Science.gov

Sample records for 3-day uterotrophic assay

  1. Ovariectomized mouse uterotrophic assay of 36 chemicals.

    PubMed

    Ohta, Ryo; Takagi, Atsuya; Ohmukai, Hideo; Marumo, Hideki; Ono, Atsushi; Matsushima, Yuko; Inoue, Tohru; Ono, Hiroshi; Kanno, Jun

    2012-01-01

    The concern over endocrine disruptors prompted international establishment of a strategic framework for the identification of the estrogenic compounds. OECD has launched the Conceptual Framework tool box containing various screening and testing methods including the uterotrophic assay. The (anti)estrogenicity of 36 chemicals suspected to be estrogen-receptor interactive by in silico and/or in vitro screening in the Extended Scheme for Endocrine Disruptor Screening and Testing of the Ministry of Health, Labour and Welfare, Japan, were monitored by the uterotrophic assay using C57BL/6J ovariectomized adult female mice after a 7-day exposure by oral gavage (po) and subcutaneous injection (sc). Ethynyl estradiol was used as reference for agonist and antagonist detection. In addition, Bisphenol A (sc) and Genistein (po) were tested for the comparison to rat assays. Among the 36, 2-[Bis(4-hydroxy-phenyl)methyl]benzylalcohol, 2,2',4,4'-Tetrahydroxybenzophenone, 2,4-Dihydroxybenzophenone, 3,3',5-Triiodothyroacetic acid, New fuchsin and alpha-Naphtholbenzein, showed both estrogenic agonistic and antagonistic activities; first two showed U-shaped dose-response in antagonistic studies. N,N-Diphenyl-p-phenylenediamine, 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone, n-Butyl 4-hydroxybenzoate, and Reserpine were agonistic by sc. Benzo [a] pyrene, Benz [a] anthracene, Dibenz [a,h] anthracene, 2-(2H-Benzotriazol-2-yl)-4,6-di(t-pentyl)phenol, Rosemarinic acid, meta-Thymol, 6-Gingerol, Colchicine, Malachite green base, Fenbuconazole, and Lead acetate were antagonistic. The rest, i.e. n-Heptyl 4-hydroxybenzoate, Tetrazolium violet, Pravastatin sodium salt, Physostigmine, salicylate (1:1), Nordihydroguaiaretic acid, o-Cresolphthalein, 1,3-Dinitrobenzene, C.I. Pigment orange, Tetrabromobis-phenol-A, 2-Hydroxy-4-methoxybenzophenone, Ethylparaben, Propyl p-hydroxybenzoate, Kaempferol, 2-(2-Benzotriazolyl)-p-cresol and Phenolphthalein were negative for both effects. Taking together with in

  2. Key Learnings from the Endocrine Disruptor Screening Program (EDSP) Tier 1 Rodent Uterotrophic and Hershberger Assays

    PubMed Central

    Marty, M Sue; O'Connor, John C

    2014-01-01

    In 2009, companies began screening compounds using the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP). EDSP has two tiers: Tier 1 includes 11 assays to identify compounds with potential endocrine activity. This article describes two laboratories' experiences conducting Tier 1 uterotrophic and Hershberger assays. The uterotrophic assay detects estrogen receptor agonists through increases in uterine weight. The advantages of the uterotrophic rat models (immature vs. adult ovariectomized) and exposure routes are discussed. Across 29 studies, relative differences in uterine weights in the vehicle control group and 17α-ethynylestradiol–positive control group were reasonably reproducible. The Hershberger assay detects androgen receptor (AR) agonists, antagonists, and 5α-reductase inhibitors through changes in accessory sex tissue (AST) weights. Across 23 studies, AST weights were relatively reproducible for the vehicle groups (baseline), testosterone propionate (TP) groups (androgenic response), and flutamide + TP groups (antiandrogenic response). In one laboratory, one and four compounds were positive in the androgenic and antiandrogenic portions of the assay, respectively. Each compound was also positive for AR binding. In the other laboratory, three compounds showed potential antiandrogenic activity, but each compound was negative for AR binding and did not fit the profile for 5α-reductase inhibition. These compounds induced hepatic enzymes that enhanced testosterone metabolism/clearance, resulting in lower testosterone and decreased capacity to maintain AST weights. The Hershberger androgenic and antiandrogenic performance criteria were generally attainable. Overall, the uterotrophic and Hershberger assays were easily adopted and function as described for EDSP screening, although the mode of action for positive results may not be easily determined. PMID:24515841

  3. Positive control study for the intact immature Swiss-Webster mouse uterotrophic assay

    NASA Astrophysics Data System (ADS)

    Alisjahbana, Arlisa; Yusuf, Ayda T.

    2014-03-01

    Endocrine disruptors are chemicals in the environment that can disrupt the normal functions of hormones in the body. A majority of these endocrine disruptors are estrogenic. The most commonly used in vivo method to test the estrogenicity of substances is the uterotrophic assay. This assay has many procedural variations, but works on the principle of measuring increase of uterine weight (uterotrophic response) as a response to estrogenic chemicals in animals with low levels of endogenous estrogens. Positive control study should be performed before using this method for the first time. The Developmental Biology laboratory in ITB plans to test pollutants for estrogenicity, therefore this study is conducted. In this study, intact immature Swiss-Webster mice (Mus musculus L.) were used. Twenty four female mice were weaned at 21 days old and divided into four groups. Each group were given 0, 5, 50, or 500 μg/kg bw 17β-estradiol daily for three days. 17β-estradiol dissolved in 10% ethanol-corn oil were administered via subcutaneous injection to the dorsoscapular and lumbar region with an injection volume of 0,05 mL/10 g bw. Twenty four hours after the last injection, the mice were killed by cervical dislocation and then dissected. The uterus is removed and weighted to obtain the uterine wet weight and blotted weight, and then processed for histological observation. Uterine gland number were calculated form transverse sections of the uterus, luminal epithelium height and cell number were measured form longitudinal sections. Both wet and blotted uterine weights per body weight ratio increased in a dose dependent manner with significant differences to controls (p< 0.05). Uterine gland number and luminal epithelium cell height also increase in a dose dependent manner with significant differences to controls (p<0.05). Cell number showed no significant difference to controls. The results are consistent with the hypothesis except for cell number which did not differ

  4. Uterotrophic and Hershberger assays for endocrine disruption properties of plastic food contact materials polypropylene (PP) and polyethylene terephthalate (PET).

    PubMed

    Chung, Bu Young; Kyung, Minji; Lim, Seong Kwang; Choi, Seul Min; Lim, Duck Soo; Kwack, Seung Jun; Kim, Hyung Sik; Lee, Byung-Mu

    2013-01-01

    Plasticizers or plastic materials such as phthalates, bisphenol-A (BPA), and styrene are widely used in the plastic industry and are suspected endocrine-disrupting chemicals (EDC). Although plastic materials such as polypropylene (PP) and polyethylene terephthalate (PET) are not EDC and are considered to be safe, their potential properties as EDC have not been fully investigated. In this study, plastic samples eluted from plastic food containers (PP or PET) were investigated in Sprague-Dawley rats using Hershberger and uterotrophic assays. In the Hershberger assay, 6-wk-old castrated male rats were orally treated for 10 consecutive days with plastic effluent at 3 different doses (5 ml/kg) or vehicle control (corn oil, 1 ml/100 g) to determine the presence of both anti-androgenic and androgenic effects. Testosterone (0.4 mg/ml/kg) was subcutaneously administered for androgenic evaluation as a positive control, whereas testosterone (0.4 mg/ml/kg) and flutamide (3 mg/kg/day) were administered to a positive control group for anti-androgenic evaluation. The presence of any anti-androgenic or androgenic activities of plastic effluent was not detected. Sex accessory tissues such as ventral prostate or seminal vesicle showed no significant differences in weight between treated and control groups. For the uterotrophic assay, immature female rats were treated with plastic effluent at three different doses (5 ml/kg), with vehicle control (corn oil, 1 ml/100 g), or with ethinyl estradiol (3 μg/kg/d) for 3 d. There were no significant differences between test and control groups in vagina or uterine weight. Data suggest that effluents from plastic food containers do not appear to produce significant adverse effects according to Hershberger and uterotrophic assays. PMID:23862761

  5. Estrogenicity of parabens revisited: impact of parabens on early pregnancy and an uterotrophic assay in mice.

    PubMed

    Shaw, Jordan; deCatanzaro, Denys

    2009-07-01

    Parabens, a class of preservatives routinely added to cosmetics, pharmaceuticals, and foods, have estrogenic properties. Given that intrauterine implantation of fertilized ova in inseminated females can be disrupted by minute levels of exogenous estrogens, we assessed the impact of parabens upon early gestation. In Experiment 1, butylparaben was administered subcutaneously in several doses ranging from 0.05 to 35 mg/animal/day to inseminated CF-1 mice on days 1-4 of pregnancy. Butylparaben exposure did not affect litter size, the number of pups born, postnatal day 3 litter weights, or the number of pups surviving to postnatal day 5. In contrast, administration of 500 ng/animal/day 17beta-estradiol terminated all pregnancies. In Experiment 2, propylparaben was subcutaneously administered to inseminated CF-1 mice on gestational days 1-4. Dams were sacrificed on gestation day 6 and the number of implantation sites was counted. Propylparaben had no impact on the number of implantation sites observed. Since Experiments 1 and 2 did not yield the anticipated results, an uterotrophic assay was conducted in Experiment 3 to re-evaluate the in vivo estrogenicity of parabens. Ovariectomized CF-1 and CD-1 mice were administered butylparaben in doses ranging from 0.735 to 35 mg per animal for three consecutive days. Mice were sacrificed on the fourth day, and uterine mass was recorded. There was no effect of butylparaben on uterine wet or dry mass at any dose in either strain. In contrast, administration of 17beta-estradiol consistently increased uterine mass in both strains. These data indicate that the estrogen-sensitive period of implantation is not vulnerable to paraben exposure, and that the in vivo estrogenicity of parabens may not be as potent as previously reported.

  6. A critical assessment of the OECD collaborative study to validate the uterotrophic assay for the detection of oestrogenic and anti-oestrogenic chemicals.

    PubMed

    Combes, Robert D

    2003-11-01

    The design and execution of a recently completed validation study on the rat uterotrophic assay for detecting oestrogens and anti-oestrogens, managed by the OECD, are critically assessed with respect to internationally agreed criteria for the validation of new in vitro and in vivo toxicity test methods. It is concluded that, while the design of the study did not take account of several important criteria for validation, the uterotrophic assay appears to reliably detect the strong and weak oestrogenic substances used in the study, which act via binding to the oestrogen receptor in vivo. However, the reliability of the assay has not been substantiated for detecting anti-oestrogens that act as antagonists, due to the involvement of an insufficient number of experiments and test chemicals. Moreover, the data do not permit an assessment of the accuracy of the prediction of oestrogenicity, and the protocols have not been sufficiently optimised with regard to controlling variables. This problem has been exacerbated by a wish to introduce as much flexibility as possible into the protocols during the formal validation phase of the study, rather than during a separate prevalidation stage. In addition, the choice between surgically treated and/or immature animals, and details of housing and husbandry conditions that are necessary for increasing the sensitivity and efficiency of the assay, need to be clarified. The assay also lacks a well-defined prediction model by which the overall relevance of the data to toxicity, and especially to human hazard, can be assessed, and no performance criteria have been established. The results of this analysis of the study indicate that it would be premature to produce an OECD test guideline for the uterotrophic assay at this time, before some of the above issues have been satisfactorily resolved. PMID:15598176

  7. A Curated Database of Rodent Uterotrophic Bioactivity

    PubMed Central

    Kleinstreuer, Nicole C.; Ceger, Patricia C.; Allen, David G.; Strickland, Judy; Chang, Xiaoqing; Hamm, Jonathan T.; Casey, Warren M.

    2015-01-01

    Background: Novel in vitro methods are being developed to identify chemicals that may interfere with estrogen receptor (ER) signaling, but the results are difficult to put into biological context because of reliance on reference chemicals established using results from other in vitro assays and because of the lack of high-quality in vivo reference data. The Organisation for Economic Co-operation and Development (OECD)-validated rodent uterotrophic bioassay is considered the “gold standard” for identifying potential ER agonists. Objectives: We performed a comprehensive literature review to identify and evaluate data from uterotrophic studies and to analyze study variability. Methods: We reviewed 670 articles with results from 2,615 uterotrophic bioassays using 235 unique chemicals. Study descriptors, such as species/strain, route of administration, dosing regimen, lowest effect level, and test outcome, were captured in a database of uterotrophic results. Studies were assessed for adherence to six criteria that were based on uterotrophic regulatory test guidelines. Studies meeting all six criteria (458 bioassays on 118 unique chemicals) were considered guideline-like (GL) and were subsequently analyzed. Results: The immature rat model was used for 76% of the GL studies. Active outcomes were more prevalent across rat models (74% active) than across mouse models (36% active). Of the 70 chemicals with at least two GL studies, 18 (26%) had discordant outcomes and were classified as both active and inactive. Many discordant results were attributable to differences in study design (e.g., injection vs. oral dosing). Conclusions: This uterotrophic database provides a valuable resource for understanding in vivo outcome variability and for evaluating the performance of in vitro assays that measure estrogenic activity. Citation: Kleinstreuer NC, Ceger PC, Allen DG, Strickland J, Chang X, Hamm JT, Casey WM. 2016. A curated database of rodent uterotrophic bioactivity. Environ

  8. A Demonstration of the Uncertainty in Predicting the Estrogenic Activity of Individual Chemicals and Mixtures From an In Vitro Estrogen Receptor Transcriptional Activation Assay (T47D-KBluc) to the In Vivo Uterotrophic Assay Using Oral Exposure.

    PubMed

    Conley, Justin M; Hannas, Bethany R; Furr, Johnathan R; Wilson, Vickie S; Gray, L Earl

    2016-10-01

    In vitro estrogen receptor assays are valuable tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently unable to fully account for absorption, distribution, metabolism, and excretion. To explore this issue, we calculated relative potency factors (RPF), using 17α-ethinyl estradiol (EE2) as the reference compound, for several chemicals and mixtures in the T47D-KBluc estrogen receptor transactivation assay. In vitro RPFs were used to predict rat oral uterotrophic assay responses for these chemicals and mixtures. EE2, 17β-estradiol (E2), benzyl-butyl phthalate (BBP), bisphenol-A (BPA), bisphenol-AF (BPAF), bisphenol-C (BPC), bisphenol-S (BPS), and methoxychlor (MET) were tested individually, while BPS + MET, BPAF + MET, and BPAF + BPC + BPS + EE2 + MET were tested as equipotent mixtures. In vivo ED50 values for BPA, BPAF, and BPC were accurately predicted using in vitro data; however, E2 was less potent than predicted, BBP was a false positive, and BPS and MET were 76.6 and 368.3-fold more active in vivo than predicted from the in vitro potency, respectively. Further, mixture ED50 values were more accurately predicted by the dose addition model using individual chemical in vivo uterotrophic data (0.7-1.5-fold difference from observed) than in vitro data (1.4-86.8-fold). Overall, these data illustrate the potential for both underestimating and overestimating in vivo potency from predictions made with in vitro data for compounds that undergo substantial disposition following oral administration. Accounting for aspects of toxicokinetics, notably metabolism, in in vitro models will be necessary for accurate in vitro-to-in vivo extrapolations.

  9. Uterotrophic activity of bisphenol A in the immature rat.

    PubMed Central

    Ashby, J; Tinwell, H

    1998-01-01

    Bisphenol A (BPA) is active in immature AP rat uterotrophic assays when evaluated using either the oral or the subcutaneous (sc) injection routes of exposure (three daily administrations of 400-800 mg/kg BPA). Premature vaginal opening was seen for 8 of 14 animals exposed to 600 and 800 mg/kg BPA by sc injection. Vaginal opening was not produced by BPA in the gavage studies. These results are consistent with those of Dodds and Lawson [Nature 137:96 (1936)] who found that BPA induces persistent vaginal cornification in ovariectomized rats exposed to three twice-daily injections of 85 mg/kg BPA (total daily dose 170 mg/kg), but they conflict with the reported inactivity of BPA in the immature mouse uterotrophic assay. The uterotrophic activity of diethylstilbestrol in the rat is also established (0.04 mg/kg/day for three days). PMID:9799186

  10. Uterotrophic assay of two concentrations of migrates from each of 23 polystyrenes administered orally (by gavage) to immature female Wistar rats.

