of the study was to investigate further the usefulness of, and relationships between, MenB immunologic assays. A phase II trial of the OMV vaccine, MenBvac, with proven efficacy was initiated to compare immunologic assays incorporating the vaccine and six heterologous strains. Correlations were achieved between the SBA assay, OMV ELISA, and OPA using human polymorphonuclear leukocytes and human complement but not between an OPA using HL60 phagocytic cells and baby rabbit complement. Correlations between the surface labeling assay, the SBA assay, and the OMV ELISA were promising, although target strain dependent. Correlations between the salivary antibody ELISA and other assays were poor. Correlations to the WBA were prevented since many samples had results greater than the range of the assay. The study confirmed the immunogenicity and benefit of a third dose of MenBvac against the homologous vaccine strain using a variety of immunologic assays. These results emphasize the need for standardized methodologies that would allow a more robust comparison of assays between laboratories and promote their further evaluation as correlates of protection against MenB disease.

  7. Comparison of isolated cranberry (Vaccinium macrocarpon Ait.) proanthocyanidins to catechin and procyanidins A2 and B2 for use as standards in the 4-(dimethylamino)cinnamaldehyde assay.

    PubMed

    Feliciano, Rodrigo P; Shea, Michael P; Shanmuganayagam, Dhanansayan; Krueger, Christian G; Howell, Amy B; Reed, Jess D

    2012-05-09

    The 4-(dimethylamino)cinnamaldehyde (DMAC) assay is currently used to quantify proanthocyanidin (PAC) content in cranberry products. However, this method suffers from issues of accuracy and precision in the analysis and comparison of PAC levels across a broad range of cranberry products. Current use of procyanidin A2 as a standard leads to an underestimation of PACs content in certain cranberry products, especially those containing higher molecular weight PACs. To begin to address the issue of accuracy, a method for the production of a cranberry PAC standard, derived from an extraction of cranberry (c-PAC) press cake, was developed and evaluated. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were 2.2 times higher than those determined by procyanidin A2. Increased accuracy is critical for estimating PAC content in relationship to research on authenticity, efficacy, and bioactivity, especially in designing clinical trials for determination of putative health benefits.

  8. Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays

    PubMed Central

    Vollmer, Tanja; Diekmann, Juergen; Eberhardt, Matthias; Knabbe, Cornelius; Dreier, Jens

    2016-01-01

    Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes. PMID:27556482

  9. Comparison of a gel microcolumn assay with the conventional tube test for red blood cell alloantibody titration.

    PubMed

    Finck, Rachel; Lui-Deguzman, Carrie; Teng, Shih-Mao; Davis, Rebecca; Yuan, Shan

    2013-04-01

    Titration is a semiquantitative method used to estimate red blood cell (RBC) alloantibody reactivity. The conventional tube test (CTT) technique is the traditional method for performing titration studies. The gel microcolumn assay (GMA) is also a sensitive method to detect RBC alloantibodies. The aim of this study was to compare a GMA with the CTT technique in the performance of Rh and K alloantibody titration. Patient serum samples that contained an RBC alloantibody with a singular specificity were identified by routine blood bank workflow. Parallel titration studies were performed on these samples by both the CTT method and a GMA (ID-Micro Typing System anti-IgG gel card, Micro Typing Systems, Inc., an Ortho-Clinical Diagnostics Company). Forty-eight samples were included, including 11 anti-D, five anti-c, 13 anti-E, one anti-C, three anti-e, and 15 anti-K. Overall, the two methods generated identical results in 21 of 48 samples. For 42 samples (87.5%) the two methods generated results that were within one serial dilution, and for the remaining six samples, results were within two dilutions. GMA systems may perform comparably to the CTT in titrating alloantibodies to Rh and Kell antigens. © 2012 American Association of Blood Banks.

  10. Diagnostic tests for amoebic liver abscess: comparison of enzyme-linked immunosorbent assay (ELISA) and counterimmunoelectrophoresis (CIE).

    PubMed

    Restrepo, M I; Restrepo, Z; Elsa Villareal, C L; Aguirre, A; Restrepo, M

    1996-01-01

    The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoelectrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results in both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100%) than that of the CIE technique (66%).

  11. Bluetongue in Bosnia: comparisons of competitive enzyme-linked immunosorbent assay and standard agar gel immunodiffusion tests.

    PubMed

    Velić, L; Velić, R; Bajrović, T; Dukić, B; Camo, D

    2004-01-01

    At the end of August 2002, clinical symptoms of bluetongue (BT) (fever between 39 degrees C and 41 degrees C, muco-purulent or bloody nasal discharge, oedema of the lips and the intramandibular space, foot lesions including laminitis and coronitis in some cases, diarrhoea and dysentery) were recorded in Pramenka sheep flocks in north-east Bosnia in August 2002. A total of 9 599 serum samples (ovine: 8 967; bovine: 632) from 40 communities of Bosnia and Herzegovina were tested for the presence of anti-bluetongue virus (BTV) antibodies using competitive enzyme-linked immunosorbent assay (c-ELISA) and the standard agar gel immunodiffusion (AGID) test. The c-ELISA revealed BTV-seropositive reactions in 187 (1.94%) samples and the AGID test detected 141 (1.53%) cases. Complete agreement was recorded between the c-ELISA and AGID test results for bovine sera. These results indicate that the ability of c-ELISA to detect anti-bluetongue virus antibodies in ovine sera was superior to that of the AGID. All positive sera were collected from animals in the river areas of Bosnia and Herzegovina.

  12. Analysis and comparison of the microflora isolated from fresco surface and from surrounding air environment through molecular and biodegradative assays.

    PubMed

    Pangallo, Domenico; Kraková, Lucia; Chovanová, Katarína; Simonovičová, Alexandra; De Leo, Filomena; Urzì, Clara

    2012-05-01

    The aim of this study was to find a correlation among the environmental isolated microflora and the fresco colonizators through the investigation of their biodegradative abilities and DNA characteristics. A molecular technique named RAMP (Random Amplified Microsatellite Polymorphisms) was utilized in order to analyze the DNA diversity of bacterial and fungal species isolated from fresco as well as from air samples. The RAMP-PCR results were combined with the screening of some biodegradative properties obtained through the use of specific agar plate assays detecting the proteolytic, solubilization and biomineralization abilities of the isolated microflora. This comparative analysis showed that only in few cases a direct link among the fresco and airborne isolates of specific microbial group existed. The investigation clearly evidenced that colonization of surface of Ladislav's fresco occurred in different time and by different strains than those observed at the moment of sampling campaign. Furthermore, the microflora investigation permitted the identification of taxonomically interesting bacteria with particular biodegradative properties, which had been less studied until now.

  13. [Nursing-home-acquired pneumococcal pneumonia--comparison of sputum cultures with Binax NOW Streptococcus pneumoniae urinary antigen assay].

    PubMed

    Rikimaru, Toru; Nishiyama, Mamoru; Yonemitsu, Junko; Nagabuchi, Masako; Shimada, Akiko; Koga, Takeharu; Aizawa, Hisamichi

    2008-11-01

    To clarify the clinical significance of Pneumococcal pneumonia in nursing-home-acquired pneumonia, we examined the positive disease rate of using sputum cultures and the Binax NOW Streptococcus pneumoniae urinary antigen assay in 154 nursing-home patients with pneumonia. These included 54 males and 100 females with a mean age of 86.2 years. Bacteriological findings for sputum culture in 130 patients showed Streptococcus pneumoniae to be cultured in 11 cases (8%). In 72 in whom the Streptococcus pneumoniae-urinary antigen test (Binax NOW) was done, the urinary-antigen-positive rate (26/72 ; 36%) was higher than the culture positive rate for S. pneumoniae. Both examinations were done in 64 patients, among whom 5 in whom S. pneumoniae was cultured also had positive results for the urinary antigen test. Almost half of those undergoing percutaneous endoscopic gastroscopy (PEG) tube nutrition had positive results for the urinary antigen test, but not all such patients had positive cultures for S. pneumoniae. Although the culture-positive rate for S. pneumoniae in sputum was low, we concluded that S. pneumoniae was frequently linked to nursing-home-acquired pneumonia, especially in "total-care" patients.

  14. Comparison of BGM and PLC/PRC/5 Cell Lines for Total Culturable Viral Assay of Treated Sewage▿

    PubMed Central

    Rodríguez, Roberto A.; Gundy, Patricia M.; Gerba, Charles P.

    2008-01-01

    The objective of this study was to compare PLC/PRF/5 and BGM cell lines for use in a total culturable viral assay (TCVA) of treated sewage effluents. Samples were collected before and after chlorination from an activated sludge wastewater treatment plant and from the effluent of a high-rate enhanced flocculation system, followed by UV light disinfection. Cell monolayers were observed for cytopathic effect (CPE) after two passages of 14 days each. Monolayers exhibiting viral CPE were tested for the presence of adenoviruses and enteroviruses by PCR or reverse transcription-PCR. Eight percent of the samples exhibited CPE on BGM cells, and 57% showed CPE on PLC/PRF/5 cells. Only enteroviruses were detected on the BGM cells, while 30% and 52% of the samples were positive for enteroviruses and adenoviruses, respectively, on the PLC/PRF/5 cells. Thirty percent of the samples were positive for both adenoviruses and enteroviruses in chlorinated activated sludge effluent. Thirty percent of the samples were positive for adenoviruses in the UV treatment effluent, but no enteroviruses were detected. In conclusion, the PLC/PRF/5 cells were more susceptible than BGM cells to viruses found in treated sewage. The use of BGM cells for TCVA may underestimate viral concentration in sewage effluent samples. The PLC/PRF/5 cells were more susceptible to adenoviruses, which is important in the evaluation of UV disinfection systems because adenoviruses are highly resistant to UV inactivation. PMID:18326686

  15. Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection.

    PubMed

    Oh, Jin Sik; Song, Dae Sub; Yang, Jeong Sun; Song, Ju Young; Moon, Hyoung Joon; Kim, Tae Yung; Park, Bong Kyun

    2005-12-01

    An indirect porcine epidemic diarrhea (PED) virus (PEDV) enzyme-linked immunosorbent assay (ELISA) was compared with the serum neutralization (SN) test by testing 46 samples from experimentally infected sows, 73 samples from naive sows, and 1,024 field sow samples from 48 commercial swine farms of undefined PED status. The SN test and the ELISA were performed using PEDV, KPEDV-9 strain. Viral proteins as a coating antigen of PEDV ELISA were extracted from the cytoplasm of PEDV-infected Vero cells using a non-ionic detergent, Triton X-100, and a simple protocol of PEDV ELISA was followed. The presence of antibodies in these experimental samples was confirmed by SN and ELISA in which the sensitivity of the ELISA was 89.1%, and the corresponding specificity was 94.5%. On testing 1,024 field samples, an overall agreement of 84.2% was generated between the SN and ELISA. This study demonstrates that the PEDV ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against PEDV.

  16. Comparison of axillary bud growth and patatin accumulation in potato leaf cuttings as assays for tuber induction

    NASA Technical Reports Server (NTRS)

    Wheeler, R. M.; Hannapel, D. J.; Tibbitts, T. W.

    1988-01-01

    Single-node leaf cuttings from potatoes (Solanum tuberosum L.) cvs. Norland, Superior, Norchip, and Kennebec, were used to assess tuber induction in plants grown under 12, 16, and 20 h daily irradiation (400 micromol s-1 m-2 PPF). Leaf cuttings were taken from plants at four, six and 15 weeks after planting and cultured for 14 d in sand trays in humid environments. Tuber induction was determined by visually rating the type of growth at the attached axillary bud, and by measuring the accumulation of the major tuber protein, patatin, in the base of the petioles. Axillary buds from leaf cuttings of plants grown under the 12 h photoperiod consistently formed round, sessile tubers at the axils for all four cultivars at all harvests. Buds from cuttings of plants grown under the 16 and 20 h photoperiods exhibited mixed tuber, stolon, and leafy shoot growth. Patatin accumulation was highest in petioles of cuttings taken from 12 h plants for all cultivars at all harvests, with levels in 16 and 20 h cuttings approx. one-half that of the 12 h cuttings. Trends, both in visual ratings of axillary buds and in petiole patatin accumulation, followed the harvest index (ratio of tuber to total plant dry matter), suggesting that either method is an acceptable assay for tuber induction in the potato.