    PubMed

    Bachmann, S; Hellwig, J; Jäckh, R; Christian, M S

    1998-01-01

    The Styrene Steering Committee (SSC) of the European Chemical Industry Council (CEFIC) sponsored this work to address any concern that styrene dimers and trimers that might migrate from polystyrene containers into food could possess some estrogenic activity and thus possibly affect human health. All phases of the study were conducted in conformance with GLP regulations and without knowledge of the oligomer migrates tested. All activities were managed and audited under a third-party contract between the SSC and Argus International. Low and high doses of the styrene oligomer migrates of 23 polystyrene samples [i.e. 9 general purpose polystyrenes (GPPS), 8 high impact polystyrenes (HIPS) and 6 expandable polystyrenes (EPS)] were tested for estrogenicity in an in vivo uterotrophic assay (immature female rat model). This model is considered to be the "gold standard" for use in screening for estrogenic effects because it evaluates both direct and indirect potential effects. The two concentrations of migrates of each of the 23 polystyrenes tested were selected to simulate daily human consumption of a low and high amount of food. Representative dimer and trimer concentrations were obtained in conformance with EEC Council Directives and calculated to be at levels simulating human consumption of 0.5 or 5 kg of food for the GPPS and the HIPS samples and of 0.5 or 3.15 kg of food for the EPS samples, respectively. The study was conducted in a series of three blocks. Each block included concurrent untreated control (negative control), vehicle control (25% ethanol, 20 ml/kg/day) and positive control (diethylstilbestrol-dipropionate, DES-DP, 5 micrograms/kg/day) groups, and low and high doses of each of 7 (1 block) or 8 (2 blocks) polystyrene oligomer migrates. Each group in each block consisted of 10 immature Wistar (Chbb: THOM-SPF) female rats. Beginning when the rats were 22 +/- 1 days of age, each rat was appropriately handled (untreated control group) or administered twice

  11. In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay

    EPA Science Inventory

    Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms o...

  12. In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay##

    EPA Science Inventory

    Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms of...

  13. Testing the Uterotrophic Activity of Perfluorooctanoic Acid (PFOA) in the Immature CD-1 Mouse

    EPA Science Inventory

    The uterotrophic assay is an in vivo screening tool used to determine the estrogenic or anti-estrogenic potential of an exogenously administered compound. Recent studies reported that PFOA increased activity of estrogen-responsive genes in fish, some in association with liver tum...

  14. Assessment of 3-nitro-1,2,4-triazol-5-one as a potential endocrine disrupting chemical in rats using the Hershberger and uterotrophic bioassays.

    PubMed

    Quinn, M J; Bannon, D I; Jackovitz, A M; Hanna, T L; Shiflett, A A; Johnson, M S

    2014-01-01

    The explosive 3-nitro-1,2,4-triazol-5-one (NTO) is an insensitive formulation developed to replace high energetics that are susceptible to accidental detonation from heat, shock, and impact. Although studies have shown NTO to be nontoxic at acute exposures, recent subacute and subchronic tests have demonstrated effects on testes and subsequent sperm production in rats. This study assessed endocrine disruption as a potential mechanism for these reproductive effects via the Hershberger and uterotrophic bioassays. These assays are 2 of the US Environmental Protection Agency's tier 1 in vivo screens for the Endocrine Disruptor Screening Program that measure differences in androgen- and estrogen-sensitive tissue weights in castrated and ovariectomized rats. The gonadectomized rats were orally exposed to NTO in a corn oil vehicle at doses of 250, 500, or 1000 mg/kg body weight (bw)/d for 10 and 3 days for the Hershberger and uterotrophic assays, respectively, according to standard protocols. Male rats also received testosterone (0.2 mg/kg/d, subcutaneous) and antiandrogenic flutamide (3mg/kg/d, oral) as negative and positive controls, and females received 17 α-ethynyl estradiol (0.3 µg/d, subcutaneous) as positive controls. 3-Nitro-1,2,4-triazol-5-one caused neither a decrease in androgen-sensitive male reproductive selected tissue (seminal vesicles with fluid/without fluid, glans penis, Cowper gland, ventral prostrate, and levator ani-bulbocavernosus) weights nor a change in uterine weights. The results of this study provide no evidence to suggest that NTO acts like an estrogenic or antiandrogenic endocrine disruptor in rats at these doses.

  15. The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses.

    PubMed Central

    Owens, William; Ashby, John; Odum, Jenny; Onyon, Lesley

    2003-01-01

    Many commercial laboratory diets have detectable levels of isoflavones (e.g., phytoestrogens such as genistein [GN]) that have weak estrogenic activity both in vitro and in vivo. During validation studies of the uterotrophic bioassay, diet samples from 20 participating laboratories were collected and analyzed for three major phytoestrogens: GN, daidzein (DN), and coumestrol (CM). Soy phytoestrogens GN and DN were found at total phytoestrogen levels from 100 to 540 microg/g laboratory diet; a forage phytoestrogen, CM, ranged from nondetectable to 4 microg/g laboratory diet. The phytoestrogen levels were compared with both baseline uterine weights of the control groups and with the relative uterine weight increase of groups administered two weak estrogen agonists: bisphenol A (BPA) and nonylphenol (NP). The comparison uses a working assumption of additivity among the phytoestrogens, despite several significant qualifications to this assumption, to estimate total genistein equivalents (TGE). Some evidence was found that phytoestrogen levels in the diet > 325-350 microg/g TGE could diminish the responsiveness of the uterotrophic bioassay to weak agonists. This was especially true for the case of the intact, immature female version of the uterotrophic bioassay, where higher food consumption relative to body weight leads to higher intakes of dietary phytoestrogens versus ovariectomized adults. This dietary level is sufficient in the immature female to approach a biological lowest observable effect level for GN of 40-50 mg/kg/day. These same data, however, show that low to moderate levels of dietary phytoestrogens do not substantially affect the responsiveness of the assay with weak estrogen receptor agonists such as NP and BPA. Therefore, laboratories conducting the uterotrophic bioassay for either research or regulatory purposes may routinely use diets containing levels of phytoestrogens < 325-350 microg/g TGE without impairing the responsiveness of the bioassay. PMID

  16. Raw drone milk of honeybees elicits uterotrophic effect in rats: evidence for estrogenic activity.

    PubMed

    Seres, Adrienn B; Ducza, Eszter; Báthori, Mária; Hunyadi, Attila; Béni, Zoltán; Dékány, Miklós; Gáspár, Róbert

    2013-05-01

    Numerous honeybee products are used in medicine, but the literature furnishes no information concerning the effects of the drone milk (DM), although drone brood, which is similar to DM, was reported to elicit a hormone-like strengthening effect. In certain countries, DM is traditionally used to treat infertility and to promote vitality in both men and women. The aim of this study was to determine the putative estrogen hormone-like effect of raw DM in rats and to identify the effective compounds. Uterotrophic assays revealed that DM increased the relative weight of the immature rat uterus. This effect was confirmed by reverse transcription polymerase chain-reaction and Western blot methods, in which the mRNA and protein expression of the estrogen-dependent peptide complement component C3 was determined. Column chromatography and uterotrophic assays were used to fractionate and check bioactivity, respectively. The active compound after the last fractionation was identified by the nuclear magnetic resonance and mass spectrometry techniques as E-dec-2-enedioic acid, which is very similar to the fatty acids with estrogenic activity that were previously isolated from royal jelly. These results lead us to suppose that E-dec-2-enedioic acid is responsible for the estrogen-like effect of DM. This appears to be the first report on the pharmacological effects of DM and E-dec-2-enedioic acid in mammals.

  17. Raw drone milk of honeybees elicits uterotrophic effect in rats: evidence for estrogenic activity.

    PubMed

    Seres, Adrienn B; Ducza, Eszter; Báthori, Mária; Hunyadi, Attila; Béni, Zoltán; Dékány, Miklós; Gáspár, Róbert

    2013-05-01

    Numerous honeybee products are used in medicine, but the literature furnishes no information concerning the effects of the drone milk (DM), although drone brood, which is similar to DM, was reported to elicit a hormone-like strengthening effect. In certain countries, DM is traditionally used to treat infertility and to promote vitality in both men and women. The aim of this study was to determine the putative estrogen hormone-like effect of raw DM in rats and to identify the effective compounds. Uterotrophic assays revealed that DM increased the relative weight of the immature rat uterus. This effect was confirmed by reverse transcription polymerase chain-reaction and Western blot methods, in which the mRNA and protein expression of the estrogen-dependent peptide complement component C3 was determined. Column chromatography and uterotrophic assays were used to fractionate and check bioactivity, respectively. The active compound after the last fractionation was identified by the nuclear magnetic resonance and mass spectrometry techniques as E-dec-2-enedioic acid, which is very similar to the fatty acids with estrogenic activity that were previously isolated from royal jelly. These results lead us to suppose that E-dec-2-enedioic acid is responsible for the estrogen-like effect of DM. This appears to be the first report on the pharmacological effects of DM and E-dec-2-enedioic acid in mammals. PMID:23631495

  18. AHTN and HHCB show weak estrogenic--but no uterotrophic activity.

    PubMed

    Seinen, W; Lemmen, J G; Pieters, R H; Verbruggen, E M; van der Burg, B

    1999-12-20

    The ubiquitous presence of the polycyclic musks AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetraline) and HHCB (1,2,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-gamma-2-b enzopyreen) in surface waters and their identification in human milk fat together with their polycyclic nature, which makes them potential candidates for interference with estrogen receptors, prompted us to assess these compounds for their potential estrogenic effects. We therefore investigated the effects of AHTN and HHCB in ERalpha- and ERbeta-dependent gene transcription assays with Human Embryonal Kidney 293 (HEK293) cells, which have proven to be very suitable to estimate the estrogenic activity of compounds with low binding activity (Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252-4264). Both AHTN and HHCB were found to induce a slight but dose-dependent stimulation of transcriptional activity in the transiently ERalpha transfected HEK293 cells. This weak estrogenic response was not observed in the ERbeta transiently transfected cells. However, these cells were less responsive to estradiol than the ERalpha transfected HEK293 cells. Also, no significant increase in transcriptional activity was observed in HEK293 cell lines, permanently expressing the same estrogen-responsive reporter gene construct and either ERalpha or ERbeta. In the classical uterine weight assay performed in juvenile Balb/c mice, no uterotrophic activity of AHTN and HHCB was noted at relatively high dietary exposure levels up to 50 and 300 ppm, respectively, at which levels an increase in liver weight was evident. Also the vitellogenin production by carp hepatocytes, a sensitive marker of estrogenic activity, was not affected by these two fragrance materials (Smeets, J.M.W., Rouhani Rankouhi, T., Nichols, K.M., Komen, H., Kaminsky, N

  19. Comparative Uterotrophic Effects of Endoxifen and Tamoxifen in Ovariectomized Sprague Dawley Rats

    PubMed Central

    Schweikart, Karen M.; Eldridge, Sandy R.; Safgren, Stephanie L.; Parman, Toufan; Reid, Joel M.; Ames, Matthew M.; Goetz, Matthew P.; Davis, Myrtle A.

    2014-01-01

    Endoxifen (4-hydroxyl-N-desmethyl tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for three days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was three-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen. PMID:24670817

  20. Does the Medicare 3-Day Rule Increase Length of Stay?

    PubMed

    Hernandez, Victor H; Ong, Alvin; Post, Zachary; Orozco, Fabio

    2015-09-01

    Medicare will only cover a stay in a skilled nursing facility (SNF) after TKA if the patient stays for at least 3 days at the inpatient hospital. The 3-day stay rule was instituted in 1965, to prevent over utilization of Medicare. We retrospectively reviewed 800 consecutive TKA, identifying patients that were discharged to rehab after surgery. 322 patients were discharged to SNF after surgery (209 Medicare, 113 private insurances). The LOS was 2.3 days for privately insured patients and 3.02 for Medicare recipients (P<0.05). No difference was found with regard to age, BMI, and ASA score. The Medicare 3-day rule independently increased the LOS in patients who required inpatient rehab, leading to increased cost. We suggest that this rule must be revised.

  1. Congenital aganglionosis in a 3-day-old Holstein calf

    PubMed Central

    2005-01-01

    Abstract Necropsy of a 3-day-old Holstein heifer revealed proximal megacolon and distal colorectal hypoplasia. Histologically, the hypoplastic distal colon and rectum lacked submucosal and myenteric ganglia. Clinical history, physical examination, and pathologic findings were consistent with intestinal aganglionosis, a congenital anomaly well documented in humans and foals but not previously reported in cattle. PMID:15943121

  2. Craniomandibular System and Postural Balance after 3-Day Dry Immersion

    PubMed Central

    Treffel, Loïc; Dmitrieva, Liubov; Gauquelin-Koch, Guillemette; Custaud, Marc-Antoine; Blanc, Stéphane; Gharib, Claude; Millet, Catherine

    2016-01-01

    The objective of the study was to determine the influence of simulated microgravity by exposure to dry immersion on the craniomandibular system. Twelve healthy male volunteers participated in a 3-day dry immersion study. Before and immediately after exposure we measured maximal bite force using piezoresistive sensors. The mechanical properties of the jaw and cervical muscles were evaluated before, during, and after dry immersion using MyotonPRO. Because recent studies reported the effects of jaw motor activity on the postural stability of humans, stabilometric measurements of center of pressure were performed before and after dry immersion in two mandibular positions: rest position without jaw clenching, and intercuspidal position during voluntary teeth clenching. Results revealed no significant changes of maximal bite force after dry immersion. All postural parameters were significantly altered by dry immersion. There were however no significant differences in stabilometric data according to mandibular position. Moreover the masseter tonicity increased immediately after the end of dry immersion period. Dry immersion could be used as a valid model for studying the effects of microgravity on human subjects. However, 3 days appear insufficient in duration to evaluate the effects of weightlessness on maximal bite force. Our research suggests a link between postural disturbance after dry immersion and masseter tonicity. PMID:26913867

  3. Craniomandibular System and Postural Balance after 3-Day Dry Immersion.

    PubMed

    Treffel, Loïc; Dmitrieva, Liubov; Gauquelin-Koch, Guillemette; Custaud, Marc-Antoine; Blanc, Stéphane; Gharib, Claude; Millet, Catherine

    2016-01-01

    The objective of the study was to determine the influence of simulated microgravity by exposure to dry immersion on the craniomandibular system. Twelve healthy male volunteers participated in a 3-day dry immersion study. Before and immediately after exposure we measured maximal bite force using piezoresistive sensors. The mechanical properties of the jaw and cervical muscles were evaluated before, during, and after dry immersion using MyotonPRO. Because recent studies reported the effects of jaw motor activity on the postural stability of humans, stabilometric measurements of center of pressure were performed before and after dry immersion in two mandibular positions: rest position without jaw clenching, and intercuspidal position during voluntary teeth clenching. Results revealed no significant changes of maximal bite force after dry immersion. All postural parameters were significantly altered by dry immersion. There were however no significant differences in stabilometric data according to mandibular position. Moreover the masseter tonicity increased immediately after the end of dry immersion period. Dry immersion could be used as a valid model for studying the effects of microgravity on human subjects. However, 3 days appear insufficient in duration to evaluate the effects of weightlessness on maximal bite force. Our research suggests a link between postural disturbance after dry immersion and masseter tonicity. PMID:26913867

  4. Endocrine Activity of Bisphenol S (BPS) Using In Vitro Estrogenic/Anti-Androgenic Transcriptional Activation Assays and the In Vivo Uterotrophic Assay

    EPA Science Inventory

    Bisphenol A (BPA) is gradually being phased out of many consumer products and processes leading to potential increases in human and environmental exposures to relatively understudied replacement compounds, including Bisphenol S (BPS). Research from our lab has shown that BPA and...

  5. "All in the mind"? Brain-targeting chemical delivery system of 17β-estradiol (Estredox) produces significant uterotrophic side effect.