  17. Comparison of the immunofluorescence assay with RT-PCR and nested PCR in the diagnosis of canine distemper.

    PubMed

    Jóźwik, A; Frymus, T

    2005-05-01

    Two pairs of primers were prepared, both localized within the sequences of the nucleoprotein gene (NP) of canine distemper virus (CDV). A number of experiments were done to optimize the conditions of RT-PCR and nested PCR methods. The nucleic acids of the Onderstepoort, Rockborn, Snyder Hill and Lederle strains of CDV could be detected with these primers. However, they did not react with the sequences of the Edmonston strain of the measles virus. The detection limit for RT-PCR was 10 TCID50 and for nested PCR 0.1 TCID50 of CDV. The RT-PCR was able to demonstrate the nucleic acid of CDV in the blood of all seven puppies vaccinated with a modified live virus. Blood samples of 23 dogs clinically suspected of distemper were examined by RT-PCR combined with nested PCR, and the results were compared with the detection of the CDV antigen in the smears from the mucous membranes by the direct immunofluorescence (IF) test. Of the 23 dogs, 12 were positive in nested PCR, six in the IF assay, and only two in single RT-PCR. It is concluded that nested PCR seems to be the most sensitive method for ante-mortem diagnosis of canine distemper, especially in its subacute or chronic forms.

  18. Comparison between Culture and a Multiplex Quantitative Real-Time Polymerase Chain Reaction Assay Detecting Ureaplasma urealyticum and U. parvum

    PubMed Central

    Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

    2014-01-01

    A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR. PMID:25047036

  19. Comparison of axillary bud growth and patatin accumulation in potato leaf cuttings as assays for tuber induction

    NASA Technical Reports Server (NTRS)

    Wheeler, R. M.; Hannapel, D. J.; Tibbitts, T. W.

    1988-01-01

    Single-node leaf cuttings from potatoes (Solanum tuberosum L.) cvs. Norland, Superior, Norchip, and Kennebec, were used to assess tuber induction in plants grown under 12, 16, and 20 h daily irradiation (400 micromol s-1 m-2 PPF). Leaf cuttings were taken from plants at four, six and 15 weeks after planting and cultured for 14 d in sand trays in humid environments. Tuber induction was determined by visually rating the type of growth at the attached axillary bud, and by measuring the accumulation of the major tuber protein, patatin, in the base of the petioles. Axillary buds from leaf cuttings of plants grown under the 12 h photoperiod consistently formed round, sessile tubers at the axils for all four cultivars at all harvests. Buds from cuttings of plants grown under the 16 and 20 h photoperiods exhibited mixed tuber, stolon, and leafy shoot growth. Patatin accumulation was highest in petioles of cuttings taken from 12 h plants for all cultivars at all harvests, with levels in 16 and 20 h cuttings approx. one-half that of the 12 h cuttings. Trends, both in visual ratings of axillary buds and in petiole patatin accumulation, followed the harvest index (ratio of tuber to total plant dry matter), suggesting that either method is an acceptable assay for tuber induction in the potato.

  20. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.

    PubMed

    Kodedová, Marie; Sychrová, Hana

    2016-09-10

    New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species.

  1. Comparison of rapid immunodiagnosis assay kit with molecular and immunopathological approaches for diagnosis of rabies in cattle

    PubMed Central

    Ahmad, Ajaz; Singh, C. K.

    2016-01-01

    Aim: Presently, diagnosis of rabies is primarily based on, conventional fluorescent antibody technique (FAT), immunopathological and molecular techniques. Recently, rapid immunodiagnostic assay (RIDA) - A monoclonal antibody-based technique has been introduced for rapid diagnosis of rabies. The present investigation is envisaged to study the efficacy of RIDA kit for the diagnosis of rabies in cattle. Materials and Methods: About 11 brain samples from cattle, clinically suspected for rabies, were screened by the FAT, Heminested reverse transcriptase polymerase chain reaction (HnRT-PCR), Immunohistochemistry (IHC), and RIDA. Results: The sensitivity for detection of rabies from brain tissue by RIDA was 85.7% as compared to 100% by IHC as well as HnRT-PCR. The accuracy of detection of rabies by RIDA was 91.6% as compared to 100% that of IHC and HnRT-PCR, whereas specificity of RIDA was 100% like that of the IHC and HnRT-PCR. Conclusion: Despite a comparatively low-sensitivity and accuracy of RIDA, latter can still be useful in screening a large number of field samples promptly. However, it is recommended that negative results with RIDA in cattle need to be authenticated by suitable alternative diagnostic approaches. PMID:27051193

  2. Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use.

    PubMed

    Ruberu, Shiyamalie R; Langlois, Gregg W; Masuda, Melisa; Kittredge, Clive; Perera, S Kusum; Kudela, Raphael M

    2017-09-08

    The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg(-1) to 13,000 µg STX equiv. kg(-1) by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg(-1) on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg(-1)) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.

  3. Comparison of Estrogen Receptor Assay Results from Pathology Reports with Results from Central Laboratory Testing: Implications for Population-Based Studies of Breast Cancer

    PubMed Central

    Collins, LC; Marotti, J; Baer, HJ; Deitz, AC; Colditz, GA; Tamimi, RM

    2014-01-01

    Population-based studies of women with breast cancer commonly utilize information culled from pathology reports rather than central pathology review. The reliability of this information, particularly with regard to tumor biomarker results, is of concern. To address this, we evaluated the concordance between estrogen receptor (ER) results as determined from the original pathology reports and ER results obtained on the same specimens following testing in a single laboratory. Tissue microarrays (TMAs) were constructed from paraffin blocks of 3,167 breast cancers that developed in women enrolled in the Nurses’ Health Study. ER immunostains were performed on all TMA sections in single run. Results of ER immunostains performed on the TMA sections were compared with ER assay results abstracted from pathology reports. Among 1,851 cases of invasive breast cancer in which both ER results from pathology reports and central ER test results were available, the reported ER status and the ER status as determined from immunostains on TMAs were in agreement in 1,651 cases (87.3 %; kappa value 0.64, p<0.0001). When the comparison was restricted to ER assays originally performed by immunohistochemistry, the agreement rate increased to 92.3% (kappa value 0.78, p<0.0001). These results provide a framework for the accuracy of ER results abstracted from clinical records. Further, they suggest that utilizing ER assay results from pathology reports is a reasonable, albeit imperfect, alternative to central laboratory ER testing for large, population-based studies of patients with breast cancer. PMID:18230800

  4. Comparison of indirect immunofluorescence assays for diagnosis of scrub typhus and murine typhus using venous blood and finger prick filter paper blood spots.

    PubMed

    Phetsouvanh, Rattanaphone; Blacksell, Stuart D; Jenjaroen, Kemajittra; Day, Nicholas P J; Newton, Paul N

    2009-05-01

    We performed indirect immunofluorescence assays (IFAs) to compare levels of IgM and IgG antibodies to Orientia tsutsugamushi and Rickettsia typhi in admission-phase serum samples and filter paper blood spots (assayed immediately and stored at 5.4 degrees C and 29 degrees C for 30 days) collected on the same day from 53 adults with suspected scrub typhus and murine typhus admitted to Mahosot Hospital Vientiane, Lao People's Democratic Republic. The sensitivities and specificities of admission-phase filter paper blood spots in comparison to paired sera were between 91% and 95% and 87% and 100%, respectively, for the diagnosis of scrub typhus and murine typhus. The classification of patients as having or not having typhus did not significantly differ after storage of the blood spots for 30 days (P > 0.4) at 5.4 degrees C and 29 degrees C. Because filter paper blood samples do not require sophisticated and expensive storage and transport, they may be an appropriate specimen collection technique for the diagnosis of rickettsial disease in the rural tropics.

  5. Enzyme-linked immunosorbent assay for group A Streptococcal anti-DNase B in human sera, using recombinant proteins - Comparison to the DNA methyl green micromethod.

    PubMed

    Das, Sarita; Dileepan, T; Johnson, D R; Kaplan, E L; Patrick Cleary, P

    2017-09-19

    Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM). DNAs from various Streptococcal species were screened for presence of dnaseB gene by PCR. Measurements of ADB in sera collected from subjects belonging to different ages, and ethnic groups were used to compare the two methods. dnaseB was not detected by PCR in DNA samples isolated from different strains of group B (GBS), C (GCS) and G (GGS) Streptococci. The ADB based ELISA proved to be highly sensitive and more responsive to changes in antibody concentration than DMGM. Use of recombinant DNase B eliminates the variability associated with the enzyme, partially purified from Streptococcal culture supernatants from various commercial sources and may provide a more reliable source of antigen to a wider group of laboratories concerned with GAS diagnosis. Copyright © 2017. Published by Elsevier B.V.

  6. Comparison of PCR assays targeting the multi-copy targets B1 gene and 529 bp repetitive element for detection of Toxoplasma gondii in swine muscle.

    PubMed

    Veronesi, Fabrizia; Santoro, Azzurra; Milardi, Giovanni Luigi; Diaferia, Manuela; Branciari, Raffaella; Miraglia, Dino; Cioffi, Attilia; Gabrielli, Simona; Ranucci, David

    2017-05-01

    The comparison of the sensitivities of two molecular assays designed to target the multi-copy sequences of the Toxoplasma gondii genomic B1 region and 529 bp-RE respectively, in detecting T. gondii in swine muscle was assessed. Diaphragm pillars were obtained from 498 slaughtered pigs managed in intensive farms in Central Italy. Genomic DNA was extracted from the tissues and T. gondii-B1 and 529 bp-RE sequences were amplified by specific PCR protocols. Toxoplasma gondii DNA was detected in 165 samples (33.13%). There was a good correlation (κ = 0.77) between the results obtained targeting the two different genetic markers, however the 529 bp RE-PCR assay overall detected a significantly higher (P < 0.05) number of T. gondii-positive samples (150 samples) than the B1-PCR protocol (134). Our results show that: i) standardized B1 and 529 bp-RE PCRs applied to muscle tissues can detect a high rate of T. gondii-infection; ii) a multi-target PCR approach is recommended for the accurate diagnosis of infection in swine and can also be used in food testing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Development and evaluation of a four-tube real time multiplex PCR assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts.

    PubMed

    Jansen, Rogier R; Schinkel, Janke; Koekkoek, Sylvie; Pajkrt, Dasja; Beld, Marcel; de Jong, Menno D; Molenkamp, Richard

    2011-07-01

    Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, like culture and antigen detection. However, comprehensive data on sensitivity, specificity and performance of the multiplex PCR compared to the single target PCR's is limited for most published respiratory multiplex real time PCR assays. Development and extensive analysis of an internally controlled multiplex real time rt-PCR for detection of respiratory viruses. The assay was validated in comparison to single-target PCR's using plasmid targets and prospectively collected nasopharyngeal aspirates. Using plasmid targets the multiplex format was found to be as least as sensitive and specific as the single-target PCR and no competition was observed when different targets were present at different amounts in one tube. Clinical validation showed high concordance for all viruses tested except for samples with low levels of enterovirus. This multiplex showed excellent specificities for all 14 respiratory viruses and sensitivity was high except for clinical samples with low levels of enterovirus. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

    PubMed

    Selvaraju, Suresh B; Bambach, Adrienne V; Leber, Amy L; Patru, Maria-Magdalena; Patel, Anami; Menegus, Marilyn A

    2014-09-01

    The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

  9. Comparison of immunodiffusion and enzyme linked immunosorbent assay in the detection of abnormal antibodies in pigeon breeder's disease.

    PubMed Central

    Simpson, C.; Shirodaria, P. V.; Evans, J. P.; Simpson, D. I.; Stanford, C. F.