    PubMed

    Prokai-Tatrai, Katalin; Szarka, Szabolcs; Nguyen, Vien; Sahyouni, Fatima; Walker, Cary; White, Shastazia; Talamantes, Tatjana; Prokai, Laszlo

    2012-01-01

    Here we revisit the peculiarly named redox chemical delivery system concept. This unique prodrug approach has long been claimed to be capable of targeting 17β-estradiol (E2), which has numerous beneficial central effects, into the brain without detrimental peripheral hormonal exposure. Using a well-established protocol to monitor E2's antidepressant-like effect, we show that the administration of this chemical delivery system incorporated into hydroxypropyl-β-cyclodextrin (i.e., Estredox), indeed, triggers a transient antidepressant-like behavior in ovariectomized mice. At the same time, even an acute dose of the carefully purified chemical delivery system produces significant circulating E2 levels and uterotrophic side effects for several days after drug administration. For the first time, we also unequivocally show by liquid chromatography coupled with tandem mass spectrometry that the uterus of the Estredox-treated animals contains a large quantity of E2 compared to that of the control group. These thus far unexposed yet consequential peripheral side effects brought about by Estredox call for a thorough and unbiased reassessment of the extent of brain-targeting of the hormone via the chemical delivery system approach. PMID:24380028

  6. “All in the mind”? Brain-targeting chemical delivery system of 17β-estradiol (Estredox) produces significant uterotrophic side effect

    PubMed Central

    Prokai-Tatrai, Katalin; Szarka, Szabolcs; Nguyen, Vien; Sahyouni, Fatima; Walker, Cary; White, Shastazia; Talamantes, Tatjana; Prokai, Laszlo

    2013-01-01

    Here we revisit the peculiarly named redox chemical delivery system concept. This unique prodrug approach has long been claimed to be capable of targeting 17β-estradiol (E2), which has numerous beneficial central effects, into the brain without detrimental peripheral hormonal exposure. Using a well-established protocol to monitor E2’s antidepressant–like effect, we show that the administration of this chemical delivery system incorporated into hydroxypropyl-β-cyclodextrin (i.e., Estredox), indeed, triggers a transient antidepressant-like behavior in ovariectomized mice. At the same time, even an acute dose of the carefully purified chemical delivery system produces significant circulating E2 levels and uterotrophic side effects for several days after drug administration. For the first time, we also unequivocally show by liquid chromatography coupled with tandem mass spectrometry that the uterus of the Estredox-treated animals contains a large quantity of E2 compared to that of the control group. These thus far unexposed yet consequential peripheral side effects brought about by Estredox call for a thorough and unbiased reassessment of the extent of brain-targeting of the hormone via the chemical delivery system approach. PMID:24380028

  7. Validity of a Self-Administered 3-Day Physical Activity Recall in Young Adults

    ERIC Educational Resources Information Center

    Han, Jennifer L.; Dinger, Mary K.

    2009-01-01

    Background: Most physical activity recall questionnaires assess activity over a 7-day period. However, questionnaires have been validated in adolescents and adults using shorter recall timeframes. Purpose: The purpose of this study was to assess the validity of a self-administered 3-day physical activity recall instrument (3DR) in young adults.…

  8. Object Individuation in 3-Day-Old Chicks: Use of Property and Spatiotemporal Information

    ERIC Educational Resources Information Center

    Fontanari, Laura; Rugani, Rosa; Regolin, Lucia; Vallortigara, Giorgio

    2011-01-01

    Object individuation was investigated in newborn domestic chicks. Chicks' spontaneous tendency to approach the larger group of familiar objects was exploited in a series of five experiments. In the first experiment newborn chicks were reared for 3 days with objects differing in either colour, shape or size. At test, each chick was presented with…

  9. Physiological responses of horses competing at a modified 1 star 3-day-event.

    PubMed

    Kohn, C W; Hinchcliff, K W; McCutcheon, L J; Geor, R; Foreman, J; Allen, A K; White, S L; Maykuth, P L; Williamson, L H

    1995-11-01

    The impending 1996 summer Olympic 3-day-event in Atlanta has focused attention on the need to determine what modifications to the demanding Endurance Test will be required to ensure safety of the horses competing. Three groups of horses participated in a Field Trial held in August of 1994 in northern Georgia to determine the safety and feasibility of conducting a modified 3-day-event in hot, humid weather. One group (TD) completed a modified 1 Star 3-day-event test, a control group (HT) completed a Horse Trial identical to the modified 1 Star test except for the omission of Phases B and C and the third group (E), comprised of European horses, completed the modified 1 Star test with a longer, faster Phase C than was used for TD. During the Endurance Test, the ambient temperature and relative humidity ranged from 24.3 degrees C and 98.9% in the morning to 30.2 degrees C and 51.6% in the afternoon. No horse failed to complete the Trial because of heat stress or fatigue. There were no significant (P < 0.05) differences detected in heart rate, rectal temperature, respiratory rate or net weight loss between HT and TD horses at any observation time. The highest rectal temperature recorded at the end of Phase C was 39.6 degrees C. These findings suggest that the modified 1 Star Endurance Test was as well tolerated by American horses as the control Horse Trial test. Rectal temperature was significantly higher for E than for TD or HT at the finish of Phase C. European horses had significantly greater decreases in weight than HT and TD at the end of Phases C and D and the next day. These findings probably reflect the faster and longer work effort of E horses during Phase C. Modification of Phase C and the rest-pause to ensure that recovery and heat dissipation occurred before the start of Phase D resulted in a 3-day-event that was safe for horses. The Field Trial provides a model for designing a modified Olympic Endurance Test. If the 1996 Olympic 3-day-event is held in

  10. A 3-Day-Old Girl Referred From Her Pediatrician for Oral Ulcerations.

    PubMed

    Neel, Mary Lauren; Kern, Jeremy; Ronis, Tova

    2016-09-01

    A 3-day-old girl was referred from her pediatrician for oral ulcerations. The patient was otherwise well appearing and afebrile. Her prenatal and antenatal courses were unremarkable, except for a failed routine hearing screen. The patient's examination was notable for several yellowish ulcers on erythematous bases located on her anterior tonsillar pillars. The patient also had a right coloboma and a II/VI systolic ejection murmur. Laboratory analyses revealed a traumatic lumbar puncture with 182 000 red blood cells and 808 white blood cells, as well as a complete blood count that showed thrombocytopenia and leukocytosis. During the patient's hospitalization, she developed a new facial rash. Her physical examination findings, along with her diagnostic evaluation and hospital course, ultimately led to 2 surprising diagnoses elaborated on in this case discussion.

  11. A 3-Day-Old Girl Referred From Her Pediatrician for Oral Ulcerations.

    PubMed

    Neel, Mary Lauren; Kern, Jeremy; Ronis, Tova

    2016-09-01

    A 3-day-old girl was referred from her pediatrician for oral ulcerations. The patient was otherwise well appearing and afebrile. Her prenatal and antenatal courses were unremarkable, except for a failed routine hearing screen. The patient's examination was notable for several yellowish ulcers on erythematous bases located on her anterior tonsillar pillars. The patient also had a right coloboma and a II/VI systolic ejection murmur. Laboratory analyses revealed a traumatic lumbar puncture with 182 000 red blood cells and 808 white blood cells, as well as a complete blood count that showed thrombocytopenia and leukocytosis. During the patient's hospitalization, she developed a new facial rash. Her physical examination findings, along with her diagnostic evaluation and hospital course, ultimately led to 2 surprising diagnoses elaborated on in this case discussion. PMID:27507895

  12. A 3-day EGCG-supplementation reduces interstitial lactate concentration in skeletal muscle of overweight subjects

    PubMed Central

    Most, Jasper; van Can, Judith G P; van Dijk, Jan-Willem; Goossens, Gijs H.; Jocken, Johan; Hospers, Jeannette J.; Bendik, Igor; Blaak, Ellen E.

    2015-01-01

    Green tea, particularly epigallocatechin-3-gallate (EGCG), may affect body weight and composition, possibly by enhancing fat oxidation. The aim of this double-blind, randomized placebo-controlled cross-over study was to investigate whether 3-day supplementation with EGCG (282mg/day) stimulates fat oxidation and lipolysis in 24 overweight subjects (age = 30 ± 2yrs, BMI = 27.7 ± 0.3 kg/m2). Energy expenditure, substrate metabolism and circulating metabolites were determined during fasting and postprandial conditions. After 6 h, a fat biopsy was collected to examine gene expression. In 12 subjects, skeletal muscle glycerol, glucose and lactate concentrations were determined using microdialysis. EGCG-supplementation did not alter energy expenditure and substrate oxidation compared to placebo. Although EGCG reduced postprandial circulating glycerol concentrations (P = 0.015), no difference in skeletal muscle lipolysis was observed. Fasting (P = 0.001) and postprandial (P = 0.003) skeletal muscle lactate concentrations were reduced after EGCG-supplementation compared to placebo, despite similar tissue blood flow. Adipose tissue leptin (P = 0.05) and FAT/CD36 expression (P = 0.08) were increased after EGCG compared to placebo. In conclusion, 3-day EGCG-supplementation decreased postprandial plasma glycerol concentrations, but had no significant effects on skeletal muscle lipolysis and whole-body fat oxidation in overweight individuals. Furthermore, EGCG decreased skeletal muscle lactate concentrations, which suggest a shift towards a more oxidative muscle phenotype. PMID:26647963

  13. Individual and mixture endocrine activity of BPS and BPC using in vitro estrogenic/anti-androgenic transcriptional activation assays and the in vivo uterotrophic assay- RAP 2.2-SSWR

    EPA Science Inventory

    Bisphenol A (BPA) is gradually being phased out of many consumer products and processes leading to potential increases in human and environmental exposures to relatively understudied replacement compounds, including Bisphenol S (BPS) and Bisphenol C (BPC).Research from our lab ha...

  14. Radar observations of a 3-day Kelvin wave in the equatorial mesosphere

    NASA Astrophysics Data System (ADS)

    Riggin, Dennis M.; Fritts, David C.; Tsuda, Toshitaka; Nakamura, Takuji; Vincent, Robert A.

    1997-11-01

    Mesospheric radars are used to investigate the characteristics of a Kelvin wave from two equatorial sites: Jakarta, Indonesia, in the western Pacific and Christmas Island in the central Pacific. Our study focuses on the time span from mid-January through mid-October 1993. A Kelvin wave with a period near 3 days was detected throughout this 9-month duration, although it underwent deep amplitude modulations on a ˜20-day timescale. A fitting procedure is applied to study the phase/amplitude behavior of the wave. The vertical wavenumber was measured by the radars and found to be small, wandering around zero with only a weak bias toward downward phase progression. The long vertical wavelength suggests that the wave was predominantly zonal wavenumber 1. The amplitude of the wave measured by the Jakarta meteor scatter radar was much larger than the amplitude measured by the MF partial reflection radar at Christmas Island. The smaller wave amplitude at Christmas Island could at least partially be due to a measurement bias associated with MF radars. The radar at Jakarta is a VHF meteor scatter radar and is not susceptible to this bias. However, the mean velocities and the amplitudes of the tidal and quasi 2-day wave components were in good agreement at the two sites. The estimated 9-month averaged zonal acceleration was ˜0.67 ms-1 day-1 over Jakarta at 94-98 km and only about half as large over Christmas Island. The magnitude of the zonal acceleration occasionally showed large enhancements which suggest the importance of refractive effects associated with vertical and temporal variations in the mean winds. The larger 3-day wave amplitudes and inferred acceleration at Jakarta may reflect its location in the western Pacific, a region of high convection, and hence an excitation region for equatorial waves. The relative phase of the wave between the two radar sites gradually shifted over a timescale of weeks. These smooth variations in relative phase are suggestive of a

  15. Rat lung metabolism after 3 days of continuous exposure to 0. 6 ppm ozone

    SciTech Connect

    Bassett, D.J.; Bowen-Kelly, E.

    1986-02-01

    Continuous exposure of rats to low concentrations of ozone has previously been associated with enhanced metabolic enzyme activities, when measured in lung homogenates. In this study, metabolic rates were measured in intact perfused lungs with altered pathology brought about by 3 days continuous exposure to 0.6 ppm ozone. Increased metabolism of ozone-exposed lungs was indicated by a twofold enhancement in glucose utilization, associated with a 62% increase in lactate formation and a 166% increase in the rate of 14CO2 production from D-(U-14C)glucose from control values of 5.2 +/- 0.5 mumol lactate and 4.4 +/- 0.6 mumol 14CO2/h per lung (+/- SE, n = 4), respectively. Mitochondrial metabolism was separately assessed by measurements of 14CO2 production from (U-14C)-pyruvate, which was found not to be significantly altered by ozone exposure, although homogenate oxygen uptake in the presence of succinate was significantly enhanced by 57%. These changes in intermediary metabolism could be correlated with increased glucose carbon incorporation into lipid and elevated activity of glucose-6-phosphate dehydrogenase. The observed elevated metabolic rates were consistent with the energy and synthetic needs of a lung during repair of ozone-induced damage.

  16. Short term /1 and 3 day/ cardiovascular adjustments to suspension antiorthostasis in rats

    NASA Technical Reports Server (NTRS)

    Musacchia, X. J.; Steffen, J. M.

    1982-01-01

    Antiorthostasis (AO) results in responses reflective of thoracic vessel loading. Initial findings included fluid and electrolyte shifts (diuresis and natriuresis) in AO but not in orthostatic (O) rats. This study aims at obtaining supportive evidence for cardiovascular responses, E.g., blood pressure and related parameters, in light of the original hypothesis. Tilting rats rapidly head-up from either horizontal (O) or head-down (AO) positions was used to assess cardiovascular sensitivities. O and AO rats were used after 1 or 3 days of suspension. Rats were controls (C), pre-tilted O and AO, tilted O and AO (rapid head-up 70-80 deg); post-tilted (to original postures). MAP in C rats was 108 +2 mmHg, in O, (117 + or - 1.02) and AO, (120 + or - 0.58). MAP, diastolic pressure (DP) and pulse pressure were consistently elevated in AO rats on day 3. With rapid head-up tilt, only MAP and DP showed significant increases. These changes were seen as cardiovascular responses to AO and support further use of this rat model for AO studies.

  17. Conformity to the opinions of other people lasts for no more than 3 days.

    PubMed

    Huang, Yi; Kendrick, Keith M; Yu, Rongjun

    2014-07-01

    When people are faced with opinions different from their own, they often revise their own opinions to match those held by other people. This is known as the social-conformity effect. Although the immediate impact of social influence on people's decision making is well established, it is unclear whether this reflects a transient capitulation to public opinion or a more enduring change in privately held views. In an experiment using a facial-attractiveness rating task, we asked participants to rate each face; after providing their rating, they were informed of the rating given by a peer group. They then rerated the same faces after 1, 3, or 7 days or 3 months. Results show that individuals' initial judgments are altered by the differing opinions of other people for no more than 3 days. Our findings suggest that because the social-conformity effect lasts several days, it reflects a short-term change in privately held views rather than a transient public compliance.

  18. Conformity to the opinions of other people lasts for no more than 3 days.

    PubMed

    Huang, Yi; Kendrick, Keith M; Yu, Rongjun

    2014-07-01

    When people are faced with opinions different from their own, they often revise their own opinions to match those held by other people. This is known as the social-conformity effect. Although the immediate impact of social influence on people's decision making is well established, it is unclear whether this reflects a transient capitulation to public opinion or a more enduring change in privately held views. In an experiment using a facial-attractiveness rating task, we asked participants to rate each face; after providing their rating, they were informed of the rating given by a peer group. They then rerated the same faces after 1, 3, or 7 days or 3 months. Results show that individuals' initial judgments are altered by the differing opinions of other people for no more than 3 days. Our findings suggest that because the social-conformity effect lasts several days, it reflects a short-term change in privately held views rather than a transient public compliance. PMID:24855020

  19. Treatment recommendations following 3-day masked continuous glucose monitoring (CGM) in youth with type 1 diabetes.

    PubMed

    Rasbach, Lisa E; Atkins, Ashley E; Milaszewski, Kerry M; Keady, Joyce; Schmidt, Lisa M; Volkening, Lisa K; Laffel, Lori M

    2014-05-01

    Glycemic control remains suboptimal in youth with type 1 diabetes. Retrospective continuous glucose monitoring (CGM) has demonstrated utility in fine-tuning diabetes management by detecting postprandial hyperglycemia and hypoglycemia. In this study, we explored the process of 3-day masked CGM use, subsequent treatment recommendations, and impact on A1c in a clinic-based sample of youth with type 1 diabetes. Over 2 years, 122 youth were referred for masked CGM. Patients/families completed a diary of blood glucose levels, insulin doses, food intake, and exercise during CGM use. A1c was assessed pre- and 2-3 months post-CGM. Treatment recommendations were formulated using data from CGM reports and diaries. Mean age was 14.3 ± 3.9 years, diabetes duration was 7.5 ± 4.7 years, and A1c was 8.5 ± 1.1% (69 ± 12 mmol/mol); 61% were pump-treated. Patients received an average of 3.1 ± 1.1 treatment recommendations following review of the CGM report. Most (80%) received reinforcement of the importance of preprandial bolusing; 37% received a recommendation regarding advanced insulin management (use of combination boluses/attend to active insulin). Receipt of the latter recommendation was related to A1c improvement ≥0.5% (OR: 4.0, P < .001). Masked CGM offers opportunities to guide advanced insulin management (by injection or pump), which may yield A1c improvements in youth with type 1 diabetes. PMID:24876612

  20. Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals

    PubMed Central

    Rotroff, Daniel M.; Dix, David J.; Houck, Keith A.; Knudsen, Thomas B.; Martin, Matthew T.; McLaurin, Keith W.; Reif, David M.; Crofton, Kevin M.; Singh, Amar V.; Xia, Menghang; Huang, Ruili

    2012-01-01

    Background: Over the past 20 years, an increased focus on detecting environmental chemicals that pose a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. Environmental Protection Agency (EPA) Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP; thus, processing these chemicals using current test batteries could require millions of dollars and decades. A need for increased throughput and efficiency motivated the development of methods using in vitro high throughput screening (HTS) assays to prioritize chemicals for EDSP Tier 1 screening (T1S). Objective: In this study we used U.S. EPA ToxCast HTS assays for estrogen, androgen, steroidogenic, and thyroid-disrupting mechanisms to classify compounds and compare ToxCast results to in vitro and in vivo data from EDSP T1S assays. Method: We implemented an iterative model that optimized the ability of endocrine-related HTS assays to predict components of EDSP T1S and related results. Balanced accuracy was used as a measure of model performance. Results: ToxCast estrogen receptor and androgen receptor assays predicted the results of relevant EDSP T1S assays with balanced accuracies of 0.91 (p < 0.001) and 0.92 (p < 0.001), respectively. Uterotrophic and Hershberger assay results were predicted with balanced accuracies of 0.89 (p < 0.001) and 1 (p < 0.001), respectively. Models for steroidogenic and thyroid-related effects could not be developed with the currently published ToxCast data. Conclusions: Overall, results suggest that current ToxCast assays can accurately identify chemicals with potential to interact with the estrogenic and androgenic pathways, and could help prioritize chemicals for EDSP T1S assays. PMID:23052129

  1. Human Monocyte Heat Shock Protein 72 Responses to Acute Hypoxic Exercise after 3 Days of Exercise Heat Acclimation

    PubMed Central

    Lee, Ben J.; Mackenzie, Richard W. A.; Cox, Valerie; James, Rob S.; Thake, Charles D.