    1992-01-01

    AIMS: To compare the sensitivity of two methods for the detection of serum antibodies to pigeon faecal antigens in patients with pigeon breeder's disease. METHODS: Serum samples stored at -20 degrees C from 50 patients with pigeon breeder's disease, 50 control samples from patients with other respiratory diseases, and 50 healthy blood donors were examined for the precipitating antibodies and IgG antibodies to antigens present in extract of pigeon droppings by immunodiffusion and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: Both antigen preparations of pigeon dropping extract were equally effective. A positive immunodiffusion reaction gave one or more precipitin lines and these antibodies were detected only in undiluted sera from 80% of the patients with pigeon breeder's disease. In the ELISA the sera were tested at a starting dilution of 1 in 100 because positive reactions were observed with sera from healthy blood donors at lower dilutions. All sera which gave optical density readings above 3 SD of the control value were considered to have IgG antibodies. These antibodies were detected in sera from all the patients with pigeon breeder's disease. The antibody titres were much higher in those patients who had precipitating antibodies (range 800-51,200) than those without (range 100-800). The antibodies were not detected in the sera of patients with respiratory diseases or healthy blood donors by either method. CONCLUSIONS: Antibodies to pigeon dropping antigens were detected by immunodiffusion and ELISA in sera from patients with pigeon breeder's disease but not in control sera. ELISA was a more sensitive method for detecting antibodies and therefore seems to have considerable potential as a routine technique in the serological diagnosis of pigeon breeder's disease. PMID:1624596

  10. Comparison of 3 type VII collagen (C7) assays for serologic diagnosis of epidermolysis bullosa acquisita (EBA).

    PubMed

    Seta, Vannina; Aucouturier, Françoise; Bonnefoy, Jonathan; Le Roux-Villet, Christelle; Pendaries, Valérie; Alexandre, Marina; Grootenboer-Mignot, Sabine; Heller, Michel; Lièvre, Nicole; Laroche, Liliane; Caux, Frédéric; Titeux, Matthias; Hovnanian, Alain; Prost-Squarcioni, Catherine

    2016-06-01

    Serologic diagnosis of epidermolysis bullosa acquisita (EBA) relies on the detection of circulating autoantibodies to type VII collagen (C7). We sought to compare the diagnostic performances of a commercialized enzyme-linked immunosorbent assay (ELISA) using C7 noncollagenous (NC) domains (C7-NC1/NC2 ELISA) and indirect immunofluorescence (IIF) biochip test on NC1-C7-expressing transfected cells (IIFT), with a full-length-C7 ELISA developed in our laboratory. C7-NC1/NC2 ELISA, IIFT, and full-length-C7 ELISA were run on 77 nonselected consecutive EBA sera. C7-NC1/NC2 ELISA, IIFT, and full-length-C7 ELISA were positive, respectively, for: 30%, 27%, and 65% of the 77 sera; 43%, 32%, and 80% of 44 sera labeling the salt-split-skin (SSS) floor (F) by IIF (SSS/F(+)); 9%, 22%, and 47% of 32 SSS/F(-) sera; 28%, 28%, and 58% of classic EBA; 41%, 41%, and 82% of inflammatory EBA; and 18%, 0%, and 55% of mucous-membrane-predominant EBA. Significant differences for all sera were found between: the 2 ELISAs for the 77 sera, SSS/F(+) and SSS/F(-) sera, and IIFT versus full-length-C7 ELISA. The retrospective design was a limitation. C7-NC1/NC2 ELISA and IIFT sensitivities for serologic diagnoses of EBA were low. Full-length-C7 ELISA was significantly more sensitive and could serve as a reference test. Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  11. Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease.

    PubMed

    Schoepfer, Alain M; Flogerzi, Beatrice; Fallegger, Silvia; Schaffer, Thomas; Mueller, Stefan; Nicod, Laurent; Seibold, Frank

    2008-11-01

    Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients. Both TST and IGRA were performed on 212 subjects (114 Crohn's disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls). Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P = 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P = 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P = 0.044) compared to the IBD group. Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.

  12. Toxoplasma gondii exposure in arctic-nesting geese: A multi-state occupancy framework and comparison of serological assays

    PubMed Central

    Elmore, Stacey A.; Huyvaert, Kathryn P.; Bailey, Larissa L.; Milhous, Jared; Alisauskas, Ray T.; Gajadhar, Alvin A.; Jenkins, Emily J.

    2014-01-01

    The zoonotic parasite, Toxoplasma gondii, has a worldwide distribution and a cosmopolitan suite of hosts. In arctic tundra regions, the definitive felid hosts are rare to absent and, while the complete transmission routes in such regions have yet to be fully elucidated, trophic and vertical routes are likely to be important. Wild birds are common intermediate hosts of T. gondii, and in the central Canadian arctic, geese are probable vectors of the parasite from temperate latitudes to the arctic regions. Our objective was to estimate seroprevalence of T. gondii in Ross’s and Lesser Snow Geese from the Karrak Lake ecosystem in Nunavut, Canada. After harvesting geese by shotgun, we collected blood on filter paper strips and tested the eluate for T. gondii antibodies by indirect fluorescent antibody test (IFAT) and direct agglutination test (DAT). We estimated seroprevalence using a multi-state occupancy model, which reduced bias by accounting for imperfect detection, and compared these estimates to a naïve estimator. Ross’s Geese had a 0.39 probability of seropositivity, while for Lesser Snow Geese the probability of positive for T. gondii antibodies was 0.36. IFAT had a higher antibody detection probability than DAT, but IFAT also had a higher probability of yielding ambiguous or unclassifiable results. The results of this study indicate that Ross’s Geese and Lesser Snow Geese migrating to the Karrak Lake region of Nunavut are routinely exposed to T. gondii at some point in their lives and that they are likely intermediate hosts of the parasite. Also, we were able to enhance our estimation of T. gondii seroprevalence by using an occupancy approach that accounted for both false-negative and false-positive detections and by using multiple diagnostic tests in the absence of a gold standard serological assay for wild geese. PMID:25161913

  13. Toxoplasma gondii exposure in arctic-nesting geese: A multi-state occupancy framework and comparison of serological assays.

    PubMed

    Elmore, Stacey A; Huyvaert, Kathryn P; Bailey, Larissa L; Milhous, Jared; Alisauskas, Ray T; Gajadhar, Alvin A; Jenkins, Emily J

    2014-08-01

    The zoonotic parasite, Toxoplasma gondii, has a worldwide distribution and a cosmopolitan suite of hosts. In arctic tundra regions, the definitive felid hosts are rare to absent and, while the complete transmission routes in such regions have yet to be fully elucidated, trophic and vertical routes are likely to be important. Wild birds are common intermediate hosts of T. gondii, and in the central Canadian arctic, geese are probable vectors of the parasite from temperate latitudes to the arctic regions. Our objective was to estimate seroprevalence of T. gondii in Ross's and Lesser Snow Geese from the Karrak Lake ecosystem in Nunavut, Canada. After harvesting geese by shotgun, we collected blood on filter paper strips and tested the eluate for T. gondii antibodies by indirect fluorescent antibody test (IFAT) and direct agglutination test (DAT). We estimated seroprevalence using a multi-state occupancy model, which reduced bias by accounting for imperfect detection, and compared these estimates to a naïve estimator. Ross's Geese had a 0.39 probability of seropositivity, while for Lesser Snow Geese the probability of positive for T. gondii antibodies was 0.36. IFAT had a higher antibody detection probability than DAT, but IFAT also had a higher probability of yielding ambiguous or unclassifiable results. The results of this study indicate that Ross's Geese and Lesser Snow Geese migrating to the Karrak Lake region of Nunavut are routinely exposed to T. gondii at some point in their lives and that they are likely intermediate hosts of the parasite. Also, we were able to enhance our estimation of T. gondii seroprevalence by using an occupancy approach that accounted for both false-negative and false-positive detections and by using multiple diagnostic tests in the absence of a gold standard serological assay for wild geese.

  14. Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

    PubMed

    Zasada, A A; Rastawicki, W; Śmietańska, K; Rokosz, N; Jagielski, M

    2013-07-01

    Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

  15. Comparison of assays for the detection of West Nile virus antibodies in equine serum after natural infection or vaccination.

    PubMed

    Joó, Kinga; Bakonyi, Tamás; Szenci, Ottó; Sárdi, Sára; Ferenczi, Emőke; Barna, Mónika; Malik, Péter; Hubalek, Zdenek; Fehér, Orsolya; Kutasi, Orsolya

    2017-01-01

    West Nile virus (WNV) mainly infects birds, horses and humans. Outcomes of the infection range from mild uncharacteristic signs to fatal neurologic disease. The main objectives of the present study were to measure serum IgG and IgM antibodies in naturally exposed and vaccinated horses and to compare results of haemagglutination inhibition test (HIT), enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralisation test (PRNT). Altogether 224 animals were tested by HIT for WNV antibodies and 41 horses were simultaneously examined by ELISA and PRNT. After primary screening for WNV antibodies, horses were vaccinated. Samples were taken immediately before and 3-5 weeks after each vaccination. McNemar's chi-squared and percent agreement tests were used to detect concordance between HIT, ELISA and PRNT. Analyses by HIT confirmed the presence of WNV antibodies in 27/105 (26%) naturally exposed horses. Sera from 57/66 (86%) vaccinated animals were positive before the first booster and from 11/11 (100%) before the second booster. HIT was less sensitive for detecting IgG antibodies. We could detect postvaccination IgM in 13 cases with IgM antibody capture ELISA (MAC-ELISA) and in 7 cases with HIT. WNV is endemic in Hungary and regularly causes natural infections. Protective antibodies could not be measured in some of the cases 12 months after primary vaccinations; protection is more reliable after the first yearly booster. Based on our findings it was not possible to differentiate infected from recently vaccinated horses using MAC-ELISA. HIT cannot be used as a substitute for ELISA or PRNT when detecting IgG, but it proved to be a useful tool in this study to gain statistical information about the tendencies within a fixed population of horses. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. A Comparison of Microscopy and Enzyme Linked Immunosorbent Assay for Diagnosis of Giardia lamblia in Human Faecal Specimens.

    PubMed

    Jahan, Noor; Khatoon, Razia; Ahmad, Siraj

    2014-11-01

    Giardia lamblia, a flagellate protozoa, is a common causative agent of parasitic diarrhoeal diseases of humans. Laboratory diagnosis mainly consists of direct microscopic examination of stool specimen for trophozoite and cysts of Giardia. However, due to intermittent faecal excretion of parasite, the case may be miss diagnosed and the patient may continue excreting the parasite and infecting others. Therefore, other mode of diagnosis should be looked for, which overcome the above drawbacks of microscopy used alone for diagnosis. The present study was done to evaluate the efficacy of RIDASCREEN Giardia (ELISA) test in comparison to direct microscopy in the diagnosis of Giardia lamblia in stool specimens from patients with diarrhea and other gastrointestinal symptoms. A total of 1680 patients were included in the study and three faecal specimens were taken from each patient which was divided into two parts. One part was used for direct wet mount examination and second part was used to put ELISA by using RIDASCREEN Giardia test. Out of 1680 stool samples, 380 specimens (22.6%) were found to be positive for Giardia lamblia. Maximum cases were detected by RIDASCREEN Giardia (ELISA) test with sensitivity of 100% and specificity of 91.5%. Maximum cases of giardiasis were detected in children less than 10 y of age (12.8%). RIDASCREEN Giardia test is a rapid and effective method with high sensitivity and specificity and detects Giardia antigens in stool specimens even when the count of parasite is low, thus reducing the chances of missing even the asymptomatic cases.

  17. Radioimmunoassay for somatomedin C: comparison with radioreceptor assay in patients with growth-hormone disorders, hypothyroidism, and renal failure

    SciTech Connect

    Baxter, R.C.; Brown, A.S.; Turtle, J.R.

    1982-03-01

    An antiserum (Tr4) was raised in rabbits against a basic somatomedin C-like peptide preparation. Using high-immunoreactivity somatomedin C tracer, we compared the performance of radioimmunoassays in which we used the Tr4 antiserum distributed by the National Pituitary Agency (NPA) with that of the human placental-membrane somatomedin radioreceptor asay (RRA). In their cross reactivity towards various somatomedin-like and unrelated peptides, the two radioimmunoassay methods were almost identical, although NPA antiserum, with about fourfold higher titer than Tr4 antiserum, showed a slightly greater sensitivity for most peptides tested. Radioimmunoassay of acid-ethanol-extracted plasma samples from normal persons and acromegalic, hypopituitary, hypothyroid, and renal-failure patients revealed no analytical differences between the antisera (for 122 samples, r = 0.979 between methods). Somatomedin values for acromegalic and hypopituitary samples showed no overlap with normals. Values for hypothyroid and pre-dialysis renal-failure samples were significantly lower than normal. By comparison, the RRA showed greater cross reactivity towards some somatomedin-like peptides and gave significantly lower values than radioimmunoassay for acromegalic and hypothyroid plasma extracts, and significantly higher values for hypopituitary and renal-failure samples. We conclude that the radioimmunoassay methods clearly are of greater diagnostic value than RRA for clinical somatomedin measurement.