    2015-01-01

    The aim of this study was to determine whether short-term heat acclimation (STHA) could confer increased cellular tolerance to acute hypoxic exercise in humans as determined via monocyte HSP72 (mHSP72) expression. Sixteen males were separated into two matched groups. The STHA group completed 3 days of exercise heat acclimation; 60 minutes cycling at 50% V˙O2peak in 40°C 20% relative humidity (RH). The control group (CON) completed 3 days of exercise training in 20°C, 40% RH. Each group completed a hypoxic stress test (HST) one week before and 48 hours following the final day of CON or STHA. Percentage changes in HSP72 concentrations were similar between STHA and CON following HST1 (P = 0.97). STHA induced an increase in basal HSP72 (P = 0.03) with no change observed in CON (P = 0.218). Basal mHSP72 remained elevated before HST2 for the STHA group (P < 0.05) and was unchanged from HST1 in CON (P > 0.05). Percent change in mHSP72 was lower after HST2 in STHA compared to CON (P = 0.02). The mHSP72 response to hypoxic exercise was attenuated following 3 days of heat acclimation. This is indicative of improved tolerance and ability to cope with the hypoxic insult, potentially mediated in part by increased basal reserves of HSP72. PMID:25874231

  2. Cellulase Assays

    NASA Astrophysics Data System (ADS)

    Zhang, Y. H. Percival; Hong, Jiong; Ye, Xinhao

    Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and β-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture.

  3. Relative validity of adolescent dietary patterns: comparison of a food frequency questionnaire and 3-day food record

    PubMed Central

    Ambrosini, Gina L; O’Sullivan, Therese A; de Klerk, Nicholas H; Mori, Trevor A; Beilin, Lawrence J; Oddy, Wendy H

    2012-01-01

    Interest in empirically derived dietary patterns has increased over the past decade. However, relatively few studies have evaluated dietary patterns using different dietary methods, or in young populations. We quantitatively compared dietary patterns from a food frequency questionnaire (FFQ) with those from a 3-day food record (FR) in a cohort of adolescents. Subjects from the Western Australian Pregnancy Cohort (Raine) Study completed a semi-quantitative FFQ and a 3-day FR at 14 y of age (n=783). Major dietary patterns were identified using exploratory factor analysis on 38 food groups. Dietary pattern z-scores were compared using 95% limits of agreement (LOA) and Spearman’s r. Two major dietary patterns were identified in the FFQ and FR. A ‘Healthy’ pattern was high in fresh fruit, vegetables, whole grains and grilled or canned fish. A ‘Western’ pattern was high in takeaway foods, confectionery, soft drinks, crisps and fried potato. The nutrient profiles of these dietary patterns were similar when estimated by the FFQ and FR. The LOA between dietary pattern scores from the FFQ and FR were -1.69 to 1.75 (‘Healthy’) and -1.89 to 1.82 (‘Western’). Minor differences in agreement were observed when boys and girls were analysed separately. Spearman’s correlation coefficients between the FFQ and FR were r=0.45 (‘Healthy’) and r=0.36 (‘Western’). Comparable dietary patterns may be obtained from a FFQ and FR using exploratory factor analysis. This supports the use of major dietary patterns identified using a FFQ in this adolescent cohort. PMID:21269548

  4. Quantitation of flaviviruses by fluorescent focus assay.

    PubMed

    Payne, Anne F; Binduga-Gajewska, Iwona; Kauffman, Elizabeth B; Kramer, Laura D

    2006-06-01

    An indirect immunofluorescence assay for quantitation of flaviviruses was developed as an alternative to the standard plaque assay. The assay was validated with West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Dengue virus (DENV) types 1-4. Vero cells were plated in 8-well chamber slides, and infected with 10-fold serial dilutions of virus. About 1-3 days after infection, cells were fixed, incubated with specific monoclonal antibody, and stained with a secondary antibody labeled with a fluorescent tag. Fluorescent foci of infection were observed and counted using a fluorescence microscope, and viral titers were calculated as fluorescent focus units (FFU) per ml. The optimal time for performing the fluorescent focus assay (FFA) on Vero cells was 24 h for WNV, and 48 h for SLEV and the four DENV serotypes. In contrast, the time required to complete a standard Vero cell plaque assay for these viruses range from 3 days for WNV to 11 days for DENV-1. Thus, the FFA method of virus titration is useful for viruses whose plaques develop slowly. In addition, these viruses can be quantitated by FFA on a mosquito cell line (C6/36), which does not support plaque formation. The FFA for flaviviruses was validated for accuracy, precision, specificity, and robustness of the assay.

  5. Cloud Based Surveys to Assess Patient Perceptions of Health Care: 1000 Respondents in 3 days for US $300

    PubMed Central

    Bardos, Jonah; Friedenthal, Jenna; Spiegelman, Jessica

    2016-01-01

    Background There are many challenges in conducting surveys of study participants, including cost, time, and ability to obtain quality and reproducible work. Cloudsourcing (an arrangement where a cloud provider is paid to carry out services that could be provided in-house) has the potential to provide vastly larger, less expensive, and more generalizable survey pools. Objective The objective of this study is to evaluate, using Amazon's Mechanical Turk (MTurk), a cloud-based workforce to assess patients’ perspectives of health care. Methods A national online survey posted to Amazon's MTurk consisted of 33 multiple choice and open-ended questions. Continuous attributes were compared using t tests. Results We obtained 1084 responses for a total cost of US $298.10 in less than 3 days with 300 responses in under 6 hours. Of those, 44.74% (485/1084) were male and 54.80% (594/1084) female, representing 49 out of 50 states and aged 18 to 69 years. Conclusions Amazon’s MTurk is a potentially useful survey method for attaining information regarding public opinions and/or knowledge with the distinct advantage of cost, speed, and a wide and relatively good representation of the general population, in a confidential setting for respondents. PMID:27554915

  6. Comparison of SAFER behavior assessment results in shelter dogs at intake and after a 3-day acclimation period.

    PubMed

    Bennett, Sara L; Weng, Hsin-Yi; Walker, Sheryl L; Placer, Margaret; Litster, Annette

    2015-01-01

    In this study, it was hypothesized that different results would be obtained by canine behavior assessments performed within 24 hr of shelter intake (Day 0) and after a 3-day acclimation period (Day 3). Safety Assessment for Evaluating Rehoming assessments were performed on 33 dogs at 2 municipal shelters. Agreements between Day 0 and Day 3 varied among subtests, and no consistent temporal patterns were observed. Weighted kappa statistics for each subtest ranged from .28 to .78, and percentage discordance was 0% to 18%. In a 2nd analysis, subtests skipped due to serious aggression were replaced with scores corresponding to serious aggression, and missing values for the Food subtest were replaced with scores for no aggression if the dog did not eat. For subtests skipped due to severe aggression, more than 50% of the dogs had scores indicating low aggression on the other assessment. Eight of 16 dogs who did not eat on Day 0 ate on Day 3; 2 showed aggression. Until the ideal time to test can be identified, it should be based on the individual dog's welfare status, and testing of dogs showing severe stress should be avoided.

  7. Comparison of SAFER behavior assessment results in shelter dogs at intake and after a 3-day acclimation period.

    PubMed

    Bennett, Sara L; Weng, Hsin-Yi; Walker, Sheryl L; Placer, Margaret; Litster, Annette

    2015-01-01

    In this study, it was hypothesized that different results would be obtained by canine behavior assessments performed within 24 hr of shelter intake (Day 0) and after a 3-day acclimation period (Day 3). Safety Assessment for Evaluating Rehoming assessments were performed on 33 dogs at 2 municipal shelters. Agreements between Day 0 and Day 3 varied among subtests, and no consistent temporal patterns were observed. Weighted kappa statistics for each subtest ranged from .28 to .78, and percentage discordance was 0% to 18%. In a 2nd analysis, subtests skipped due to serious aggression were replaced with scores corresponding to serious aggression, and missing values for the Food subtest were replaced with scores for no aggression if the dog did not eat. For subtests skipped due to severe aggression, more than 50% of the dogs had scores indicating low aggression on the other assessment. Eight of 16 dogs who did not eat on Day 0 ate on Day 3; 2 showed aggression. Until the ideal time to test can be identified, it should be based on the individual dog's welfare status, and testing of dogs showing severe stress should be avoided. PMID:25603466

  8. Motility parameters and intracellular ATP content of rabbit spermatozoa stored for 3 days at 15 degrees C.

    PubMed

    Gogol, Piotr

    2013-01-01

    The effect of semen storage duration on motility parameters and ATP content of rabbit spermatozoa were investigated. Ejaculates were collected from 9 New Zealand White male rabbits and diluted with a commercial rabbit semen extender Galap. Semen was stored at 15 degrees C for 3 days. On each day of storage sperm motility and intracellular ATP content were evaluated. Sperm motility parameters were assessed using the computer-assisted sperm analysis (CASA) system and ATP content using the bioluminescence method. The time of storage had a significant effect on sperm motility parameters (except straight-line velocity) and ATP content. A significant correlation was observed between motility parameters and sperm ATP content. The motility parameters most strongly correlated with ATP content were total motile spermatozoa (r = 0.6364), progressively motile spermatozoa (r = 0.529), amplitude of lateral head displacement (r = 0.4178), curvilinear velocity (r = 0.4111) and average path velocity (r = 0.3743). Results show that motility parameters determined using the CASA system and intracellular ATP content are sensitive indicators of sperm quality during in vitro storage and may be useful for estimation of in vivo fertilizing ability of rabbit semen. PMID:23767298

  9. Treatment of intracerebral hemorrhage in rats with 12 h, 3 days and 6 days of selective brain hypothermia.

    PubMed

    Fingas, Matthew; Penner, Mark; Silasi, Gergely; Colbourne, Frederick

    2009-09-01

    Intracerebral hemorrhage (ICH) is a devastating stroke with no proven treatment to reduce brain injury. In this study we modeled ICH by injecting 100 microL of autologous blood into the striatum of rats. We then tested whether hypothermia would reduce brain injury and improve recovery as has been repeatedly observed for ischemic and traumatic brain damage. Aside from reducing blood-brain barrier disruption, inflammation and edema, hypothermia has not consistently improved behavioral or histological outcome after ICH in animal studies. As this might relate to the choice of cooling method and the duration of hypothermia, we used a system that selectively cooled the injured hemisphere to approximately 32 degrees C (striatum) while the body remained normothermic. Cooling (vs. normothermia) started 1 h after ICH and lasted for 12 h, 3 days or 6 days followed by slow re-warming (approximately 1 degrees C/h). Functional impairment was evaluated from 2 to 3 weeks post-ICH at which time brain injury was determined. The ICH caused significant impairment on a neurological deficit scale and in tests of walking (horizontal ladder), skilled reaching (tray task) and spontaneous limb usage (cylinder test). Only the limb use asymmetry deficit was significantly mitigated by hypothermia, and then only by the longest treatment. Lesion volume, which averaged 16.9 mm3, was not affected. These results, in conjunction with earlier studies, suggest that prolonged mild hypothermia will not be a profound neuroprotectant for patients with striatal ICH, but it may nonetheless improve functional recovery in addition to its use for treating cerebral edema. PMID:19445934

  10. Effects of 3-day bed rest on physiological responses to graded exercise in athletes and sedentary men

    NASA Technical Reports Server (NTRS)

    Smorawinski, J.; Nazar, K.; Kaciuba-Uscilko, H.; Kaminska, E.; Cybulski, G.; Kodrzycka, A.; Bicz, B.; Greenleaf, J. E.

    2001-01-01

    To test the hypotheses that short-term bed-rest (BR) deconditioning influences metabolic, cardiorespiratory, and neurohormonal responses to exercise and that these effects depend on the subjects' training status, 12 sedentary men and 10 endurance- and 10 strength-trained athletes were submitted to 3-day BR. Before and after BR they performed incremental exercise test until volitional exhaustion. Respiratory gas exchange and heart rate (HR) were recorded continuously, and stroke volume (SV) was measured at submaximal loads. Blood was taken for lactate concentration ([LA]), epinephrine concentration ([Epi]), norepinephrine concentration ([NE]), plasma renin activity (PRA), human growth hormone concentration ([hGH]), testosterone, and cortisol determination. Reduction of peak oxygen uptake (VO(2 peak)) after BR was greater in the endurance athletes than in the remaining groups (17 vs. 10%). Decrements in VO(2 peak) correlated positively with the initial values (r = 0.73, P < 0.001). Resting and exercise respiratory exchange ratios were increased in athletes. Cardiac output was unchanged by BR in all groups, but exercise HR was increased and SV diminished in the sedentary subjects. The submaximal [LA] and [LA] thresholds were decreased in the endurance athletes from 71 to 60% VO(2 peak) (P < 0.001); they also had an earlier increase in [NE], an attenuated increase in [hGH], and accentuated PRA and cortisol elevations during exercise. These effects were insignificant in the remaining subjects. In conclusion, reduction of exercise performance and modifications in neurohormonal response to exercise after BR depend on the previous level and mode of physical training, being the most pronounced in the endurance athletes.

  11. Pharmacokinetics of oritavancin in plasma and skin blister fluid following administration of a 200-milligram dose for 3 days or a single 800-milligram dose.

    PubMed

    Fetterly, Gerald J; Ong, Christine M; Bhavnani, Sujata M; Loutit, Jeffrey S; Porter, Steven B; Morello, Lisa G; Ambrose, Paul G; Nicolau, David P

    2005-01-01

    Oritavancin is a novel glycopeptide currently being developed for the treatment of complicated skin and skin structure infections (cSSSI), including those caused by multidrug resistant gram-positive pathogens. The disposition of oritavancin in skin structures was investigated using a cantharide-induced blister fluid model. Seventeen healthy male subjects received oritavancin, but only 16 subjects were evaluated after one subject discontinued study drug. Each subject (eight per dose group) received 200 mg of oritavancin once a day for 3 days (group A) or 800 mg as one single dose (group B). Group A plasma samples and exudates from blister fluid were collected on days 3, 4, 7, 9, and 12 and on days 3, 4, 7, and 9, respectively. Group B samples and exudates were collected on days 1, 2, 5, 7, and 10 and on days 1, 2, 5, and 7, respectively. Drug concentrations were determined using a liquid chromatography-tandem mass spectrometry assay and, subsequently, pharmacokinetic analysis was performed. Differences between treatment groups in ratios for area under the concentration-time curve for blister fluid and plasma (AUC(blister fluid)/AUC(plasma) ratios) were evaluated using a t test (alpha = 0.05). Mean maximum concentration of drug in plasma or blister fluid was approximately 8-fold and 11-fold higher in plasma than in blister fluid following the 200- or 800-mg doses of oritavancin, respectively. Mean AUC(blister fluid)/AUC(plasma) ratios at 24 h were 0.190 (standard deviation [SD], 0.052) and 0.182 (SD, 0.062) for groups A and B, respectively (P = 0.791). To place these results in a clinical context, mean drug concentrations in blister fluid exceed the oritavancin MIC at which 90% of strains are inhibited of Staphylococcus aureus (2 microg/ml) by approximately 2- to 5.5-fold at 12 h and 1.5- to 3-fold at 24 h following administration of both dosing regimens. These results support the potential use of oritavancin for the treatment of cSSSI.

  12. Editor’s Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment

    PubMed Central

    Miller, Michelle M.; Alyea, Rebecca A.; LeSommer, Caroline; Doheny, Daniel L.; Rowley, Sean M.; Childs, Kristin M.; Balbuena, Pergentino; Ross, Susan M.; Dong, Jian; Sun, Bin; Andersen, Melvin A.

    2016-01-01

    A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERβ, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments. PMID:27503385

  13. Cross-species conservation of endocrine pathways: a critical analysis of tier 1 fish and rat screening assays with 12 model chemicals.

    PubMed

    Ankley, Gerald T; Gray, L Earl

    2013-04-01

    Many structural and functional aspects of the vertebrate hypothalamic-pituitary-gonadal (HPG) axis are known to be highly conserved, but the significance of this from a toxicological perspective has received comparatively little attention. High-quality data generated through development and validation of Tier 1 tests for the U.S. Environmenal Protection Agency Endocrine Disruptor Screening Program (EDSP) offer a unique opportunity to compare responses of mammals versus fish to chemicals that may affect shared pathways within the HPG axis. The present study focuses on data generated with model chemicals that act (primarily) as estrogen receptor agonists (17α-ethynylestradiol, methoxychlor, bisphenol A), androgen receptor agonists (methyltestosterone, 17β-trenbolone), androgen receptor antagonists (flutamide, vincolozolin, p,p'-DDE), or inhibitors of different steroidogenic enzymes (ketoconazole, fadrozole, fenarimol, prochloraz). All 12 chemicals had been tested in the EDSP fish short-term (21 d) reproduction assay and in one or more of the four in vivo Tier 1 screens with rats (uterotrophic, Hershberger, male and female pubertal assays). There was a high concordance between the fish and rat assays with respect to identifying chemicals that impacted specific endocrine pathways of concern. Although most chemicals were detected as positive in both rat and fish assays, eliminating data from one class of vertebrate or the other would weaken the battery. For example, the effects of competitive inhibitors of steroid hormone synthesis were far more obvious in the fish assay, whereas the activity of androgen receptor antagonists was clearer in mammalian assays. The observations are significant both to the cross-species extrapolation of toxicity of HPG-active substances and the optimization of screening and testing frameworks for endocrine-disrupting chemicals.

  14. Acetylsalicylic Acid Daily vs Acetylsalicylic Acid Every 3 Days in Healthy Volunteers: Effect on Platelet Aggregation, Gastric Mucosa, and Prostaglandin E2 Synthesis.

    PubMed

    Ferreira, Plinio Minghin Freitas; Gagliano-Jucá, Thiago; Zaminelli, Tiago; Sampaio, Marinalva Ferreira; Blackler, Rory Willian; Trevisan, Miriam da Silva; Novaes Magalhães, Antônio Frederico; De Nucci, Gilberto

    2016-07-01

    Substantial platelet inhibition was observed 3 days after a single administration of acetylsalicylic acid 81 mg to healthy volunteers. Here we investigate prostaglandin E2 (PGE2 ) antrum concentrations and gastrointestinal symptoms in two treatment groups: one receiving losartan and acetylsalicylic acid every day and the other receiving losartan every day and acetylsalicylic acid every 3 days. Twenty-eight healthy volunteers from both sexes received either 50 mg losartan and acetylsalicylic acid 81 mg daily or 50 mg losartan and acetylsalicylic acid 81 every 3 days with placebo on the other days. Therapy was delivered for 30 days for both groups. Gastric endoscopy was performed before and after treatment period. Biopsies were collected for PGE2 quantification. Platelet function tests were carried out before and during treatment and TXB2 release on platelet rich plasma was measured. The every 3 day low-dose acetylsalicylic acid regimen produced complete inhibition of platelet aggregation compared to the daily treatment. Thromboxane B2 release was substantially abolished for both groups during treatment. There was no significant difference on the endoscopic score of both treatment groups after the 30-day treatment (P = .215). There was over 50% suppression of antrum PGE2 content on volunteers receiving acetylsalicylic acid daily (P = .0016), while for the every 3 day dose regimen there was no significant difference between pre and post-treatment antrum PGE2 dosages (P = .4193). Since PGE2 is involved in gastric healing, we understand that this new approach could be safer and as efficient as the standard daily therapy on a long-term basis.

  15. Rocuronium and sugammadex in a 3 days old neonate for draining an ovarian cyst. Neuromuscular management and review of the literature.

    PubMed

    Carlos, Ricardo Vieira; Torres, Marcelo Luis Abramides; de Boer, Hans D

    2016-01-01

    A case is reported in which a 3-days old neonate with a giant ovarian cyst was scheduled for surgery. The patient received a dose of sugammadex to reverse a rocuronium-induced neuromuscular block. A fast and efficient recovery from neuromuscular block was achieved within 90s. No adverse events or other safety concerns were observed. Furthermore, a review of the literature on the use of sugammadex in neonates was performed.

  16. [Rocuronium and sugammadex in a 3 days old neonate for draining an ovarian cyst. Neuromuscular management and review of the literature].

    PubMed

    Carlos, Ricardo Vieira; Torres, Marcelo Luis Abramides; de Boer, Hans D

    2016-01-01

    A case is reported in which a 3-days old neonate with a giant ovarian cyst was scheduled for surgery. The patient received a dose of sugammadex to reverse a rocuronium-induced neuromuscular block. A fast and efficient recovery from neuromuscular block was achieved within 90s. No adverse events or other safety concerns were observed. Furthermore, a review of the literature on the use of sugammadex in neonates was performed.

  17. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  18. Concentrations of salinomycin in eggs and tissues of laying chickens fed medicated feed for 14 days followed by withdrawal for 3 days.

    PubMed

    Akhtar, A H; abou el-Sooud, K; Shehata, M A

    1996-01-01

    Laying chickens were fed medicated feed containing various concentrations of sodium salinomycin (SAL) for 14 days followed by a 3 day withdrawal period. Eggs, collected during treatment and withdrawal, tissues and ovarian yolk of birds slaughtered after 0, 1, and 3 days' withdrawal were extracted and analysed by high performance liquid chromatography (HPLC). Tissues, ovarian yolk and freeze-dried egg albumen and yolk were extracted with acetone, followed by partitioning with petroleum ether and HPLC analysis. Albumen was extracted with methanol and analysed without further clean-up. Salinomycin was detected at 520 nm after post-column reaction with vanillin at 95 degrees C. Recoveries of fortified salinomycin from freeze-dried eggs (albumen and yolk) and tissue, premix and feed were nearly quantitative (> 90%), except liver which was < 85%. The detection limit was estimated to be 5 ng g-1, with the practical quantifiable limit being about 10 ng g-1. Highest SAL concentrations were in the more fatty components such as egg yolk, ovarian yolk and subcutaneous fat. SAL residues in other tissues were generally low and followed the order liver, kidney, thigh and breast muscles. SAL residues were dependent on the SAL concentration in feed and declined rapidly during withdrawal. PMID:8950111

  19. An evaluation of the lower coverage anti-G suit without an abdominal bladder after 3 days of 7 deg head down tilt

    NASA Technical Reports Server (NTRS)

    Stegmann, B. J.; Krutz, R. W.; Burton, R. R.; Sawin, C. F.

    1992-01-01

    Twelve healthy male subjects were initially deconditioned by 3 days of 7 deg head down tilt bed rest followed by donning the reentry anti-G suit and being centrifuged using a simulated Shuttle reentry profile. For 6 of the 12 subjects, anti-G suit pressure was increased in 0.5 psig increments if eye level blood pressure dropped below 70 mmHg. The second half of the subjects had their G-suits inflated in 0.5 psig increments if they reported 50 percent peripheral light dimming. Results show that the maximum allowable pressure for either exposure was 2.5 psig, which is the operational limit of the Shuttle anti-G suit controller.

  20. Evaluation of short-term clinical efficacy of 3-day therapy with azithromycin in comparison with 5-day cefcapene-pivoxyl for acute streptococcal tonsillopharyngitis in primary care.

    PubMed

    Koga, Takeharu; Rikimaru, Toru; Tokunaga, Naoto; Higashi, Toshihiro; Nakamura, Masahiro; Ichikawa, Yoichiro; Matsuo, Kazuhiko

    2011-08-01

    Group A streptococcal (GAS) tonsillopharyngitis is one of the few conditions for which antibiotics are advocated among common upper respiratory infections. Although a 3-day course of azithromycin is attracting attention as a treatment of choice for the condition, it is not clear if the efficacy of the treatment is comparable with that of treatment with cephalosporins. A prospective, randomized, comparative multicenter study was conducted to compare the efficacy of azithromycin (AZM) given once daily for 3 days with that of cefcapene-pivoxyl (CFPN-PI) divided into three daily doses for 5 days. 88 patients (male: 38, mean age: 16.5) were treated with AZM and 69 (male: 34, mean age: 16.9) with CFPN-PI. The symptoms of all but 5 (2 for AZM and 3 for CFPN-PI) of the patients were resolved by the 8th day of the treatment. By the 4th day of the treatment, criteria for clinical efficacy were fulfilled in 71 (80.7%) subjects who were treated with AZM and in 48 (67.6%) of those treated with CFPN-PI (p = 0.07). The same figures on the 8th day of the treatment were 86 (97.7%) and 68 (95.8%), respectively (p = 0.66), confirming there was no significant difference in clinical efficacy between the two treatments. Mild adverse reactions were reported by two patients treated with AZM and by none treated with CFPN-PI. The clinical efficacy of a 3-day course with AZM was comparable with that of a 5-day course of CFPN-PI for GAS tonsillopharyngitis.

  1. Absolute nuclear material assay

    DOEpatents

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

    2012-05-15

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

  2. Absolute nuclear material assay

    DOEpatents

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

    2010-07-13

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

  3. Validation of four questions on food habits from the Swedish board of health and social welfare by 3-day food records in medical and nursing students

    PubMed Central

    Fredriksson, Ellinor; Brekke, Hilde K.; Ellegård, Lars

    2014-01-01

    Background The Swedish board for health and social welfare (SoS) has presented four questions on dietary habits as indicators of adherence to dietary recommendations. However, these questions have not been evaluated. Objective To evaluate if four questions on dietary habits correlate with dietary intake assessed by food records. Design A total of 279 medical and nursing students, 170 women and 109 men, completed four questions on usual consumption frequency of vegetables, fruits, fish, and sweets. Depending on scoring from 0 to 12 points, subjects were classified as having low (0–4 points), average (5–8 points), or high (9–12 points) adherence to dietary recommendations as proposed by SoS. Nutrient intake was calculated from 3-day food records. Mean dietary intake, expressed per 10 MJ of fibre, ascorbic acid, folate, vitamin D, sucrose, fish, and fruits and vegetables, was analysed for each group and differences assessed by ANOVA. Results Energy intake was 11.8±3.0 MJ in male and 8.5±2.2 MJ in female students. Most students, 64%, were classified as average adherers to dietary recommendations, whereas only 6% were classified as low and 30% as high. Dietary intake of fibre, ascorbic acid, and folate was significantly higher in the high adherence group compared to both the other groups (p<0.01), but vitamin D significantly so only compared to the average group (p=0.002). Intake of fruits and vegetables was significantly different between all groups (p<0.003), with increasing amounts with increasing adherence. The low adherence group had higher intake of sucrose than the other groups (p<0.005). Median fish intake was nil in the low and average adherence groups, with significant difference between high and average adherence groups (p=0.001). Conclusions Four questions on the consumption frequency of vegetables, fruits, fish, and sweets correlate well with the dietary intake of fibre, ascorbic acid, folate, vitamin D, fish, sucrose, and fruits and vegetables as

  4. Resonance enhancement in the accelerator transmutation of 1.3-day {sup 232}Pa and 2.1-day {sup 238}Np

    SciTech Connect

    Moore, M. S.; Danon, Y.

    1995-09-15

    The suggestion that the transmutation of actinide waste into fission products might best be done with thermalized spallation neutrons and odd-odd target materials such as {sup 238}Np has been studied. During the 1993 LAMPF/PSR cycle, we measured the fission cross section of 1.3-day {sup 232}Pa and 2.1-day {sup 238}Np from 0.01 eV to 40 keV at the LANSCE facility, and have carried out a preliminary resonance analysis of the observed structure and of the thermal region, with a 1/v representation above a few eV. In the present study, we calculate the reaction rates of these two species and {sup 247}Cm in a 'resonance reactor', an accelerator-driven assembly whose slowing-down properties are well known. Our model is a 1.8 m{sup 3} block of lead with a helium-cooled tungsten target in the center, i.e., the Rensselaer Intense Neutron Source (RINS). We include the effects of adding moderator outside an idealized lead slowing-down assembly, giving resonance enhancement factors for {sup 232}Pa and {sup 238}Np, and present parameters for the accelerator required to drive such an assembly to accomplish actinide burnup of these species.

  5. Mainstream partial nitritation and anammox in a 200,000 m3/day activated sludge process in Singapore: scale-down by using laboratory fed-batch reactor.

    PubMed

    Yeshi, Cao; Hong, Kwok Bee; van Loosdrecht, Mark C M; Daigger, Glen T; Yi, Png Hui; Wah, Yuen Long; Chye, Chua Seng; Ghani, Yahya Abd

    2016-01-01

    A laboratory fed-batch reactor has been used to study under controlled conditions the performance of partial nitritation/anammox for the 200,000 m(3)/day step-feed activated sludge process at the Changi Water Reclamation Plant, Singapore. The similarity of the concentrations of NH(4), NO(2), NO(3), PO(4), suspended chemical oxygen demand (sCOD), pH, and alkalinity (ALK) between the on-site process and laboratory reactor illustrates that the laboratory fed-batch reactor can be used to simulate the site performance. The performance of the reactor fed by primary effluent illustrated the existence of anammox and heterotrophic denitrification and apparent excessive biological phosphorus removal as observed from the site. The performance of the reactor fed by final effluent proved the presence of anammox process on site. Both the laboratory reactor and on-site process showed that higher influent 5-day biochemical oxygen demand/total nitrogen (BOD(5)/TN) (COD/TN) ratio increases the nitrogen removal efficiency of the process.

  6. Use of a nationwide call center for Ebola response and monitoring during a 3-day house-to-house campaign - Sierra Leone, September 2014.

    PubMed

    Miller, Leigh Ann; Stanger, Emily; Senesi, Reynold Gb; DeLuca, Nick; Dietz, Patricia; Hausman, Leslie; Kilmarx, Peter H; Mermin, Jonathan

    2015-01-16

    During May 23, 2014-January 10, 2015, Sierra Leone reported 7,777 confirmed cases of Ebola virus disease (Ebola). In response to the epidemic, on August 5, Sierra Leone's Emergency Operations Center established a toll-free, nationwide Ebola call center. The purpose of the call center is to encourage public reporting of possible Ebola cases and deaths to public health officials and to provide health education about Ebola to callers. This information also functions as an "alert" system for public health officials and supports surveillance efforts for the response. National call center dispatchers call district-level response teams composed of surveillance officers and burial teams to inform them of reported deaths and possible Ebola cases. Members of these response teams investigate cases and conduct follow-up actions such as transporting ill persons to Ebola treatment units or providing safe, dignified medical burials as resources permit. The call center continues to operate. This report describes calls received during a 3-day national campaign and reports the results of an assessment of the call center operation during the campaign.

  7. Mainstream partial nitritation and anammox in a 200,000 m3/day activated sludge process in Singapore: scale-down by using laboratory fed-batch reactor.

    PubMed

    Yeshi, Cao; Hong, Kwok Bee; van Loosdrecht, Mark C M; Daigger, Glen T; Yi, Png Hui; Wah, Yuen Long; Chye, Chua Seng; Ghani, Yahya Abd

    2016-01-01

    A laboratory fed-batch reactor has been used to study under controlled conditions the performance of partial nitritation/anammox for the 200,000 m(3)/day step-feed activated sludge process at the Changi Water Reclamation Plant, Singapore. The similarity of the concentrations of NH(4), NO(2), NO(3), PO(4), suspended chemical oxygen demand (sCOD), pH, and alkalinity (ALK) between the on-site process and laboratory reactor illustrates that the laboratory fed-batch reactor can be used to simulate the site performance. The performance of the reactor fed by primary effluent illustrated the existence of anammox and heterotrophic denitrification and apparent excessive biological phosphorus removal as observed from the site. The performance of the reactor fed by final effluent proved the presence of anammox process on site. Both the laboratory reactor and on-site process showed that higher influent 5-day biochemical oxygen demand/total nitrogen (BOD(5)/TN) (COD/TN) ratio increases the nitrogen removal efficiency of the process. PMID:27386982

  8. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

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

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  9. Lateral flow assays.