  18. Comparison of interferon gamma release assay & tuberculin skin tests for diagnosis of latent tuberculosis in patients on maintenance haemodialysis.

    PubMed

    Agarwal, Sanjay K; Singh, Urvashi B; Zaidi, Sabahat H; Gupta, Sanjay; Pandey, Ravinder M

    2015-04-01

    Tuberculosis (TB) is a common infection in patients on haemodialysis. There is a definite role of treatment of latent TB (LTB) in these patients. However, diagnosis of LTB in these patients by tuberculin skin test (TST) is unreliable. There is suggestion that interferon gamma release assay (IGRA) will be more reliable test for diagnosis of LTB in this setting. Thus, we evaluated value of IGRA and TST for the diagnosis of LTB in patients on dialysis in an Indian setting. Patients with end stage kidney disease on dialysis were included. Patients with active TB were excluded. Each patient was subjected to TST (induration of ≥10 mm was taken as positive) and QuantiFERON TB Gold In-Tube test (QFT-GIT) for diagnosis of LTB. A total of 185 patients were included; 129 (69.7%) were males and mean age was 36.7 ± 12.3 yr. Past history of TB was present in 18 (9.7%) patients. One hundred and thirty four (72.4%) patients had scar of BCG vaccination. QFT-GIT test was positive in 66 (36%), TST in 32 (17%) and both in 13 (7%) patients. Of the 66 patients positive with QFT-GIT, only 13 (19.6%) were positive for TST. Of the 32 patients positive with TST, only 13 (40.6%) were positive with QFT-GIT; 100 (54%) patients were negative for both the tests. Overall, 85 (45.9%) patients were positive for either of the two tests. Poor agreement was shown between the two methods. On logistic regression analysis, odds of QFT-GIT to be positive in patients with BCG vaccination was 1.23 and with history of TB 0.99, both being insignificant. odds of tuberculin skin test to be positive in patients with BCG vaccination was 1.04 and with history of TB 0.99, both again being insignificant. Our findings showed that more number of patients (36%) on haemodialysis were positive for QuantiFERON Gold In-Tube test as compared to TST (17%). There was poor agreement between the two tests. No significant effect of BCG vaccination and history of TB in past was observed on both tests.

  19. Determination of free insulin-like growth factor-I in human serum: comparison of ultrafiltration and direct immunoradiometric assay.

    PubMed

    Frystyk, J; Ivarsen, P; Støving, R K; Dall, R; Bek, T; Hagen, C; Ørskov, H

    2001-04-01

    Two fundamentally different methods are currently used for the determination of free insulin-like growth factor-I (IGF-I): ultrafiltration by centrifugation (UF) and direct immunoradiometric assay (IRMA). The aim was to evaluate a commercial IRMA (DSL, Webster, TX, USA) and to compare it with UF. In the IRMA it is recommended that samples be incubated for 2 h at 5;C. When comparing samples (n = 8) incubated for 1 and 2 h, levels increased by 27 +/- 5% (P< 0.0001). When incubating samples at 22;C instead of 5;C, levels increased by 192 +/- 32% (P< 0.0001). Addition of IGF-binding protein-1 (IGFBP-1) to normal sera (n = 6) dose-dependently decreased ultrafiltered free IGF-I only (P< 0.0007). Similarly, UF was more sensitive than IRMA to addition of IGFBP-2 (P< 0.05). In healthy subjects (n = 35) IRMA yielded 20% higher levels than UF (1.09 +/- 0.09 vs 0.91 +/- 0.12 microg/L; P< 0.0001). IRMA and UF yielded similar results in healthy subjects treated with IGF-I (n = 5) or growth hormone (n = 7) and in acromegalic patients (n = 6) before and after somatostatin analogue treatment. However, marked differences were observed in conditions with elevated IGFBP-1 and -2. In type-1 diabetics (n = 23) ultrafiltered free IGF-I was more reduced than IRMA free IGF-I (38 +/- 9 vs 76 +/- 7% of matched controls (n = 13); P< 0.0001). In patients with chronic renal failure (n = 25), IRMA free IGF-I was identical to control levels (n = 13), whereas ultrafiltered free IGF-I was decreased by 51 +/- 7% (P< 0.0001). Similarly, women with anorexia nervosa (n = 9) studied before and after weight gain showed significant changes in ultrafiltered free IGF-I only (P< 0.03). In conclusion, IRMA was not very robust with respect to variations in sample incubation and this may bias results. IRMA generally yielded higher levels than UF, in accordance with the knowledge that IRMA measures free plus readily dissociable IGF-I. IRMA was less affected than UF by added IGFBP-1 and -2, and reductions in free

  20. Direct Comparison of Xpert MTB/RIF Assay with Liquid and Solid Mycobacterial Culture for Quantification of Early Bactericidal Activity

    PubMed Central

    Kayigire, Xavier A.; Friedrich, Sven O.; Venter, Amour; Dawson, Rodney; Gillespie, Stephen H.; Boeree, Martin J.; Heinrich, Norbert; Hoelscher, Michael

    2013-01-01

    The early bactericidal activity of antituberculosis agents is usually determined by measuring the reduction of the sputum mycobacterial load over time on solid agar medium or in liquid culture. This study investigated the value of a quantitative PCR assay for early bactericidal activity determination. Groups of 15 patients were treated with 6 different antituberculosis agents or regimens. Patients collected sputum for 16 h overnight at baseline and at days 7 and 14 after treatment initiation. We determined the sputum bacterial load by CFU counting (log CFU/ml sputum, reported as mean ± standard deviation [SD]), time to culture positivity (TTP, in hours [mean ± SD]) in liquid culture, and Xpert MTB/RIF cycle thresholds (CT, n [mean ± SD]). The ability to discriminate treatment effects between groups was analyzed with one-way analysis of variance (ANOVA). All measurements showed a decrease in bacterial load from mean baseline (log CFU, 5.72 ± 1.00; TTP, 116.0 ± 47.6; CT, 19.3 ± 3.88) to day 7 (log CFU, −0.26 ± 1.23, P = 0.2112; TTP, 35.5 ± 59.3, P = 0.0002; CT, 0.55 ± 3.07, P = 0.6030) and day 14 (log CFU, −0.55 ± 1.24, P = 0.0006; TTP, 54.8 ± 86.8, P < 0.0001; CT, 2.06 ± 4.37, P = 0.0020). The best discrimination between group effects was found with TTP at day 7 and day 14 (F = 9.012, P < 0.0001, and F = 11.580, P < 0.0001), followed by log CFU (F = 4.135, P = 0.0024, and F = 7.277, P < 0.0001). CT was not significantly discriminative (F = 1.995, P = 0.091, and F = 1.203, P = 0.316, respectively). Culture-based methods are superior to PCR for the quantification of early antituberculosis treatment effects in sputum. PMID:23596237

  1. Direct comparison of Xpert MTB/RIF assay with liquid and solid mycobacterial culture for quantification of early bactericidal activity.

    PubMed

    Kayigire, Xavier A; Friedrich, Sven O; Venter, Amour; Dawson, Rodney; Gillespie, Stephen H; Boeree, Martin J; Heinrich, Norbert; Hoelscher, Michael; Diacon, Andreas H

    2013-06-01

    The early bactericidal activity of antituberculosis agents is usually determined by measuring the reduction of the sputum mycobacterial load over time on solid agar medium or in liquid culture. This study investigated the value of a quantitative PCR assay for early bactericidal activity determination. Groups of 15 patients were treated with 6 different antituberculosis agents or regimens. Patients collected sputum for 16 h overnight at baseline and at days 7 and 14 after treatment initiation. We determined the sputum bacterial load by CFU counting (log CFU/ml sputum, reported as mean ± standard deviation [SD]), time to culture positivity (TTP, in hours [mean ± SD]) in liquid culture, and Xpert MTB/RIF cycle thresholds (C(T), n [mean ± SD]). The ability to discriminate treatment effects between groups was analyzed with one-way analysis of variance (ANOVA). All measurements showed a decrease in bacterial load from mean baseline (log CFU, 5.72 ± 1.00; TTP, 116.0 ± 47.6; C(T), 19.3 ± 3.88) to day 7 (log CFU, -0.26 ± 1.23, P = 0.2112; TTP, 35.5 ± 59.3, P = 0.0002; C(T), 0.55 ± 3.07, P = 0.6030) and day 14 (log CFU, -0.55 ± 1.24, P = 0.0006; TTP, 54.8 ± 86.8, P < 0.0001; C(T), 2.06 ± 4.37, P = 0.0020). The best discrimination between group effects was found with TTP at day 7 and day 14 (F = 9.012, P < 0.0001, and F = 11.580, P < 0.0001), followed by log CFU (F = 4.135, P = 0.0024, and F = 7.277, P < 0.0001). C(T) was not significantly discriminative (F = 1.995, P = 0.091, and F = 1.203, P = 0.316, respectively). Culture-based methods are superior to PCR for the quantification of early antituberculosis treatment effects in sputum.

  2. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  3. Partial comparison of the NxTAG Respiratory Pathogen Panel Assay with the Luminex xTAG Respiratory Panel Fast Assay V2 and singleplex real-time polymerase chain reaction for detection of respiratory pathogens.

    PubMed

    Esposito, Susanna; Scala, Alessia; Bianchini, Sonia; Presicce, Maria Lory; Mori, Alessandro; Sciarrabba, Calogero Sathya; Fior, Giulia; Principi, Nicola

    2016-09-01

    In this study, 185 nasopharyngeal swabs were tested to compare the sensitivity and specificity of the Luminex NxTAG (NxTAG) Respiratory Pathogen Panel (RPP) Assay with those of the Luminex Respiratory Virus Panel (RVP) Fast Assay v2 and singleplex real-time polymerase chain reaction (PCR). The NxTAG Assay identified at least one infectious agent in 164 (88.7%) of the swabs. In 91 (6.2%) tests with negative results with the RVP Fast Assay v2, a virus was identified by the NxTAG (P < 0.001). With the NxTAG Assay, the detection rates were significantly higher for respiratory syncytial virus (P = 0.003), human metapneumovirus (P < 0.001), human rhinovirus/human enterovirus (P = 0.009) and human adenovirus (P < 0.001). Finally, the NxTAG Assay identified M. pneumoniae in 32 of 44 (72.7%) PCR-positive samples. However, the concordance with real-time PCR results was low for both assays. In conclusion, the results indicate that the NxTAG Assay overcomes some of the limitations of previous Luminex assays, although further studies are needed for a more complete evaluation of the new assay.

  4. Reliability of the Siemens Enzygnost and Novagnost Epstein-Barr virus assays for routine laboratory diagnosis: agreement with clinical diagnosis and comparison with the Merifluor Epstein-Barr virus immunofluorescence assay.

    PubMed

    Kreuzer, Christina; Nabeck, Klaus Udo; Levy, H Roma; Daghofer, Elisabeth

    2013-06-03

    Diagnosis of Epstein-Barr virus (EBV) infection is routinely conducted by clinical laboratories, especially to diagnose infectious mononucleosis. At an estimated general population incidence of 1:200, this represents a potentially significant testing burden. We evaluated the reliability of the Siemens Novagnost® and Enzygnost® EBV microtiter assays measuring VCA IgM and IgG, and EBNA-1 IgG for clinical diagnosis of EBV-related infectious mononucleosis. Remnant sera from 537 patients tested for EBV infection were used to compare the Siemens assays to each other and to the Merifluor assay. The Siemens assays are qualitative/semiquantitative, automatable enzyme immunoassays. The Merifluor assays are manual, qualitative indirect immunofluorescent assays. Testing was conducted on the Siemens and Merifluor assays in parallel. All assays were conducted and interpreted according to each manufacturer's specifications. Agreement of serostatus between each of the three assays was assessed. Discrepant results were resolved using a third method (Mikrogen recomLine). Final EBV serostatus indicated 2.9% of the population had an acute infection, 89.6% had a past infection, and 7.5% were EBV naive. All three assays demonstrated 100% agreement with acute infection. Agreement with past-infection serostatus was 99.1% for Enzygnost, between 86% and 98.8% for Novagnost, and 98.1% for Merifluor. Seronegative agreement was 100% for Enzygnost, 89.7% for Novagnost, and 92.3% for Merifluor. The Siemens Enzygnost and Novagnost EBV microtiter assays are suitable for clinical rule-in of acute EBV infection and for identifying EBV-naive individuals. Both assays also adequately identify remote EBV infections. Because these assays can be automated, they can improve speed and efficiency of EBV testing, especially in high-volume laboratories.

  5. Comparison of a Flow Assay for Brucellosis Antibodies with the Reference cELISA Test in West African Bos indicus

    PubMed Central

    Bronsvoort, Barend M. deC.; Koterwas, Bronwyn; Land, Fiona; Handel, Ian G.; Tucker, James; Morgan, Kenton L.; Tanya, Vincent N.; Abdoel, Theresia H.; Smits, Henk L.

    2009-01-01

    Brucellosis is considered by the Food and Agricultural Organisation and the World Health Organisation as one of the most widespread zoonoses in the world. It is a major veterinary public health challenge as animals are almost exclusively the source of infection for people. It is often undiagnosed in both human patients and the animal sources and it is widely acknowledged that the epidemiology of brucellosis in humans and animals is poorly understood, particularly in sub-Saharan Africa. It is therefore important to develop better diagnostic tools in order to improve our understanding of the epidemiology and also for use in the field for disease control and eradication. As with any new diagnostic test, it is essential that it is validated in as many populations as possible in order to characterise its performance and improve the interpretation of its results. This paper describes a comparison between a new lateral flow assasy (LFA) for bovine brucellosis and the widely used cELISA in a no gold standard analysis to estimate test performance in this West African cattle population. A Bayesian formulation of the Hui-Walter latent class model incorporated previous studies' data on sensitivity and specificity of the cELISA. The results indicate that the new LFA is very sensitive (∼87%) and highly specific (∼97%). The analysis also suggests that the current cut-off of the cELSIA may not be optimal for this cattle population but alternative cut-offs did not significantly change the estimates of the LFA. This study demonstrates the potential usefulness of this simple to use test in field based surveillance and control which could be easily adopted for use in developing countries with only basic laboratory facilities. PMID:19381332

  6. [Comparison of prediction performance of PAHs carcinogenicity between a BALB/c-E6E7 cell transformation assay and a BALB/c 3T3 cell transformation assay].

    PubMed

    Wu, Shuang; Li, Jin-Tao; Zhong, Ru-Gang; Zeng, Yi

    2012-10-01

    To predict the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) by cell transformation assay using BALB/c 3T3 cells and HPV16-E6E7-transfected BALB/c 3T3 cells (BALB/c-E6E7 cells). The cell transformation assays induced by PAHs using BALB-E6E7 cells and BALB/c 3T3 cells. The initiating and promoting activities of PAHs examined in a BALB-E6E7 cell transformation assay were similar to in a BALB/c 3T3 cell transformation assay, which was up to the standard of agents classified by the IARC. There were much more transformed foci appeared and much shorter time consumed to accomplish phenotypic alterations in the BALB/c-E6E7 cell transformation assay than in the BALB/c 3T3 cell transformation assay. The BALB/c-E6E7 cell transformation assay was superior to the BALB/c 3T3 cell transformation assay in cost and labor performance, the sensitivity of transformation response. The BALB/c-E6E7 cell transformation assay, with a satisfied prediction performance of initiating activity and promoting activity, would improve the overall process of safety and risk assessment of carcinogenicity.

  7. Comparison of Schistosoma mansoni Prevalence and Intensity of Infection, as Determined by the Circulating Cathodic Antigen Urine Assay or by the Kato-Katz Fecal Assay: A Systematic Review

    PubMed Central

    Kittur, Nupur; Castleman, Jennifer D.; Campbell, Carl H.; King, Charles H.; Colley, Daniel G.

    2016-01-01

    The relationship between results from Kato-Katz (KK) fecal microscopy and urine-based point-of-care circulating cathodic antigen (POC-CCA) assays for Schistosoma mansoni infection remains a critical issue. This systematic literature review of 25 published papers compares prevalence of S. mansoni infection by KK with that by the POC-CCA assay. Nineteen published studies met our inclusion criteria for data extraction and analysis. Above a prevalence of 50% by KK, KK and POC-CCA results yielded essentially the same prevalence. Below 50% prevalence by KK, the prevalence by the POC-CCA assay was between 1.5- and 6-fold higher and increased as prevalence by KK decreased. Five of nine publications met inclusion criteria for extractable data on intensity of S. mansoni infection by KK assay and visual band density using the POC-CCA assay. A clear positive relationship exists between intensity by the KK and POC-CCA assays. This systematic review indicates that below 50% prevalence, the POC-CCA assay is much more sensitive than the KK assay. However, the existing data are inadequate to precisely define the relationship between POC-CCA and KK at lower levels of KK prevalence. More studies directly comparing the two assays in low-prevalence areas are essential to inform decision-making by national schistosomiasis control programs. PMID:26755565

  8. Comparison of Schistosoma mansoni Prevalence and Intensity of Infection, as Determined by the Circulating Cathodic Antigen Urine Assay or by the Kato-Katz Fecal Assay: A Systematic Review.

    PubMed

    Kittur, Nupur; Castleman, Jennifer D; Campbell, Carl H; King, Charles H; Colley, Daniel G

    2016-03-01

    The relationship between results from Kato-Katz (KK) fecal microscopy and urine-based point-of-care circulating cathodic antigen (POC-CCA) assays for Schistosoma mansoni infection remains a critical issue. This systematic literature review of 25 published papers compares prevalence of S. mansoni infection by KK with that by the POC-CCA assay. Nineteen published studies met our inclusion criteria for data extraction and analysis. Above a prevalence of 50% by KK, KK and POC-CCA results yielded essentially the same prevalence. Below 50% prevalence by KK, the prevalence by the POC-CCA assay was between 1.5- and 6-fold higher and increased as prevalence by KK decreased. Five of nine publications met inclusion criteria for extractable data on intensity of S. mansoni infection by KK assay and visual band density using the POC-CCA assay. A clear positive relationship exists between intensity by the KK and POC-CCA assays. This systematic review indicates that below 50% prevalence, the POC-CCA assay is much more sensitive than the KK assay. However, the existing data are inadequate to precisely define the relationship between POC-CCA and KK at lower levels of KK prevalence. More studies directly comparing the two assays in low-prevalence areas are essential to inform decision-making by national schistosomiasis control programs.

  9. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  10. Comparison of Benchtop Microplate Beta Counters with the Traditional Gamma Counting Method for Measurement of Chromium-51 Release in Cytotoxic Assays

    PubMed Central

    Wallace, Dora; Hildesheim, Allan; Pinto, Ligia A.

    2004-01-01

    The most traditional method used to measure the lytic activity of cytotoxic T lymphocytes or natural killer (NK) cells is the chromium release assay (CRA). No study has been reported that systematically compares the traditional gamma counting method with various benchtop microplate scintillation formats to measure chromium release. Here we investigated the utilization of microplate beta counters in comparison with the traditional gamma counting method to quantitate antigen-specific cytolysis, lymphokine-activated killer (LAK) activity, and NK activity in the CRA. Supernatants from standard CRA (n = 7) were directly transferred to a 96-well microplate containing either a solid scintillant (Lumaplate) or a liquid scintillant (flexible beta plate). Samples were quantified by using two benchtop microplate beta counters, Wallac Microbeta Trilux (Lumalux and Trilux methods, respectively) and Packard TopCount instruments (TopCount method). These results were then compared with data from an identical assay run in parallel using the traditional gamma counting method (LKB). The lytic activity for influenza virus-stimulated effectors measured in the benchtop microplate beta counters using Lumalux and Trilux methods exhibited excellent correlations with the one measured in the traditional LKB (r = 0.967 and 0.968, respectively). The TopCount method demonstrated a similar correlation (r = 0.966). Similar findings were observed for LAK and NK activity. The 96-well microplate format, specifically the dry-scintillant Lumaplates, offers several advantages over the traditional gamma counting format. Most notable are the reductions in sample volume needed and in the total sample preparation and counting time. Furthermore, this system reduces the amount of dry and mixed radioactive waste generated while using the same instrument for gamma- and beta-emitting isotopes. PMID:15013972

  11. Development and Comparison of TaqMan-Based Real-Time PCR Assays for Detection and Differentiation of Ralstonia solanacearum strains.

    PubMed

    Stulberg, Michael J; Rascoe, John; Li, Wenbin; Yan, Zonghe; Nakhla, Mark K; Huang, Qi

    2016-10-01

    Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.

  12. Predicting in vivo cardiovascular properties of β-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses

    PubMed Central

    Baker, Jillian G.; Kemp, Philip; March, Julie; Fretwell, Laurice; Hill, Stephen J.; Gardiner, Sheila M.

    2011-01-01

    β-Adrenoceptor antagonists differ in their degree of partial agonism. In vitro assays have provided information on ligand affinity, selectivity, and intrinsic efficacy. However, the extent to which these properties are manifest in vivo is less clear. Conscious freely moving rats, instrumented for measurement of heart rate (β1; HR) and hindquarters vascular conductance (β2; HVC) were used to measure receptor selectivity and ligand efficacy in vivo. CGP 20712A caused a dose-dependent decrease in basal HR (P<0.05, ANOVA) at 5 doses between 6.7 and 670 μg/kg (i.v.) and shifted the dose-response curve for isoprenaline to higher agonist concentrations without altering HVC responses. In contrast, at doses of 67 μg/kg (i.v.) and above, ICI 118551 substantially reduced the HVC response to isoprenaline without affecting HR responses. ZD 7114, xamoterol, and bucindolol significantly increased basal HR (ΔHR: +122±12, +129±11, and +59±11 beats/min, respectively; n=6), whereas other β-blockers caused significant reductions (all at 2 mg/kg i.v.). The agonist effects of xamoterol and ZD 7114 were equivalent to that of the highest dose of isoprenaline. Bucindolol, however, significantly antagonized the response to the highest doses isoprenaline. An excellent correlation was obtained between in vivo and in vitro measures of β1-adrenoceptor efficacy (R2=0.93; P<0.0001).—Baker, J. G., Kemp, P., March, J., Fretwell, L., Hill, S. J., Gardiner, S. M. Predicting in vivo cardiovascular properties of β-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses. PMID:21865315

  13. Comparison of a commercial real-time PCR assay, RealCycler® PJIR kit, progenie molecular, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections.

    PubMed

    Guillaud-Saumur, Thibaud; Nevez, Gilles; Bazire, Amélie; Virmaux, Michèle; Papon, Nicolas; Le Gal, Solène

    2017-04-01

    We compared the RealCycler® PJIR kit (Progenie Molecular), available in Europe, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. Excellent agreement was found (concordance rate, 97.4%; Cohen's kappa, 0.918>0.8) showing that this commercial assay represents an alternative method for the diagnosis of P. jirovecii infections.

  14. Quality assurance/quality control of foot and mouth disease solid phase competition enzyme-linked immunosorbent assay--Part II. Quality control: comparison of two charting methods to monitor assay performance.

    PubMed

    Goris, N; De Clercq, K

    2005-12-01

    Diagnostic laboratories are increasingly required to meet stringent quality standards, and validated assays are needed to achieve formal accreditation. Validation of test methods is often considered to be finalised when the assay parameters and characteristics have been established. However, like any process, diagnostic assays are subject to random variation resulting in shifts in the mean test values. Continuous monitoring of assays using control charts will alert the interpreter of changes in performance. For this purpose, several charting methods have been developed and implemented. This paper compares the Shewhart and the exponentially weighted moving average (EWMA) control charts with respect to the day to day monitoring of internal quality control samples for the foot and mouth disease solid phase competition enzyme-linked immunosorbent assay. Both chart types are equally sensitive to shifts, but the EWMA method seems to provide the best balance between false rejection and false acceptance.

  15. Validation of a fluorescence polarization assay (FPA) performed in microplates and comparison with other tests used for diagnosing B. melitensis infection in sheep and goats.