    PubMed

    Koczula, Katarzyna M; Gallotta, Andrea

    2016-06-30

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

  10. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

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

  11. Tube-Forming Assays.

    PubMed

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

    2016-01-01

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

  12. New Rapid Spore Assay

    NASA Astrophysics Data System (ADS)

    Kminek, Gerhard; Conley, Catharine

    2012-07-01

    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  13. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  14. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  15. Development and validity of a 3-day smartphone-assisted 24-hour recall to assess beverage consumption in a Chinese population: a randomized cross-over study

    PubMed Central

    Smith, Lindsey P.; Hua, Jenna; Seto, Edmund; Du, Shufa; Zang, Jiajie; Zou, Shurong; Popkin, Barry M.; Mendez, Michelle A.

    2014-01-01

    This paper addresses the need for diet assessment methods that capture the rapidly changing beverage consumption patterns in China. The objective of this study was to develop a 3-day smartphone-assisted 24-hour recall to improve the quantification of beverage intake amongst young Chinese adults (n=110) and validate, in a small subset (n=34), the extent to which the written record and smartphone-assisted recalls adequately estimated total fluid intake, using 24-hour urine samples. The smartphone-assisted method showed improved validity compared to the written-assisted method, when comparing reported total fluid intake to total urine volume. However, participants reported consuming fewer beverages on the smartphone-assisted method compared to the written-assisted method, primarily due to decreased consumption of traditional zero-energy beverages (i.e. water, tea) in the smartphone-assisted method. It is unclear why participants reported fewer beverages in the smartphone-assisted method than the written-assisted method. One possibility is that participants found the smartphone method too cumbersome, and responded by decreasing beverage intake. These results suggest that smartphone-assisted 24-hour recalls perform comparably but do not appear to substantially improve beverage quantification compared to the current written record based approach. In addition, we piloted a beverage screener to identify consumers of episodically consumed SSBs. As expected, a substantially higher proportion of consumers reported consuming SSBs on the beverage screener compared to either recall type, suggesting that a beverage screener may be useful in characterizing consumption of episodically consumed beverages in China’s dynamic food and beverage landscape. PMID:25516327

  16. Effect of 3-Day Bed Rest on the Basal Sympathetic Activity and Responsiveness of this System to Physiological Stimuli In Athletes and Sedentary Subjects

    NASA Technical Reports Server (NTRS)

    Smorawinski, Jerzy; Adrian, Jacek; Kaciuba-Uscilko, Hanna; Nazar, Krystyna; Greenleaf, John E.; Dalton, P. Bonnie (Technical Monitor)

    2002-01-01

    The aims of this study were: (1) to examine the effect of three days of bed rest (BR) on basal plasma epinephrine [E] and norepinephrine [NE] and the catecholamine responses to various physiological stimuli, and (2) to find out whether previous physical activity modifies effects of BR. In the first series, 29 young men (11 sedentary students, 8 endurance and 10 strength trained athletes) were submitted to oral glucose tolerance test in supine position and to active orthostatic test before and after 3 days of BR. Plasma [E] and [NE] were measured after overnight fast (basal condition), at 60, 120 and 180 min after glucose ingestion (70 a), and at the 8th min of unsupported standing. In the second series, other 22 subjects (12 sedentary students, 10 endurance and 10 strength trained athletes) were submitted to 2 min cold pressor test (CPT) and exercise. Plasma E and NE were determined in the supine position after overnight fast and at 60th and 120th s of hand cooling. Then, after breakfast followed by 2-3 hour sitting, the subjects performed cycle ergometer exercise with workload increasing until volitional exhaustion. Plasma [E] and [NE] were determined at the end of each load. Plasma catecholamines were determined made radioenzymatically. After BR, basal plasma [NE] was decreased in endurance and strength athletes (p<0.01) but not in sedentary subjects. In neither group BR affected the basal [E]. Responses of both catecholamines to glucose load were diminished after BR in all three groups (p<0.05) but the effect was most pronounced in the endurance athletes. All subjects tolerated well 8-min standing although their heart rate response was increased after BR. Plasma catecholamine responses standing were not significantly affected by BR in either group but the plasma [NE] and [E] during standing were lowered after BR in endurance athletes (p<0.01). BR did not affect blood pressure and catecholamine responses to CPT. The pre- and post-exercise plasma catecholamines

  17. Rapid screening assay for calcium bioavailability studies

    SciTech Connect

    Luhrsen, K.R.; Hudepohl, G.R.; Smith, K.T.

    1986-03-01

    Calcium bioavailability has been studied by numerous techniques. The authors report here the use of the gamma emitting isotope of calcium (/sup 47/Ca) in a whole body retention assay system. In this system, calcium sources are administered by oral gavage and subsequent counts are determined and corrected for isotopic decay. Unlike iron and zinc retention curves, which exhibit a 2-3 day equilibration period, calcium reaches equilibration after 24 hours. Autoradiographic analysis of the femurs indicate that the newly absorbed calcium is rapidly distributed to the skeletal system. Moreover, the isotope is distributed along the entire bone. Comparisons of calcium bioavailability were made using intrinsic/extrinsic labeled milk from two species i.e. rat and goat as well as CaCO/sub 3/. In addition, extrinsic labeled cow milk was examined. In the rat, the extrinsic labeled calcium from milk was better absorbed than the intrinsic calcium. This was not the case in goat milk or the calcium carbonate which exhibited no significant differences. Chromatographic analysis of the labeled milk indicates a difference in distribution of the /sup 47/Ca. From these data, the authors recommend the use of this assay system in calcium bioavailability studies. The labeling studies and comparisons indicate caution should be used, however, in labeling techniques and species milk comparison.

  18. Against vaccine assay secrecy.

    PubMed

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors.

  19. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

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

  20. Interpreting coagulation assays.

    PubMed

    Green, David

    2010-09-01

    The interpretation of coagulation assays requires knowledge of the principal clotting pathways. The activated partial thromboplastin time is sensitive to all hemostatic factors except FVII, whereas the prothrombin time reflects levels of prothrombin and FV, FVII, and FX. Using the two tests in concert is helpful in identifying hemophilia, the coagulopathy of liver disease, and disseminated intravascular coagulation. In addition, the activated partial thromboplastin time and prothrombin time are used for monitoring anticoagulant therapy with heparin and warfarin, respectively. Measurement of D-dimer is informative in patients suspected of having thrombotic disorders and determining the risk of thrombosis recurrence. Mixing tests distinguish clotting factor deficiencies from circulating anticoagulants such as heparin, the lupus anticoagulant, and antibodies directed against specific clotting factors. The modified Bethesda assay detects and provides an indication of the strength of FVIII inhibitors. However, interpreting the results of coagulation assays is not always straightforward, and expert consultation is occasionally required to resolve difficult clinical situations. PMID:20855988

  1. Against vaccine assay secrecy

    PubMed Central

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  2. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies.

  3. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  4. Lateral flow strip assay

    DOEpatents

    Miles, Robin R.; Benett, William J.; Coleman, Matthew A.; Pearson, Francesca S.; Nasarabadi, Shanavaz L.

    2011-03-08

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

  5. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  6. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  7. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  8. Radioreceptor assay for oxyphenonium.

    PubMed

    Ensing, K; de Zeeuw, R A

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. PMID:6428927

  9. C. elegans chemotaxis assay.

    PubMed

    Margie, Olivia; Palmer, Chris; Chin-Sang, Ian

    2013-01-01

    Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior. The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress (1-10). The method described here aims to address these issues by modifying the assay developed by Bargmann et al.(1). A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired. PMID:23644543

  10. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

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

  11. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  12. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

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

  13. Titration of avirulent Newcastle disease virus by the plaque assay method.

    PubMed

    Kournikakis, B; Fildes, J

    1988-08-01

    Parameters of a plaque assay for avirulent strains of Newcastle disease virus (NDV) were optimized for reproducibility and optimum titer in LLC-MK2 cells. Plaques were visible after 2 days and maximum virus titers were reached in 3 days. Virus titers were not affected by continued incubation through 6 days, although plaque size increased. An adsorption volume of 0.1 or 0.2 ml per 60 mm Petri dish was optimal, as was an adsorption time of 45 min. Trypsin (2.5 micrograms/ml) and magnesium sulfate (0.03 M) were essential requirements of the overlay medium and the presence of DEAE-dextran (0.02%) resulted in a 30% increase in titer. The use of cell monolayers, 1, 2, or 3 days old facilitated the performance of multiple assays per week and did not affect the virus titer.

  14. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  15. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  16. TOTAL CULTURABLE VIRUS QUANTAL ASSAY

    EPA Science Inventory

    This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced ...

  17. Examining triclosan-induced potentiation of the estrogen uterotrophic effect

    EPA Science Inventory

    Triclosan (TCS), a widely used antibacterial, has been shown to be an endocrine disruptor. We reported previously that TCS potentiated the estrogenic effect of ethinyl estradiol (EE) on uterine growth in rats orally administered 3 μg/kg EE and TCS (2 to 18 mg/kg) in the utero...

  18. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  19. Adipose tissue angiogenesis assay.

    PubMed

    Rojas-Rodriguez, Raziel; Gealekman, Olga; Kruse, Maxwell E; Rosenthal, Brittany; Rao, Kishore; Min, Soyun; Bellve, Karl D; Lifshitz, Lawrence M; Corvera, Silvia

    2014-01-01

    Changes in adipose tissue mass must be accompanied by parallel changes in microcirculation. Investigating the mechanisms that regulate adipose tissue angiogenesis could lead to better understanding of adipose tissue function and reveal new potential therapeutic strategies. Angiogenesis is defined as the formation of new capillaries from existing microvessels. This process can be recapitulated in vitro, by incubation of tissue in extracellular matrix components in the presence of pro-angiogenic factors. Here, we describe a method to study angiogenesis from adipose tissue fragments obtained from mouse and human tissue. This assay can be used to define effects of diverse factors added in vitro, as well as the role of endogenously produced factors on angiogenesis. We also describe approaches to quantify angiogenic potential for the purpose of enabling comparisons between subjects, thus providing information on the role of physiological conditions of the donor on adipose tissue angiogenic potential.

  20. Yeast DEL assay detects clastogens.

    PubMed

    Kirpnick, Zhanna; Homiski, Michael; Rubitski, Elizabeth; Repnevskaya, Marina; Howlett, Niall; Aubrecht, Jiri; Schiestl, Robert H

    2005-04-01

    Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in

  1. What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity.

    PubMed

    Šrut, Maja; Štambuk, Anamaria; Klobučar, Göran I V

    2013-06-01

    DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3 days to benzo[a]pyrene (B[a]P, 0.1-10 μM) and ethyl methanesulfonate (EMS, 0.1-1mM) followed by 3 days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.

  2. Re-feeding rats a high-sucrose diet after 3 days of starvation enhances histone H3 acetylation in transcribed region and expression of jejunal GLUT5 gene.

    PubMed

    Honma, Kazue; Masuda, Yuriko; Mochizuki, Kazuki; Goda, Toshinao

    2014-01-01

    Fasting for 3 days leads to reduction in the expression of GLUT5 and SGLT1 genes in jejunum. Re-feeding a high-sucrose diet in fasted rats enhanced mRNA levels and histone H3 acetylation on transcribed region of GLUT5 gene within 24 h, but not in SGLT1. Responsiveness of jejunal GLUT5 gene is associated with changes in histone H3 acetylation on transcribed region.

  3. An assay for adjuvanticity

    PubMed Central

    Dresser, D. W.

    1968-01-01

    Adult mice injected with an adequate amount of a non-immunogenic antigen progress to a specific state of immunological paralysis, unless a substance with `extrinsic' adjuvanticity is injected before the induction of paralysis is completed. Consequently incipiently paralysed mice can be used to assay substances for adjuvanticity. Conventional adjuvants such as Freund's adjuvant and pertussis possess adjuvanticity; other substances with varying degrees of adjuvanticity are listed in the tables. It has been shown that the adjuvanticity effect of an injection of pertussis lasts for only a few days, although the effect of such an injection of pertussis on phagocytosis of carbon particles does not reach a maximum until 2 weeks after the injection. The dose-effectiveness of alum precipitated (highly phagocytosable) bovine γ-globulin was greatly increased by the intraperitoneal injection of pertussis. The evidence is considered to be incompatible with increased phagocytosis being either an essential factor in the role of pertussis as a conventional adjuvant, or in the adjuvanticity effect of pertussis. PMID:4179956

  4. Nuclear criticality safety: 3-day training course

    SciTech Connect

    Schlesser, J.A.

    1992-11-01

    This compilation of notes is presented as a source reference for the criticality safety course. It represents the contributions of many people, particularly Tom McLaughlin, the course's primary instructor. At the completion of this training course, the attendee will: (1) be able to define terms commonly used in nuclear criticality safety; (2) be able to appreciate the fundamentals of nuclear criticality safety; (3) be able to identify factors which affect nuclear criticality safety; (4) be able to identify examples of criticality controls as used at Los Alamos; (5) be able to identify examples of circumstances present during criticality accidents; (6) be able to identify examples of safety consciousness required in nuclear criticality safety.

  5. Nuclear criticality safety: 3-day training course

    SciTech Connect

    Schlesser, J.A.

    1992-11-01

    This compilation of notes is presented as a source reference for the criticality safety course. It represents the contributions of many people, particularly Tom McLaughlin, the course`s primary instructor. At the completion of this training course, the attendee will: (1) be able to define terms commonly used in nuclear criticality safety; (2) be able to appreciate the fundamentals of nuclear criticality safety; (3) be able to identify factors which affect nuclear criticality safety; (4) be able to identify examples of criticality controls as used at Los Alamos; (5) be able to identify examples of circumstances present during criticality accidents; (6) be able to identify examples of safety consciousness required in nuclear criticality safety.

  6. Obamacare's (3) Day(s) in Court.

    PubMed

    Moncrieff, Abigail R

    2012-06-01

    Before the oral arguments in late March, the vast majority of legal scholars felt confident that the Supreme Court of the United States would uphold the individual mandate against the constitutional challenge that 26 states have levied against it. Since the oral arguments, that confidence has been severely shaken. This article asks why legal scholars were so confident before the argument and what has made us so concerned since the argument. The article posits that certain fundamental characteristics of health insurance, particularly its unusual role in steering health-care consumption decisions, which distinguishes health insurance from standard kinds of indemnity insurance, should make the constitutional question easy, but the Obama Administration's legal team was understandably hesitant to highlight those unique characteristics in its arguments. Because the Supreme Court justices seemed not to understand the uniqueness of health insurance without the government's help and because the justices seemed unusually willing to adopt a new constitutional constraint in this case, the individual mandate appears to be in far greater jeopardy than we legal scholars anticipated.

  7. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay.

    PubMed

    Chang, A; Dusing, S

    2006-01-01

    Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

  8. Local lymph node assay with non-radioisotope alternative endpoints.

    PubMed

    Suda, Akiko; Yamashita, Masahiro; Tabei, Mitsuyuki; Taguchi, Kazuhiko; Vohr, Hans-Werner; Tsutsui, Naohisa; Suzuki, Ritsuyoshi; Kikuchi, Katsuaki; Sakaguchi, Keisuke; Mochizuki, Kouki; Nakamura, Kazuichi

    2002-08-01

    The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria. Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of 3H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested. The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.

  9. Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure.

    PubMed

    Shin, Jae-Ho; Moon, Hyun Ju; Kang, Il Hyun; Kim, Tae Sung; Lee, Su Jung; Oh, Ji Young; Lee, Young Joo; Hong, Eui-Ju; Jeung, Eui-Bae; Han, Soon-Young

    2007-04-01

    Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.

  10. Towards an integrated in vitro strategy for estrogenicity testing.

    PubMed

    Wang, Si; Aarts, Jac M M J G; de Haan, Laura H J; Argyriou, Dimitrios; Peijnenburg, Ad A C M; Rietjens, Ivonne M C M; Bovee, Toine F H

    2014-09-01

    In order to define an in vitro integrated testing strategy (ITS) for estrogenicity, a set of 23 reference compounds representing diverse chemical classes were tested in a series of in vitro assays including proliferation and reporter gene assays. Outcomes of these assays were combined with published results for estrogen receptor (ER) binding assays and the OECD validated BG1Luc ER transcriptional activation (TA) assay and compared with the outcomes of the in vivo uterotrophic assay to investigate which assays most accurately predict the in vivo uterotrophic effect and to identify discrepancies between the in vitro assays and the in vivo uterotrophic assay. All in vitro assays used revealed a reasonable to good correlation (R(2)  = 0.62-0.87) with the in vivo uterotrophic assay but the combination of the yeast estrogen bioassay with the U2OS ERα-CALUX assay seems most promising for an ITS for in vitro estrogenicity testing. The main outliers identified when correlating data from the different in vitro assays and the in vivo uterotrophic assay were 4-hydroxytamoxifen, testosterone and to a lesser extent apigenin, tamoxifen and kepone. Based on the modes of action possibly underlying these discrepancies it becomes evident that to further improve the ITS and ultimately replace animal testing for (anti-)estrogenic effects, the selected bioassays have to be combined with other types of in vitro assays, including for instance in vitro models for digestion, bioavailability and metabolism of the compounds under investigation.