    PubMed

    Minas, A; Stournara, A; Minas, M; Stack, J; Petridou, E; Christodoulopoulos, G; Krikelis, V

    2007-03-30

    Fluorescence polarization assay (FPA) is a relatively new test for the serological diagnosis of Brucella spp. infection in animals. FPA, carried out in 96-well microplate format, was validated here for diagnosing B. melitensis infection in sheep and goats. This study included sera from 1933 sheep and goats, from animals reared in naturally infected flocks (verified by culture) and showing a positive reaction to two different tests conducted in parallel. In addition, 2154 sera originating from healthy sheep and goats, reared in areas where B. melitensis had never been isolated, were assayed. The optimum cut-off value offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 15 mP over the mean value of the buffer control used in each microplate as determined by receiver operating characteristic analysis. The DSn and DSp of the FPA for small ruminants carried out in microplates at this cut-off value were calculated to be 95.9% and 97.9% with 95% confidence intervals (95% CI) of 94.9-97.7% and 97.2-98.4%, respectively. The accuracy of the FPA, as expressed by determination of the area under the curve, was 0.991. Indirect ELISA and FPA tests offered the highest DSn when compared with the Rose Bengal test, the complement fixation test, the modified Rose Bengal test and competitive ELISA. The parallel or serial combination of FPA with indirect ELISA offers the highest DSn and DSp. As temperature can affect the results of the FPA, all reagents must be at the same temperature and the standard for comparison must always be read under the same conditions as the sera under test. FPA performed in microplates is a promising assay; the DSn and accuracy are better than those of the tests currently approved for diagnosing B. melitensis in small ruminants. Because of its simplicity, speed, and accuracy, this test can improve capacity for laboratory testing and the efficacy of an eradication program based on a test-and-slaughter policy.

  16. Evaluation of the New Siemens Tacrolimus Assay on the Dimension EXL Integrated Chemistry System Analyzer: Comparison With an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method.

    PubMed

    Bargnoux, Anne-Sophie; Sutra, Thibault; Badiou, Stéphanie; Kuster, Nils; Dupuy, Anne-Marie; Mourad, Georges; Pageaux, Georges-Philippe; Le Quintrec, Moglie; Cristol, Jean-Paul

    2016-12-01

    Many patients are maintained at the lower end of the tacrolimus (TAC) reference range (3-7 ng/mL), requiring the use of analytical methods displaying a very low limit of quantification for their follow-up. Therefore, the new Dimension TAC, based on affinity chrome-mediated immunoassay technology, was evaluated on the Dimension EXL Integrated Chemistry System (Siemens Healthcare Diagnostics Inc). The aims of this study were (1) to evaluate the analytical performances with special emphasis on sensibility at low levels of TAC, (2) to compare the results with an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method. Analytical performance (imprecision, linearity, limit of detection, and limit of quantification) was evaluated. Comparison to UPLC/MS/MS was performed on 106 whole blood samples from 88 transplant recipients using regression analysis and Bland-Altman plot analysis. Repeatability and within-laboratory coefficients of variation were <6% at mean TAC control levels of 3.7, 11.7, and 19.2 ng/mL. Linearity was confirmed between 1.0 and 22 ng/mL. Passing-Bablok regression analysis of Siemens TAC assay in comparison with UPLC/MS/MS values displayed a slope of 1.09 and an intercept of -0.42. Using Bland-Altman analysis, the mean bias was 0.27 ng/mL with 1.96 SD limits of -2.14 and 2.67 ng/mL. The new Dimension TAC immunoassay on the EXL analyzer demonstrated reliable and reproducible performances allowing routine monitoring in transplant patients, even at TAC concentrations at the lower end of the therapeutic range.

  17. Comparison of 4', 6'-diamidino-2-phenylindole and Giemsa stainings in preimplantation mouse embryos micronucleus assay including a triple dose study.

    PubMed

    Tian, Ying; Shen, Li; Gao, Yu; Yamauchi, Toru; Shen, Xiao-ming; Ma, Ning

    2007-04-01

    Analysis of micronuclei (MN) in preimplantation embryos is a good method for the evaluation of cytogenetic damage induced by occupational and environmental mutagen during early pregnancy. To examine whether conventional Giemsa staining produced the same accuracy of micronuclei as the DNA-specific 4', 6'-diamidino-2-phenylindole (DAPI) staining in preimplantation embryo induced by maternal exposure to chlorpyrifos, we conducted assays on 469 mouse (3 groups) preimplantation embryos micronucleus. Slides were stained with DAPI. After DAPI staining, the slides were de-stained and restained with Giemsa. Giemsa staining showed similar frequencies in MN to DNA-specific DAPI staining in all three groups. Both staining techniques revealed significant increases in frequency of MN in the treated group in comparison to the control group. Both methods showed a statistically significant correlation between MN frequency and the dose of chlorpyrifos. Compared with DAPI staining, the sensitivity of Giemsa staining was 85.0%, 86.0% and 90.9% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. The specificity was 97.9%, 91.4% and 96.5% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. Thus, we recommend that Giemsa staining technique be a standard staining method in detecting MN of preimplantation embryos induced by occupational or environmental hazards.

  18. Comparison of two new tests for rapid diagnosis of respiratory syncytial virus infections by enzyme-linked immunosorbent assay and immunofluorescence techniques.

    PubMed Central

    Freymuth, F; Quibriac, M; Petitjean, J; Amiel, M L; Pothier, P; Denis, A; Duhamel, J F

    1986-01-01

    The sensitivity and the specificity of two new commercial reagent tests, an indirect fluorescent antibody test (FAT) with a mouse monoclonal antibody (MAb) against respiratory syncytial virus (RSV) and an enzyme-linked immunosorbent assay (ELISA) RSV antigen detection kit, were determined by a comparison of results from these tests with those of tissue culture isolation and an indirect FAT with bovine polyclonal antibody (BPA). Of 251 nasal aspirates from infants with suspected RSV infection, positive results were found for 99 (39%) by the FAT-MAb, 93 (37%) by the FAT-BPA, and 87 (35%) by the ELISA; 69 of 240 (29%) were positive by cultures. The FAT-MAb was a more sensitive technique than cultures, with 87% sensitivity for the FAT-MAb and 84% for the ELISA. It was also more sensitive than the FAT-BPA, with 97% sensitivity for the FAT-MAb and 85% for the ELISA. This could be caused only by the distinctive volume of suspended specimens used in these tests. Of 171 negative culture specimens, positive (but not false-positive) results were found for 18% by the FAT-MAb and for 12% by the ELISA. Inversely, 13% of 69 culture positive specimens were FAT-MAb negative and 16% were ELISA negative, emphasizing the importance of tissue cultures for the maximum recovery of RSV, as well as for detection of other respiratory viruses. The FAT-MAb and ELISA were easy to perform and interpret, thus facilitating wider use. PMID:3536993

  19. The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB).

    PubMed

    Moquet, Jayne; Barnard, Stephen; Staynova, Albena; Lindholm, Carita; Monteiro Gil, Octávia; Martins, Vanda; Rößler, Ute; Vral, Anne; Vandevoorde, Charlot; Wojewódzka, Maria; Rothkamm, Kai

    2017-01-01

    Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Whole blood and separated lymphocyte samples were homogenously irradiated with (60)Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

  20. Comparison of NucliSens and Roche Monitor Assays for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma

    PubMed Central

    Dyer, John R.; Pilcher, Christopher D.; Shepard, Robin; Schock, Jody; Eron, Joseph J.; Fiscus, Susan A.

    1999-01-01

    We compared the performance of Organon Teknika’s NucliSens and Roche Diagnostic Systems’ Monitor quantitative human immunodeficiency type 1 RNA assays. Both had similar linearity and sensitivity over most of the dynamic range of the assays, although the Monitor assay was superior at the low range of RNA values while the NucliSens assay was more consistent at higher RNA values. NucliSens generally showed less interassay variability. PMID:9889240

  1. The fibrinogen antigenic turbidimetric assay (FIATA): the X2x test--the corrected chi-square comparison against the control-mean.

    PubMed

    Stief, Thomas W

    2007-01-01

    Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines,vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the 1O2 quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 microL plasma 50 microL PBS are added and the absorbance (A) at 405 nm is read. Then 50 microL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) DeltaA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a DeltaA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interessay CV values are < 4%. The normal range of FIATA is 100% +/-20% (x- +/- 1 SD). In = 321 or 344 unselected patient plasmas the FIATA (x- = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method (x- = 4.1 g/L; SD =1.7 g/L) with r = 0.755 and b) FIFTA (x- = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T)or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the two foldYates-corrected chi-square comparison against the mean value ofthe control-collective, called

  2. Comparison of Roche MONITOR and Organon Teknika NucliSens assays to quantify human immunodeficiency virus type 1 RNA in cerebrospinal fluid.

    PubMed

    Spearman, P; Fiscus, S A; Smith, R M; Shepard, R; Johnson, B; Nicotera, J; Harris, V L; Clough, L A; McKinsey, J; Haas, D W

    2001-04-01

    We compared Roche MONITOR and Organon Teknika NucliSens assays for human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF). Results of 282 assays were highly correlated (r = 0.826), with MONITOR values being 0.29 +/- 0.4 log(10) copies/ml (mean +/- standard deviation) values. Both assays can reliably quantify HIV-1 RNA in CSF.

  3. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    EPA Science Inventory

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  4. A comparison of the reliability of two gene targets in loop-mediated isothermal amplification assays for detecting leptospiral DNA in canine urine.

    PubMed

    Gentilini, Fabio; Zanoni, Renato Giulio; Zambon, Elisa; Turba, Maria Elena

    2017-01-01

    We compared 2 novel loop-mediated isothermal amplification (LAMP) assays that target either the 16S ribosomal RNA ( rrs) gene or the gene encoding a 32-kDa leptospiral lipoprotein ( lipL32) in order to assess the effect of the target on the accuracy of the LAMP assays. The most sensitive assay was the rrs assay with a limit of detection (LOD) of 1.2 × 10(1) genome equivalents per reaction. The novel lipL32 assay showed an LOD of 1.2 × 10(2) genome equivalents per reaction. Both assays showed adequate specificity when tested against a collection of bacteria commonly found in voided canine urine. However, when field samples were assayed, the rrs assays gave many false-positive results and a poor positive predictive value of 8.33%. In conclusion, even if the LAMP assay is used in low prevalence areas, the lipL32 assay would be preferable. Conversely, the higher analytical sensitivity of the rrs assay could be effectively used as a screening test in endemic areas with high disease prevalence, followed by confirmation of the positive results using the lipL32 assay.

  5. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    EPA Science Inventory

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  6. Comparison of the Luminex xTAG RVP Fast assay and the Idaho Technology FilmArray RP assay for detection of respiratory viruses in pediatric patients at a cancer hospital.

    PubMed

    Babady, N Esther; Mead, Peter; Stiles, Jeffrey; Brennan, Carrie; Li, Haijing; Shuptar, Susan; Stratton, Charles W; Tang, Yi-Wei; Kamboj, Mini

    2012-07-01

    Respiratory viruses are increasingly recognized as serious causes of morbidity and mortality in immunocompromised patients. The rapid and sensitive detection of respiratory viruses is essential for the early diagnosis and administration of appropriate antiviral therapy, as well as for the effective implementation of infection control measures. We compared the performance of two commercial assays, xTAG RVP Fast (Luminex Diagnostics, Toronto, Canada) and FilmArray RVP (FA RVP; Idaho Technology, Salt Lake City, UT), in pediatric patients at Memorial Sloan-Kettering Cancer Center. These assays detect the following viruses: respiratory syncytial virus; influenza A and B viruses; parainfluenza viruses 1, 2, 3, and 4; human metapneumovirus; adenovirus; enterovirus-rhinovirus; coronaviruses NL63, HKU1, 229E, and OC43; and bocavirus. We tested a total of 358 respiratory specimens from 173 pediatric patients previously tested by direct fluorescence assay (DFA) and viral culture. The overall detection rate (number of positive specimens/total specimens) for viruses tested by all methods was 24% for DFA/culture, 45% for xTAG RVP Fast, and 51% for FA RVP. The agreement between the two multiplex assays was 84.5%, and the difference in detection rate was statistically significant (P < 0.0001). Overall, the FA RVP assay was more sensitive than the xTAG RVP Fast assay and had a turnaround time of approximately 1 h. The sensitivity, simplicity, and random-access platform make FA RVP an excellent choice for laboratory on-demand service with low to medium volume.

  7. Comparison of antibody assays for detection of autoantibodies to Ro 52, Ro 60 and La associated with primary Sjögren's syndrome.

    PubMed

    Trier, Nicole Hartwig; Nielsen, Inger Ødum; Friis, Tina; Houen, Gunnar; Theander, Elke

    2016-06-01

    Anti-Ro(52/60) and anti-La constitute the hallmark autoantibodies in primary Sjögren's syndrome, being present in 40-70% of sera. Several anti-Ro/La assays exist, but antibody detection appears to be assay-specific, thus the aim of this study was to compare several anti-Ro/La assays. In total, 96 sera from individuals with primary Sjögren's syndrome and 114 healthy controls were tested for anti-Ro 52/60 and anti-La in 17 immunoassays. Especially the immunoassays used for detection of anti-Ro 52 differed in their sensitivity (48-79%), while only small differences in sensitivities were observed for the anti-Ro 60 (69-77%) anti-La (39-44%) assays. Concordances of 65%, 79% and 73% for the anti-Ro 52, anti-Ro 60 and anti-La assays were found, respectively. The majority of the assays yielded high specificities, primarily ranging from 97 to 100%, except from a single anti-Ro 60 assay, which yielded a specificity of 79%. Occasionally, reactivity levels were increased in a few assays, indicating that false-positive results can be obtained when applying assays of reduced specificity. In general, the commercial assays appeared to perform better than the in-house analyses. When correcting the in-house assays for background reactivity, sensitivities were reduced by approximately 7%, 17%, and 19% for anti-Ro 52, anti-Ro 60 and anti-La assays, respectively, illustrating the pitfalls when applying immunoassays for detection of autoantibodies, which in theory may apply to commercial assays as well. Finally, increased total sensitivities were obtained when combining assays. These studies contribute to clarify the clinical utility of immunoassays for detection of autoantibodies of Ro 52, Ro 60 and La and illustrate that the most efficient strategy to maximize antibody sensitivity is to combine several assays.

  8. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  9. Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-α transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

    PubMed

    Lee, Hae Kyung; Kim, Tae Sung; Kim, Chang Yeong; Kang, Il Hyun; Kim, Mi Gyeong; Jung, Ki Kyung; Kim, Hyung Sik; Han, Soon Young; Yoon, Hae Jung; Rhee, Gyu Seek

    2012-01-01

    The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17β-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17β-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.

  10. Comparison of 3 assay systems using a common probe substrate, calcein AM, for studying P-gp using a selected set of compounds.

    PubMed

    Szerémy, Péter; Pál, Akos; Méhn, Dóra; Tóth, Beáta; Fülöp, Ferenc; Krajcsi, Péter; Herédi-Szabó, Krisztina

    2011-01-01

    The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.

  11. A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.

    PubMed

    Turecek, P L; Romeder-Finger, S; Apostol, C; Bauer, A; Crocker-Buqué, A; Burger, D A; Schall, R; Gritsch, H

    2016-11-01

    Discrepancies have been previously reported for one-stage clotting and chromogenic assays for FVIII activity analysis. Inter-laboratory variations in instruments, method of clot detection, assay set-up, reference standard calibration, reagent source and reagent composition all contribute to assay variability. To characterise multilaboratory assay variability in measuring ADYNOVATE, OBIZUR and ADVATE FVIII activity in human plasma and survey multinational FVIII activity assay preferences. As samples from patients treated with either of the FVIII products are not available in the quantities required for a systematic collaborative study, haemophilia A plasma was spiked in vitro with either ADYNOVATE (PEGylated rFVIII), OBIZUR [Porcine Sequence Antihaemophilic Factor (Recombinant)] or ADVATE at high (0.80 IU or U mL(-1) ), medium (0.20 IU or U mL(-1) ) and low (0.05 IU or U mL(-1) ) FVIII concentrations, based on labelled potencies. Clinical laboratories used their routine FVIII activity assay to determine FVIII activity of each product. Thirty-five data sets using one-stage clotting assay and 11 sets using chromogenic assay were obtained. A vast majority of laboratories (98%) prefer and rely on the one-stage clotting assay. Mean recoveries across all concentrations were 113%, 120% and 127% for ADYNOVATE, OBIZUR and ADVATE respectively. Assay variation was comparable between ADVATE, ADYNOVATE and OBIZUR with inter-laboratory percent coefficients of variation (%CV) ranging from 11 to 22%. Mean chromogenic assay results were 116%, 51% and 113% for ADYNOVATE, OBIZUR and ADVATE respectively. Inter-laboratory CV's were similar for ADYNOVATE, OBIZUR and ADVATE. One-stage clotting assays can and will be used with sufficient accuracy and precision for the measurement of ADYNOVATE, OBIZUR and ADVATE in plasma samples from subjects with haemophilia A. Chromogenic assay underestimates OBIZUR potency, particularly at lower concentrations. © 2016 Baxalta Innovations Gmb

  12. Comparison of high-pressure liquid chromatography and microbiological assay for determination of ciprofloxacin tablets in human plasma employed in bioequivalence and pharmacokinetics study.

    PubMed

    Khan, Muhammad Khalid; Khan, Muhammad Farid; Mustafa, Ghulam; Sualah, Mohammed

    2012-01-01

    Ciprofloxacin was given orally to 28 healthy male volunteers for single oral dose of 500mg; Plasma samples were collected at different time's interval between 0 and 12h and analyzed both by high pressure liquid chromatography and by a microbiological assay. The detection limits (LOD) were 0.02μg/ml and 0.1μg/ml, for both methods respectively. For each method, coefficients of variation (R(2)) were 0.9995 and 0.9918 in plasma and limit of quantitation (LOQ).02 and 0.5μg/ml. The Comparison of means maximum concentration 2.68 μg/ml at 1.5 hr for test and 2.43 μg/ml are attain in HPLC method of Reference at 2hrs respectively. The plasma concentrations measured by microbiological assay of reference tablet are 3.95μg/ml (mean ± SE) at 1 hour and 3.80μg/ml (mean ± SE) at 1 hour. The concentrations in plasma measured by microbiological method were markedly higher than the high-pressure liquid chromatography values which indicates the presence of antimicrobially active metabolites. The mean ± SE values of pharmacokinetic parameters calculated by HPLC method, for total area under the curve (AUC 0-oo) were 13.11, and 11.91 h.mg/l for both test and reference tablets respectively. The mean ± SE values of clearance measured in l/h were 44.91 and 48.42 respectively. The elimination rate constant Kel [l/h] showed 0.17 l/h for test and 0.15 l/h reference tablets and likewise, absorption half-life expressed in hours shown 0.67 h for test and 1.04 h for reference respectively. The Mean Residence Time for test is 5.48 h and 5.49 h for reference. The mean ± SE values of pharmacokinetic parameters (Microbiological assay) for total area under the curve (AUC 0-oo) were 22.11 and 19.33 h.mg/l for both test and reference tablets respectively. The mean ± SE values of clearance measured in l/h were 29.02 and 31.63 respectively. The elimination rate constant Kel [l/h] showed 0.21 l/h for test and 0.20 l/h reference tablets and likewise, absorption half-life expressed in hours shown

  13. Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

    PubMed

    Hu, Jenny; Wala, Iwona; Han, Hong; Nagatani, Janice; Barger, Troy; Civoli, Francesca; Kaliyaperumal, Arunan; Zhuang, Yao; Gupta, Shalini

    2015-04-01

    Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Use of Whole-Genome Phylogeny and Comparisons for Development of a Multiplex PCR Assay To Identify Sequence Type 36 Vibrio parahaemolyticus

    PubMed Central

    Hall, Jeffrey A.; Xu, Feng; Ilyas, Saba; Siwakoti, Puskar; Cooper, Vaughn S.; Jones, Stephen H.

    2015-01-01

    Vibrio parahaemolyticus sequence type 36 (ST36) strains that are native to the Pacific Ocean have recently caused multistate outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole-genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern U.S. sources, were used to identify diagnostic loci, one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present in only one clade of closely related strains of ST36, ST59, and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two nonclade isolates and cps in four. Based on the distribution of these loci in sequenced genomes, prp identified clade strains with >99% accuracy, but the addition of one more locus increased accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus and determines the presence of both the tdh and trh hemolysin-encoding genes, which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a 4-year period in three northeastern U.S. states and 87 environmental isolates revealed that the prp and cps amplicons were detected only in clinical isolates identified as belonging to the ST36 clade and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections. PMID:25832299

  15. Comparison of enzyme-linked immunosorbent assay and gas chromatography procedures for the detection of cyanazine and metolachlor in surface water samples

    USGS Publications Warehouse

    Schraer, S.M.; Shaw, D.R.; Boyette, M.; Coupe, R.H.; Thurman, E.M.

    2000-01-01

    Enzyme-linked immunosorbent assay (ELISA) data from surface water reconnaissance were compared to data from samples analyzed by gas chromatography for the pesticide residues cyanazine (2-[[4-chloro-6-(ethylamino)-l,3,5-triazin-2-yl]amino]-2-methylpropanenitrile ) and metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide). When ELISA analyses were duplicated, cyanazine and metolachlor detection was found to have highly reproducible results; adjusted R2s were 0.97 and 0.94, respectively. When ELISA results for cyanazine were regressed against gas chromatography results, the models effectively predicted cyanazine concentrations from ELISA analyses (adjusted R2s ranging from 0.76 to 0.81). The intercepts and slopes for these models were not different from 0 and 1, respectively. This indicates that cyanazine analysis by ELISA is expected to give the same results as analysis by gas chromatography. However, regressing ELISA analyses for metolachlor against gas chromatography data provided more variable results (adjusted R2s ranged from 0.67 to 0.94). Regression models for metolachlor analyses had two of three intercepts that were not different from 0. Slopes for all metolachlor regression models were significantly different from 1. This indicates that as metolachlor concentrations increase, ELISA will over- or under-estimate metolachlor concentration, depending on the method of comparison. ELISA can be effectively used to detect cyanazine and metolachlor in surface water samples. However, when detections of metolachlor have significant consequences or implications it may be necessary to use other analytical methods.

  16. Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp.

    PubMed Central

    Ryu, Hodon; Griffith, John F.; Khan, Izhar U. H.; Hill, Stephen; Edge, Thomas A.; Toledo-Hernandez, Carlos; Gonzalez-Nieves, Joel

    2012-01-01

    Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters. PMID:22226950

  17. Comparison of BD GeneOhm Methicillin-Resistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA Assay for Screening Patients for the Presence of MRSA Strains▿

    PubMed Central

    Boyce, John M.; Havill, Nancy L.

    2008-01-01

    We compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) real-time PCR assay with the CHROMagar MRSA assay for the detection of MRSA in 286 nasal surveillance specimens. Compared with the CHROMagar MRSA assay, PCR had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 98.6%, 95.8%, and 100%, respectively. The mean PCR turnaround time was 14.5 h. PMID:18032616

  18. Comparison of Use of Cerebrospinal Fluid, Serum, and Throat Swab Specimens in Diagnosis of Enteroviral Acute Neurological Infection by a Rapid RNA Detection PCR Assay

    PubMed Central

    Andréoletti, Laurent; Blassel-Damman, Nathalie; Dewilde, Anny; Vallée, Louis; Cremer, Robin; Hober, Didier; Wattré, Pierre

    1998-01-01

    A PCR assay for detection of enterovirus RNA in multiple specimen types from patients with neurological infections was evaluated. Combined PCR assay of cerebrospinal fluid and serum (systemic specimens) was more sensitive than assaying either specimen alone in children but not in adults. Compared with PCR in systemic specimens, detection of enterovirus RNA in throat swabs showed a sensitivity of 62.5% and a specificity of 75.6%. PMID:9466785

  19. Comparison of Roche MONITOR and Organon Teknika NucliSens Assays To Quantify Human Immunodeficiency Virus Type 1 RNA in Cerebrospinal Fluid

    PubMed Central

    Spearman, Paul; Fiscus, Susan A.; Smith, Rita M.; Shepard, Robin; Johnson, Benjamin; Nicotera, Janet; Harris, Victoria L.; Clough, Lisa A.; McKinsey, Joel; Haas, David W.

    2001-01-01

    We compared Roche MONITOR and Organon Teknika NucliSens assays for human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF). Results of 282 assays were highly correlated (r = 0.826), with MONITOR values being 0.29 ± 0.4 log10 copies/ml (mean ± standard deviation) values. Both assays can reliably quantify HIV-1 RNA in CSF. PMID:11283098

  20. Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

    PubMed Central

    Di Giovanni, George D.; Rochelle, Paul A.

    2012-01-01

    This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

  1. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods.

    PubMed

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-08-13

    Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe - Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly

  2. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    PubMed Central

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-01-01

    Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot

  3. Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures.