  11. From Antenna to Assay

    PubMed Central

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  12. The assay of diphtheria toxin

    PubMed Central

    Gerwing, Julia; Long, D. A.; Mussett, Marjorie V.

    1957-01-01

    A precise assay of diphtheria toxin is described, based on the linear relationship between the diameter of the skin reaction to, and logarithm of the dose of, toxin. It eliminates the need for preliminary titrations, is economical, provides information about the slope of the log-dose response lines and, therefore, of the validity of the assay, and yields limits of error of potency from the internal evidence of the assay. A study has been made of the effects of avidity, combining power, toxicity and buffering on the assay of diphtheria toxins against the International Standards for both Diphtheria Antitoxin and Schick-Test Toxin. All the toxins assayed against the standard toxin, whatever their other properties might be, gave log-dose response lines of similar slope provided that they were diluted in buffered physiological saline. The assays were therefore valid. These experiments were repeated concurrently in non-immune and in actively immunized guinea-pigs, and comparable figures for potency obtained in both groups. The result was not significantly affected by the avidity or combining power of the toxin. However, non-avid toxins gave low values in Schick units when assayed, by the Römer & Sames technique, in terms of the International Standard for Diphtheria Antitoxin. The problem of the ultimate standard and the implications of these findings are discussed. PMID:13511133

  13. Comet Assay measurements: a perspective.

    PubMed

    Kumaravel, T S; Vilhar, Barbara; Faux, Stephen P; Jha, Awadhesh N

    2009-02-01

    The Comet Assay or single cell gel electrophoresis assay is one of the very widely used assays to microscopically detect DNA damage at the level of a single cell. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). In this assay, the shape, size and amount of DNA within the 'comet' play important roles in the determination of the level of damage. The use of a software in particular also provides a range of different parameters, many of which might not be relevant in determining the extent of DNA damage. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of 'hedgehog' or 'clouds', this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. It is suggested that judicious selection of different parameters, staining methods along with inter-laboratory validation and harmonisation of methodologies will further help in making this assay more robust and widely acceptable for scientific as well as regulatory studies.

  14. Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays.

    PubMed

    Folta, Kevin M; Kaufman, Lon S

    2006-01-01

    Isolation of transcriptionally active nuclei from plant tissues is a fundamental first step in many plant molecular biology protocols. Enriched nuclear fractions may be used in "run-on" assays to measure the rate of transcription for any given gene, adding additional resolution to assays of steady-state transcript accumulation such as RNA-gel blots, RT-PCR or microarrays. The protocols presented here streamline, adapt and optimize existing methods for use in Arabidopsis thaliana. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. This capability complements the immense body of steady-state transcript measurements and indirectly identifies instances where message turnover may have a critical and/or primary role in regulating gene expression levels.

  15. Plaque assay for murine norovirus.

    PubMed

    Gonzalez-Hernandez, Mariam B; Bragazzi Cunha, Juliana; Wobus, Christiane E

    2012-01-01

    Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50). This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration. However, its limit of detection is higher compared to a plaque assay. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of

  16. Troponin revisited 2008: assay performance.

    PubMed

    Tate, Jillian R

    2008-01-01

    Troponin quality specifications describing the pre-analytical, analytical and post-analytical performance of cardiac troponin (cTn) assays are important for both manufacturers of cTn assays and laboratories that routinely test for cTn. Pre-analytical requirements refer not only to acceptable sample type for analysis and the stability of cTn but also to the proper handling of specimens prior to analysis to avoid pre-analytical false positive results. Analytical issues that may contribute to differences between cTn assays include analytical sensitivity and imprecision at low cTn concentration, antibody specificity and immunoreactivity of plasma cTn forms, assay specificity and the presence of falsely positive and negative interferences, and for cTnI the lack of standardised measurement, all which may impact on patient cTn results. Current second generation cTnI and fourth generation cTnT assays generally have an imprecision of around 20% coefficient of variation (CV) at the 99th percentile of the reference population, which is greater than the recommended imprecision of 10% CV. As the next generation of more analytically sensitive cTn assays are developed it can be anticipated that cTn upper reference limits will decrease by approximately 10-fold. Monitoring assay imprecision at ultra low cTn concentrations will require that the laboratory uses a quality control close to this level and a negative control to monitor baseline drift. Establishment of cTn reference ranges will require reference populations to be cardio-healthy to enable differentiation from community populations who are at increased cardiovascular risk. Close collaboration between the laboratory and local clinicians is required to ensure adequate clinical validation of more sensitive cTn assays.

  17. Methods to assay Drosophila behavior.

    PubMed

    Nichols, Charles D; Becnel, Jaime; Pandey, Udai B

    2012-01-01

    Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases(1). We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials(2-4). The rapid iterative negative geotaxis (RING) assay(5) has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously

  18. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  19. Assaying DNA damage in hippocampal neurons using the comet assay.

    PubMed

    Nowsheen, Somaira; Xia, Fen; Yang, Eddy S

    2012-12-19

    A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique. The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell (1,2). Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

  20. Transversal stiffness and beta-actin and alpha-actinin-4 content of the M. soleus fibers in the conditions of a 3-day reloading after 14-day gravitational unloading.

    PubMed

    Ogneva, I V

    2011-01-01

    The aim of the work was to analyze the structural changes in different parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy in a three-day reloading after a 14-day gravity disuse, which was carried out by hind-limbs suspension. The object of the study was the soleus muscle of the Wistar rat. It was shown that after 14 days of disuse, there was a reduction of transversal stiffness of all points of the sarcolemma and contractile apparatus. Readaptation for 3 days leads to complete recovery of the values of the transversal stiffness of the sarcolemma and to partial value recovery of the contractile apparatus. The changes in transversal stiffness of sarcolemma correlate with beta-actin and alpha-actinin-4 in membrane protein fractions.

  1. International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE.

    PubMed

    Laurie, Karen L; Engelhardt, Othmar G; Wood, John; Heath, Alan; Katz, Jacqueline M; Peiris, Malik; Hoschler, Katja; Hungnes, Olav; Zhang, Wenqing; Van Kerkhove, Maria D

    2015-08-01

    The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

  2. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro.

  3. Time-Resolved Fluorescence Assays.

    PubMed

    Ma, Chen-Ting; Sergienko, Eduard A

    2016-01-01

    Fluorescence-based detection techniques are popular in high throughput screening due to sensitivity and cost-effectiveness. Four commonly used techniques exist, each with distinct characteristics. Fluorescence intensity assays are the simplest to run, but suffer the most from signal interference. Fluorescence polarization assays show less interference from the compounds or the instrument, but require a design that results in change of fluorophore-containing moiety size and usually have narrow assay signal window. Fluorescence resonance energy transfer (FRET) is commonly used for detecting protein-protein interactions and is constrained not by the sizes of binding partners, but rather by the distance between fluorophores. Time-resolved fluorescence resonance energy transfer (TR-FRET), an advanced modification of FRET approach utilizes special fluorophores with long-lived fluorescence and earns its place near the top of fluorescent techniques list by its performance and robustness, characterized by larger assay window and minimized compound spectral interference. TR-FRET technology can be applied in biochemical or cell-based in vitro assays with ease. It is commonly used to detect modulation of protein-protein interactions and in detection of products of biochemical reactions and cellular activities. PMID:27316992

  4. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery. PMID:16859410

  5. Barcoded microchips for biomolecular assays.

    PubMed

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  6. Displacement enzyme linked aptamer assay.

    PubMed

    Baldrich, Eva; Acero, Josep Lluis; Reekmans, Gunter; Laureyn, Wim; O'Sullivan, Ciara K

    2005-08-01

    Immense effort has been placed on the realization of immunoassays exploiting displacement of a suboptimum target, due to the ease of use and applicability to immunochromatographic strips and immunosensors. Most of the efforts reported to date focus on the use of a suboptimal target that is displaceable by the target toward which the antibody has higher affinity. Limited success has been achieved due to difficulty in obtaining suboptimal targets to which the antibody has enough affinity to bind while at the same time having lower levels of affinity in comparison to the target to facilitate displacement. Aptamers are synthetic oligonucleotides specifically selected to bind a certain target. Thanks to their high affinity and sensitivity, aptamers appear as alternative candidates to antibodies for analytical devices and several enzyme-linked aptamer assays and aptasensors have been reported. Aptamers, in contrast to antibodies, require the formation of a three-dimensional structure for target binding and can thus be anticipated to have a much higher affinity for binding its target rather than a modified form of the target (e.g., enzyme-labeled target). This phenomenon can be exploited for the development of a displacement assay, using enzyme-labeled target as a suboptimal displaceable molecule. Here, we report the first demonstration of the exploitation of an aptamer in an extremely rapid and highly sensitive displacement assay. Surface plasmon resonance studies demonstrated the thrombin-binding aptamer to have a lower affinity for enzyme-labeled thrombin than unmodified thrombin, with respective K(D) of 1.1 x 10(-8) and 2.9 x 10(-9) M. The assay is extremely rapid, requiring only 10 min for completion, and exhibits a detection limit lower than that obtainable with competitive enzyme-linked aptamer assays and comparable to that of hybrid aptamer-antibody assays. Optimal storage conditions for precoated microtiter plates (consisting of coated aptamer and captured

  7. Three dimensional colorimetric assay assemblies

    SciTech Connect

    Charych, D.; Reichart, A.

    2000-06-27

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  8. Assays for B lymphocyte function.

    PubMed

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  9. Bacterial mutagenicity assays: test methods.

    PubMed

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  10. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  11. Three dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichart, Anke

    2000-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  12. Recent Developments in Electrotaxis Assays.

    PubMed

    Wu, Jiandong; Lin, Francis

    2014-02-01

    Significance: A wide range of cell types can migrate in response to physiological or externally applied direct current electric field (dcEF), a process termed electrotaxis. In particular, electrotaxis of epithelial cells to wound-generated dcEF for mediating wound healing is a well-accepted mechanism. In addition, various immune cells have been demonstrated to undergo electrotaxis, suggesting a link between electrotaxis and inflammatory responses in wound healing. Electrotaxis research will generate important insight into the electrical guiding mechanism for cell migration thereby providing the scientific basis to further develop clinical applications for wound care. Development of advanced electrotaxis assays will critically enable in-depth experimental electrotaxis studies in vitro. Recent Advances: Recently, a number of new electrotaxis assays or new uses of previously developed assays for electrotaxis studies have been reported. These new developments provide improved solutions for experimental throughput, configuration of three-dimensional cell migration environments and coexisting guiding signals, measurements of collective electrotactic cell migration, and sorting electrotactic populations. Critical Issues: These new developments face the challenge of playing a more important role to better understand the biological mechanisms underlying electrotaxis, in addition to making a stronger impact on relevant applications. Future Directions: On one hand, specific electrotaxis assays should be further developed to improve its function and tested for a broader range of experimental conditions and electrotactic populations. On the other hand, joint efforts among electrotaxis researchers are needed to integrate the unique features of specific electrotaxis assays, allowing more advanced and efficient electrotaxis analyses to answer both basic science and clinical questions. PMID:24761355

  13. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    The release of a biological agent by terrorists represents a serious threat to the safety of US citizens. At present there are over 50 pathogens and toxins on various agency threat lists. Most of these pathogens are rarely seen by public health personnel so the ability to rapidly identify their infection is limited. Since many pathogenic infections have symptomatic delays as long as several days, effective treatment is often compromised. This translates into two major deficiencies in our ability to counter biological terrorism (1) the lack of any credible technology to rapidly detect and identify all the pathogens or toxins on current threat lists and (2) the lack of a credible means to rapidly diagnose thousands of potential victims. In this SI we are developing a rapid, flexible, inexpensive, high throughput, and deeply multiplex-capable biological assay technology. The technology, which we call the Liquid Array (LA), utilizes optical encoding of small diameter beads which serve as the templates for biological capture assays. Once exposed to a fluid sample these beads can be identified and probed for target pathogens at rates of several thousand beads per second. Since each bead can be separately identified, one can perform parallel assays by assigning a different assay to each bead in the encoded set. The goal for this development is a detection technology capable of simultaneously identifying 100s of different bioagents and/or of rapidly diagnosing several thousand individuals. We are pursuing this research in three thrusts. In the first we are exploring the fundamental interactions of the beads with proteins and nucleic acids in complex mixtures. This will provide us with a complete understanding of the limits of the technology with respect to throughput and complex environment. A major spin-off of this activity is in the rapidly emerging field of proteomics where we may be able to rapidly assess the interactions responsible for cell metabolism, structural

  14. Double-blind randomized study comparing the efficacies and safeties of a short (3-day) course of azithromycin and a 5-day course of amoxicillin in patients with acute exacerbations of chronic bronchitis.

    PubMed Central

    Mertens, J C; van Barneveld, P W; Asin, H R; Ligtvoet, E; Visser, M R; Branger, T; Hoepelman, A I

    1992-01-01

    The efficacies and safeties of a three-dose regimen of azithromycin (500 mg once daily for 3 days) and a 15-dose regimen of amoxicillin (500 mg three times daily for 5 days) were compared in a double-blind manner in patients with an acute exacerbation of chronic bronchitis. A total of 92% of patients suffered a type 1 exacerbation. Treatment success, defined as cure or major improvement, was achieved in all patients in the azithromycin group by day 5, compared with 23 (92%) of 25 patients in the amoxicillin group. On day 12, these data were 24 of 25 (96%) in the azithromycin group and 20 of 25 (80%) in the amoxicillin group (results were not significantly different). Several pathogens were isolated (MIC ranges [micrograms per milliliter] in parentheses): Haemophilus influenzae or Haemophilus parainfluenzae was isolated 23 times (azithromycin, less than or equal to 0.06 to 32; amoxicillin, 0.12 to 2); Streptococcus pneumoniae was isolated from 11 patients (azithromcyin, less than or equal to 0.06 greater than 256; amoxicillin, less than or equal to 0.06 to 0.25); Moraxella (Branhamella) catarrhalis was isolated from eight patients (azithromycin, less than or equal to 0.06; amoxicillin, less than or equal to 0.06 to 16); and other members of the family Enterobacteriaceae were isolated from eight patients. One patient treated with azithromycin had Legionella pneumophila pneumonia, and another in that group had a significant rise in titer of antibody against influenza A virus. One patient treated with amoxicillin also had a significant rise in titer of antibody against influenza A virus. Microbiological response rates were comparable. One patient who received azithromycin developed abnormal liver function. Two patients treated with amoxicillin developed abnormal liver functions, one developed exanthema, and one treatment was stopped because of nausea. It is concluded that a three-dose (3-day) regimen of azithromycin is as effective clinically and microbiologically as a

  15. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  16. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  17. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  18. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  19. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  20. Important Norwegian crude assays updated

    SciTech Connect

    Corbett, R.A

    1990-03-12

    New assays on two important Norwegian North Sea crude oils, Statfjord and Gullfaks, are presented. Both are high-quality, low-sulfur crudes that will yield a full range of good-quality products. All assay data came from industry-standard test procedures. The Statfjord field is the largest in the North Sea. Production started in 1979. Statfjord is a typical North Sea crude, produced from three separate platforms and three separate loading buoys with interconnecting lines. Current production is about 700,000 b/d. Gullfaks is produced from a large field in Block 34/10 of the Norwegian sector of the North Sea production area. Gullfaks crude oil is more biodegraded than other crudes from the region. Biodegradation has removed most of the waxy normal paraffins, resulting in a heavier, more naphthenic and aromatic crude.

  1. The skin-blanching assay.

    PubMed

    Smit, P; Neumann, H A M; Thio, H B

    2012-10-01

    The skin-blanching assay is used for the determination and bioequivalence of dermatologic glucocorticoids (GCs). The exact mechanism of the production of blanching is not fully understood, but it is considered that local vasoconstriction of the skin microvasculature and the consequent blood-flow reduction cause this phenomenon. Several factors influence skin blanching, including drug concentration, duration of application, nature of vehicle, occlusion, posture and location. The intensity of vasoconstriction can be measured in several ways: visual or quantitative methods, such as reflectance spectroscopy, thermography, laser Doppler velocimetry and chromametry. In literature, contradicting results in the correlation of the skin-blanching assay with different tests to determine GC sensitivity have been reported, limiting its clinical usefulness.

  2. Pulsating bead-based assay.

    PubMed

    Thompson, Jason A; Bau, Haim H

    2011-04-15

    In recent years, there has been a growing interest in using porous microbeads such as agarose beads as solid supports to bind target molecules from complex fluid samples. Porous beads have large surface area to volume ratios and high receptor concentrations, and they facilitate relatively high sensitivity detection and multiplexing. Unfortunately, to take full advantage of the porous beads' attributes, long incubation times are needed due to the relatively slow mass transfer of target molecules from the exterior solution into the beads' interior. To accelerate the mass transfer process, we propose a novel assay in which functionalized porous beads are periodically compressed and expanded. Preliminary experiments were carried out to compare the performance of the pulsating beads with that of conventional, nonpulsating beads. These experiments indicate that the pulsating beads significantly accelerate binding rates with minimal increase in nonspecific binding. Thus, pulsing has the potential of significantly reducing assay time.