    PubMed

    Calderaro, A; Martinelli, M; Motta, F; Larini, S; Arcangeletti, M C; Medici, M C; Chezzi, C; De Conto, F

    2014-08-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used ('Staphylococcus aureus/CNS', 'Enterococcus faecalis/OE', 'GNR Traffic Light' and 'Yeasts Traffic Light' PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  4. Validation and comparison of two commercial ELISA kits and three in-house developed real-time PCR assays for the detection of potentially allergenic mustard in food.

    PubMed

    Palle-Reisch, Monika; Hochegger, Rupert; Štumr, Stepan; Korycanova, Kveta; Cichna-Markl, Margit

    2015-05-01

    The study compares the applicability of two commercial mustard ELISA kits (Mustard ELISA Kit-specific and Mustard ELISA Kit-total) and three in-house developed real-time PCR assays (singleplex assay for white mustard, singleplex assay for black/brown mustard and duplex assay for the detection of white, black and brown mustard). Analyses of raw and brewed model sausages containing white and black/brown mustard in the range from 1 to 50 ppm indicate that both ELISAs and the three real-time PCR assays allow the detection of traces of mustard in raw and in brewed sausages. The ELISAs were found to be more sensitive than the real-time PCR assays. When the ELISAs and real-time PCR assays were applied to the analysis of 15 commercial foodstuffs differing in their labelling concerning mustard, in one sample mustard was detected with both ELISAs and the three real-time PCR assays although mustard was not indicated on the food ingredient list. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Use of an improved E. coli method for the measurement of cobalamin in serum: comparison with the E. gracilis assay results.

    PubMed Central

    Sourial, N A

    1981-01-01

    Owing to the higher serum cobalamin results that are obtained by R-binder radioisotopic dilution assay compared to microbiological assays (E. gracilis and L. leichmannii) it was suggested that serum contained a cobamide(s) that could not be detected by the more specific microbiological assays and that a much less specific test organism, which responds to most naturally occurring cobamides, such as the cobamide-dependent E. coli mutant, might respond to these cobamide(s) in serum. In an attempt to investigate this possibility an improved and simplified E. coli assay for the measurement of cobamide in serum was developed. The method is described, and the results obtained in normal subjects, in patients with megaloblastic anemia, and in anaemic pregnant women not suffering from megaloblastic anaemia are reported and compared with E. gracilis assay results. PMID:6787097

  6. Comparison of the Luminex Respiratory Virus Panel fast assay with in-house real-time PCR for respiratory viral infection diagnosis.

    PubMed

    Gadsby, Naomi J; Hardie, Alison; Claas, Eric C J; Templeton, Kate E

    2010-06-01

    The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.

  7. Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping.

    PubMed

    Lee, Hyo-Pyo; Cho, Woojae; Bae, Jae-Man; Shin, Ji Young; Shin, Soo-Kyung; Hwang, Sun Young; Min, Kyung Tae; Kim, Soo Nyung; Lee, Sun Joo; Kim, Soo-Ok; Yoo, Wang Don; Hong, Sun Pyo

    2013-06-01

    The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. We evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. The RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. Overall, 216 specimens (93.1%, κ = 0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9-3173.3) was higher than the LA assay (67.0, 95% CI: 8.2-2887.0). The RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis ▿

    PubMed Central

    Gadsby, Naomi J.; Hardie, Alison; Claas, Eric C. J.; Templeton, Kate E.

    2010-01-01

    The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens. PMID:20357215

  9. Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody.

    PubMed

    Fricke, W A; Brinkhous, K M; Garris, J B; Roberts, H R

    1985-09-01

    An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil-T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF-binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF-binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those

  10. A comparison of two West Nile virus detection assays (TaqMan reverse transcriptase polymerase chain reaction and VecTest antigen assay) during three consecutive outbreaks in northern Illinois.

    PubMed

    Lampman, Richard L; Krasavin, Nina M; Szyska, Michael; Novak, Robert J

    2006-03-01

    Mosquitoes identified as female Culex (Culex) species, primarily mixtures or uniform batches of Culex pipiens and Culex restuans, were collected daily from gravid traps by 2 mosquito abatement districts (MADs) in Cook County, Illinois. From 2002 through 2004, batches (pools) of mosquitoes were tested by the MADs for West Nile virus (WNV) by using VecTest WNV antigen assays and the same samples were retested, usually within 1-2 wk, for WNV RNA by the TaqMan reverse transcriptase polymerase chain reaction (RT-PCR). There were 952 TaqMan-positive pools out of 3,953 pools over the 3 years, and about one half of that number were VecTest-positive. The difference between the 2 detection assays varied between and within years. The VecTest assays detected about 57% and 69% of the TaqMan RT-PCR-positive pools from Des Plaines Valley MAD and Northwest MAD in 2002, but only about 40% and 46% in 2003, and 36% and 55% in 2004, respectively. Based on a subset of the 2004 data, a linear relationship was found between VecTest detection of WNV and TaqMan cycle threshold between 18 and 28 cycles. A temporal decrease in the difference between the 2 assays was observed in 2003 and 2004, which we conjecture is due, at least partially, to a seasonal decline in the proportion of recently infected mosquitoes. This trend was not observed in 2002 because infection rates indicated a high likelihood of more than 1 infected mosquito per pool at the peak of transmission. Unlike a previous study, the 95% confidence intervals of infection rates based on the 2 detection methods did not always overlap. The highest infection rates occurred in 2002 when mean monthly temperatures were above average.

  11. Detection of anti-infliximab antibodies is impacted by antibody titer, infliximab level and IgG4 antibodies: a systematic comparison of three different assays

    PubMed Central

    Afonso, Joana; Lopes, Susana; Gonçalves, Raquel; Caldeira, Paulo; Lago, Paula; Tavares de Sousa, Helena; Ramos, Jaime; Gonçalves, Ana Rita; Ministro, Paula; Rosa, Isadora; Vieira, Ana Isabel; Coelho, Rosa; Tavares, Patrícia; Soares, João; Sousa, Ana Lúcia; Carvalho, Diana; Sousa, Paula; da Silva, João Pereira; Meira, Tânia; Silva Ferreira, Filipa; Dias, Cláudia Camila; Chowers, Yehuda; Ben-Horin, Shomron; Magro, Fernando

    2016-01-01

    Background: There is scant information on the accuracy of different assays used to measure anti-infliximab antibodies (ADAs), especially in the presence of detectable infliximab (IFX). We thus aimed to evaluate and compare three different assays for the detection of IFX and ADAs and to clarify the impact of the presence of circulating IFX on the accuracy of the ADA assays. Methods: Blood samples from 79 ulcerative colitis (UC) patients treated with infliximab were assessed for IFX levels and ADAs using three different assays: an in-house assay and two commercial kits, Immundiagnostik and Theradiag. Sera samples with ADAs and undetectable levels of IFX were spiked with exogenous IFX and analyzed for ADAs. Results: The three assays showed 81–96% agreement for the measured IFX level. However, the in-house assay and Immundiagnostik assays detected ADAs in 34 out of 79 samples, whereas Theradiag only detected ADAs in 24 samples. Samples negative for ADAs with Theradiag, but ADA-positive in both the in-house and Immundiagnostik assays, were positive for IFX or IgG4 ADAs. In spiking experiments, a low concentration of exogenous IFX (5 µg/ml) hampered ADA detection with Theradiag in sera samples with ADA levels of between 3 and 10 µg/ml. In the Immundiagnostik assay detection interference was only observed at concentrations of exogenous IFX higher than 30 µg/ml. However, in samples with high levels of ADAs (>25 µg/ml) interference was only observed at IFX concentrations higher than 100 µg/ml in all three assays. Binary (IFX/ADA) stratification of the results showed that IFX+/ADA- and IFX-/ADAs+ were less influenced by the assay results than the double-positive (IFX+/ADAs+) and double-negative (IFX-/ADAs-) combination. Conclusions: All three methodologies are equally suitable for measuring IFX levels. However, erroneous therapeutic decisions may occur when patients show double-negative (IFX-/ADAs-) or double-positive (IFX+/ADAs+) status, since agreement between

  12. Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line).

    PubMed

    Zwolak, Iwona

    2015-08-01

    This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO(3) dose (=50 µM). Also, NaVO(3) cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO(3) at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO(3) concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO(3) on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO(3)-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO(3) cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity

  13. SU-E-T-427: Cell Surviving Fractions Derived From Tumor-Volume Variation During Radiotherapy for Non-Small Cell Lung Cancer: Comparison with Predictive Assays

    SciTech Connect

    Chvetsov, A; Schwartz, J; Mayr, N; Yartsev, S

    2014-06-01

    Purpose: To show that a distribution of cell surviving fractions S{sub 2} in a heterogeneous group of patients can be derived from tumor-volume variation curves during radiotherapy for non-small cell lung cancer. Methods: Our analysis was based on two data sets of tumor-volume variation curves for heterogeneous groups of 17 patients treated for nonsmall cell lung cancer with conventional dose fractionation. The data sets were obtained previously at two independent institutions by using megavoltage (MV) computed tomography (CT). Statistical distributions of cell surviving fractions S{sup 2} and cell clearance half-lives of lethally damaged cells T1/2 have been reconstructed in each patient group by using a version of the two-level cell population tumor response model and a simulated annealing algorithm. The reconstructed statistical distributions of the cell surviving fractions have been compared to the distributions measured using predictive assays in vitro. Results: Non-small cell lung cancer presents certain difficulties for modeling surviving fractions using tumor-volume variation curves because of relatively large fractional hypoxic volume, low gradient of tumor-volume response, and possible uncertainties due to breathing motion. Despite these difficulties, cell surviving fractions S{sub 2} for non-small cell lung cancer derived from tumor-volume variation measured at different institutions have similar probability density functions (PDFs) with mean values of 0.30 and 0.43 and standard deviations of 0.13 and 0.18, respectively. The PDFs for cell surviving fractions S{sup 2} reconstructed from tumor volume variation agree with the PDF measured in vitro. Comparison of the reconstructed cell surviving fractions with patient survival data shows that the patient survival time decreases as the cell surviving fraction increases. Conclusion: The data obtained in this work suggests that the cell surviving fractions S{sub 2} can be reconstructed from the tumor volume

  14. A comparison of different sample matrices for evaluating functional sensitivity, imprecision and dilution linearity of the Abbott ARCHITECT i2000 TSH assay.

    PubMed

    Quinn, Frank A; Scopp, Richard; Lach, Agnes; Drake, Christopher; Mo, May; Albright, John; Trimpe, Kevin

    2002-07-01

    Important performance characteristics for any thyroid-stimulating hormone (TSH) assay include low-end sensitivity, precision across a wide dynamic range, and linear specimen dilution. Laboratories often characterize the performance of their TSH assay using many different sample matrices (e.g. native human serum, synthetic buffer, processed human serum, etc.). However, this can lead to possible confusion as the relationships between sample matrix and assay performance are often poorly understood. The purpose of our study was to evaluate the performance of the ARCHITECT i2000 TSH assay using a variety of different sample matrices. Functional sensitivity was found to be essentially equivalent in both hyperthyroid patient sera (0.0035 microIU/ml) and TSH affinity-stripped (0.0038 microIU/ml) sample matrices. Assay imprecision was also independent of sample matrix, with total imprecision < or = 5.3% for both synthetic buffer and serum matrices. Similarly, specimens were found to dilute linearly over a wide range in both serum and synthetic buffer matrices. We conclude that the i2000 TSH assay measures TSH similarly in a variety of sample matrices. This is an important design feature for this immunoassay which provides assurance that analytical performance assessed with matrices other than unprocessed human serum are representative of assay performance seen with patient specimens.

  15. [Identification of 23 mycobacterial species by Invader assay with targeting 16S rRNA gene and ITS-1 region--comparison with DDH method in clinical isolates].

    PubMed

    Nagano, Makoto; Ichimura, Sadahiro; Ito, Nobuko; Tomii, Takayuki; Kazumi, Yuko; Takei, Katsuaki; Abe, Chiyoji; Sugawara, Isamu

    2008-07-01

    The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested. The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay. These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.

  16. Comparison of real-time PCR assays for detection of pathogenic Leptospira spp. in blood and identification of variations in target sequences.

    PubMed

    Bourhy, Pascale; Bremont, Sylvie; Zinini, Farida; Giry, Claude; Picardeau, Mathieu

    2011-06-01

    Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10(2) and 10(3) bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, i