  3. Comet Assay in Cancer Chemoprevention.

    PubMed

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

    2016-01-01

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

  4. Fathead minnow FHM cells for use in in vitro cytotoxicity assays of aquatic pollutants

    SciTech Connect

    Babich, H.; Borenfreund, E.

    1987-08-01

    The suitability of the fathead minnow (FHM) epithelial cell line for use as the target (indicator) system in in vitro cytotoxicity assays was evaluated using several endpoints. The organometal diethyltin dichloride served as the representative test agent. The concentration of diethyltin dichloride which resulted in a midpoint toxicity was 3.5 microM in a 3-day cell growth assay, 3.8 microM in the 24-hr neutral red assay, and 16.5 microM in a 4-hr cell detachment assay. The neutral red assay was used to compare the relative sensitivities of the FHM cells (exposed at 34/sup 0/C) with those of bluegill sunfish (BF-2) cells, a fibroblastic cell culture (exposed at 26 degrees C), in the presence of different classes of test agents frequently occurring as aquatic pollutants. For both fish species the sequence of potencies of the test agents was in the order of organometals greater than pesticides approximately equal to polychlorinated biphenyls greater than polynuclear aromatic hydrocarbons greater than phenolics. Overall, the FHM cells were more sensitive than were the BF-2 cells. However, there was a better correlation between the in vitro cytotoxicity data for the BF-2 cell culture and LC50 data for bluegill sunfish than between similar data for the FHM cell line and fathead minnows.

  5. Evaluation of nonstructural 1 antigen assays for the diagnosis and surveillance of dengue in Singapore.

    PubMed

    Pok, Kwoon-Yong; Lai, Yee-Ling; Sng, Joshua; Ng, Lee-Ching

    2010-12-01

    Early and accurate diagnosis of dengue is imperative for disease surveillance, which helps in the control of dengue in endemic countries. In this study, we evaluated the performance of three commercially available dengue nonstructural 1 (NS1) antigen assays (Bio-Rad Platelia™ Dengue NS1 Antigen ELISA, PanBio Dengue Early ELISA, and Bio-Rad Dengue NS1 Antigen Strip test) and compared them with reverse-transcription polymerase chain reaction (RT-PCR) and other commercially available serological assays for the diagnosis of dengue. The analysis showed RT-PCR to be the most sensitive and specific (100%) diagnostic method during the first 3 days of fever. The overall sensitivity of dengue NS1 antigen assays within the same period was 81.7%, indicating their potential role as a cost-effective and convenient alternative method to RT-PCR for the diagnosis of dengue fever in a primary healthcare setting. However, reduced sensitivity in detecting secondary dengue infections was one of the drawbacks of dengue NS1 antigen assays. Nonetheless, it remains a useful assay for the early detection of dengue and hence could play an important role in routine surveillance efforts to control dengue outbreaks in Singapore.

  6. Danish North Sea crude assayed

    SciTech Connect

    Rhodes, A.K.

    1994-09-12

    Danish North Sea blend was assayed earlier this year. The light, sweet crude comprises crude oil from 10 fields. The crude is piped from offshore production facilities to the A/S Dansk Shell refinery at Fredericia, Denmark. Fig. 1 shows the boiling point curve for the crude, and Fig. 2 illustrates the metals content (vanadium, nickel, and iron), as a function of distillation temperature. The table lists properties of the crude and its fractions.

  7. Two offshore Australian crudes assayed

    SciTech Connect

    Rhodes, A.K.

    1994-05-09

    Two light, sweet crudes from offshore Australia have been assayed. Gippsland crude, also called Bass Strait, is produced off the coast of Victoria, in southeastern Australia. The 47 API, 0.09% sulfur crude was analyzed in mid-1993. Skua, a 42 API, 0.06 wt % sulfur crude, is produced in the Timor Sea. Data are given on the whole crude and fractions for both deposits. Both chemical and physical properties are listed.

  8. Method for selecting exposure levels for the Drosophila sex-linked recessive lethal assay

    SciTech Connect

    Thompson, E.D.; Reeder, B.A.

    1987-01-01

    The use of the Drosophila sex-linked recessive lethal assay for detecting mutagenicity of chemicals is well established. When compounds are tested by feeding adult flies, the National Toxicology Program protocol specifies a 3-day feeding regimen at an exposure level that produces about 30% mortality. Uptake of the test compound is monitored by feeding behavior, amount of excretion, or abdomen size. An alternate method for determining uptake is to add radiolabeled sucrose to the feeding solution and then to determine the amount of radioactivity in the flies. We have found that the addition of radiolabeled sucrose underestimates consumption for feeding exposures longer than 24 hr because sucrose is metabolized and as much as 30% of the label is excreted, presumably as /sup 14/CO/sub 2/ or /sup 3/H/sub 2/O. Here we describe a method for determining uptake of chemicals by adding /sup 14/C-leucine to the feeding solution. The incorporation of /sup 14/c-leucine is essentially linear over the 3-day feeding period, which permits accurate estimates of food consumption. Use of this method demonstrates that lower exposure levels of a chemical that do not produce mortality actually results in higher consumption by the flies. The method is proposed as a prescreen to select the appropriate exposure level for the sex-linked recessive lethal assay.

  9. The in vitro micronucleus assay.

    PubMed

    Doherty, Ann T

    2012-01-01

    The in vitro micronucleus test detects genotoxic damage in interphase cells. The in vitro micronucleus test provides an alterative to the chromosome aberration test, and because the in vitro micronucleus test examines cells at interphase, the assessment of micronuclei can be scored faster, as the analysis of damage is thought to be less subjective and is more amenable to automation.Micronuclei may be the result of aneugenic (whole chromosome) or clastogenic (chromosome breakage) damage. This chapter provides methods for mononucleate and binucleate micronucleus tests and the addition of centromeric labelling and a non-disjunction assay to investigate any potential aneugenic mode of action.

  10. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  11. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  12. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  13. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  14. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  15. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

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

    2011-04-01

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

  16. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

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

  17. Serum indices: managing assay interference.

    PubMed

    Farrell, Christopher-John L; Carter, Andrew C

    2016-09-01

    Clinical laboratories frequently encounter samples showing significant haemolysis, icterus or lipaemia. Technical advances, utilizing spectrophotometric measurements on automated chemistry analysers, allow rapid and accurate identification of such samples. However, accurate quantification of haemolysis, icterus and lipaemia interference is of limited value if laboratories do not set rational alert limits, based on sound interference testing experiments. Furthermore, in the context of increasing consolidation of laboratories and the formation of laboratory networks, there is an increasing requirement for harmonization of the handling of haemolysis, icterus and lipaemia-affected samples across different analytical platforms. Harmonization may be best achieved by considering both the analytical aspects of index measurement and the possible variations in the effects of haemolysis, icterus and lipaemia interferences on assays from different manufacturers. Initial verification studies, followed up with ongoing quality control testing, can help a laboratory ensure the accuracy of haemolysis, icterus and lipaemia index results, as well as assist in managing any biases in index results from analysers from different manufacturers. Similarities, and variations, in the effect of haemolysis, icterus and lipaemia interference in assays from different manufacturers can often be predicted from the mechanism of interference. Nevertheless, interference testing is required to confirm expected similarities or to quantify differences. It is important that laboratories are familiar with a number of interference testing protocols and the particular strengths and weaknesses of each. A rigorous approach to all aspects of haemolysis, icterus and lipaemia interference testing allows the analytical progress in index measurement to be translated into improved patient care. PMID:27147624

  18. Antioxidants and the Comet assay.

    PubMed

    Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

    2009-01-01

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

  19. Assay of potentially contaminated propellant

    SciTech Connect

    Koster, J.E.; Williams, H.E. III; Scott, W.S.

    1995-02-01

    One of the decontamination and decommissioning projects within DOD is demilitarization of an aging stockpile of munitions. A large portion of the stockpile contains depleted uranium (DU) as an armor piercing core and so these munitions must be assayed for the presence of uranium in other components. The assay method must be fast and preferably easy to implement. Presence of DU is indicated by its alpha decay. The alpha particles in turn produce ions in the ambient air. If a significant fraction of these ions can escape the quantity of propellant, the ions can be detected instead of the alpha particles. As a test of the feasibility of detecting alpha emissions from DU somewhere within a cartridge of propellant, the transmission of ions through layers of real propellant was measured. The propellant is in the form of graphite-coated cylindrical pellets. A 105nun cartridge was modified for use as a pellet chamber. A check source served as an ion source. The ion detector consisted of a grid held at 300V coupled to an ammeter. Results confirm that this is a promising technique for testing the propellant for the presence of DU quickly yet with sensitivity.

  20. Rotor assembly and assay method

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1993-01-01

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor.

  1. Rotor assembly and assay method

    DOEpatents

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  2. Data transformation methods for multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  3. 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

  4. Blood bactericidal assay (Pasteurella haemolytica) comparison of morbidity in marketed feeder calves.

    PubMed

    Purdy, C W; Richards, A B; Foster, G S

    1989-02-01

    An in vitro bactericidal assay that used bovine heparinized blood was investigated for its usefulness in detecting differences in the bactericidal immunity of calves against Pasteurella haemolytica serotype 1 (Ph1). Greater than 90% of killing occurred within 30 minutes. The substitution of fetal calf serum for autologous calf plasma caused loss of bactericidal activity of the blood. Decomplemented calf serum also was low in bactericidal activity. The blood bactericidal assay appears to be opsonin antibody-dependent and complement-dependent. The coefficient of variation (CV) that can be expected with this assay was established by use of a group of 8 calves; within-day CV maximum was 0.9, and between-day CV maximum was 2.1. The blood bactericidal assay was used to evaluate 30 calves under typical market stress from 4 farms in eastern Tennessee. All calves had decreased bactericidal activity, as they moved into a feedyard in Texas. The bactericidal activity was reduced among sick calves, based on the severity of clinical signs. Morbidity was highest during the first 14 days in the feedlot. During this period, healthy calves had a decreased bactericidal index (BI) of 4 points, and calves with clinical signs of bovine respiratory tract disease for 3 days had a decreased BI of 8 points. The average reduction in the BI of calves with clinical signs of bovine respiratory tract disease for 6 or more days was 14 points. PMID:2719384

  5. Sensitive Detection Assays for Influenza RNA Do Not Reveal Viremia in US Blood Donors

    PubMed Central

    Stramer, Susan L.; Collins, Cynthia; Nugent, Thomas; Wang, Xue; Fuschino, Meghan; Heitman, John W.; Law, Jacqueline; Krysztof, David E.; Kiely, Nancy; Todd, Deborah; Vermeulen, Nicolaas M. J.; Harrington, Karen; Kamel, Hany; Kelvin, David J.; Busch, Michael P.; St. George, Kirsten; Hewlett, Indira K.; Linnen, Jeffrey M.

    2012-01-01

    (See the article by Delwart et al, on pages 875–85, and see the editorial commentary by Katz, on pages 867–9.) Background. There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays. Methods. Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested. Results. Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing. Conclusions. Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply. PMID:22293429

  6. Human Arterial Ring Angiogenesis Assay.

    PubMed

    Seano, Giorgio; Primo, Luca

    2016-01-01

    In this chapter we describe a model of human angiogenesis where artery explants from umbilical cords are embedded in gel matrices and subsequently produce capillary-like structures. The human arterial ring (hAR) assay is an innovative system that enables three-dimensional (3D) and live studies of human angiogenesis. This ex vivo model has the advantage of recapitulating several steps of angiogenesis, including endothelial sprouting, migration, and differentiation into capillaries. Furthermore, it can be exploited for (1) identification of new genes regulating sprouting angiogenesis, (2) screening for pro- or anti-angiogenic drugs, (3) identification of biomarkers to monitor the efficacy of anti-angiogenic regimens, and (4) dynamic analysis of tumor microenvironmental effects on vessel formation. PMID:27172955

  7. Predictive Assay For Cancer Targets

    SciTech Connect

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

    2005-09-19

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

  8. Development of forensic assay signatures for ebolaviruses.

    PubMed

    Song, Jian; Doggett, Norman; Wren, Melinda; Burr, Tom; Fenimore, P W; Hatcher, Eneida L; Bruno, William J; Li, Po-E; Stubben, Chris; Wolinsky, Murray

    2015-03-01

    Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan-MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 "unknown" ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high-resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.

  9. Expert system for transuranic waste assay

    SciTech Connect

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  10. Automated amperometric plutonium assay system

    SciTech Connect

    Burt, M.C.

    1985-01-01

    The amperometric titration for plutonium assay has been used in the nuclear industry for over twenty years and has been in routine use at the Hanford Engineering Development Laboratory since 1976 for the analysis of plutonium oxide and mixed oxide fuel material for the Fast Flux Test Facility. It has proven itself to be an accurate and reliable method. The method may be used as a direct end point titration or an excess of titrant may be added and a back titration performed to aid in determination of the end point. Due to the slowness of the PuVI-FeII reaction it is difficult to recognize when the end point is being approached and is very time consuming if the current is allowed to decay to the residual value after each titrant addition. For this reason the back titration in which the rapid FeII-CrVI reaction occurs is used by most laboratories. The back titration is performed by the addition of excess ferrous solution followed by two measured aliquots of standard dichromate with measurement of cell current after each addition.

  11. Assay development status report for total cyanide

    SciTech Connect

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN{sup {minus}} ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation.

  12. Matrix effects of TRU (transuranic) assays using the SWEPP PAN assay system

    SciTech Connect

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of {sup 239}Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs.

  13. Sensitive field assays for water analysis

    SciTech Connect

    Douglas, W.L.

    1984-08-01

    The goal of the project is to develop a rapid, simple, and inexpensive dry-film assay device for detection of environmental contaminants using the compound geosmin as a model. Phase I activities centered upon the immunochemical reagents necessary for the assay, development of an enzyme-cycling system that makes possible detection of substances in the parts per billion (PPB) range or lower, and demonstration of how the Immuno-Replacement-Assay can be used to detect geosmin.

  14. Multiplex assays to diagnose celiac disease.

    PubMed

    Lochman, Ivo; Martis, Peter; Burlingame, Rufus W; Lochmanová, Alexandra

    2007-08-01

    Patients with celiac disease are sensitive to the gluten fractions of wheat. Symptoms include gastrointestinal problems and a failure to thrive in children, but may range from headaches to general malaise in adults. Thus, it is difficult to diagnose celiac disease by symptoms alone. The standard diagnostic criteria include the presence of the characteristic anti-gliadin or anti-tissue transglutaminase antibodies (anti-tTG) in serum, flattened mucosa on intestinal biopsy, and improved symptoms on a gluten-free diet. Because of the ease of use of the tTG enzyme-linked immunosorbent assay (ELISA) compared to endomysial by indirect immunofluorescence assay, there has been much more screening for celiac disease in recent years. This increased screening showed that celiac disease was more prevalent than previously believed. We compared a new multiplex assay that includes a novel form of deamidated gliadin and recombinant human tTG as the antigens to other assays using standard antigens. In addition, the new assay detects the presence of selective IgA deficiency, which shows a 10-fold increase in prevalence in patients with celiac disease compared to the general population. The combination of sensitivity and specificity of the new multiplex assay was equal or better than those for standard assays. Thus the performance, ease of use, and ability to measure three clinically important parameters in a single test make the new multiplex assay a viable alternative to standard assays in a clinical lab.

  15. 233U Assay A Neutron NDA System

    SciTech Connect

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-11-17

    The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

  16. 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.

  17. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  18. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  19. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  20. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  1. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification....

  2. Statistical inference for serial dilution assay data.

    PubMed

    Lee, M L; Whitmore, G A

    1999-12-01

    Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.

  3. A simplified plaque assay for varicella vaccine.

    PubMed

    Husson-van Vliet, J; Colinet, G; Yane, F; Lemoine, P

    1987-11-01

    A simple and accurate plaque assay is described for potency testing of attenuated varicella vaccine. Assays were performed on the African green monkey kidney continuous cell line CV-1, in multidish-plates, under a semi-solid carboxymethylcellulose overlay. The test is economical and yields accurate individual titre estimates, the reliability of which may be assessed by parallel titration of reference preparations.

  4. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  5. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  6. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  7. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  8. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay....

  9. Development of an upconverting chelate assay

    NASA Astrophysics Data System (ADS)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  10. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    PubMed

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana

    2012-01-01

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  11. Dynamic, Quantitative Assays of Phagosomal Function

    PubMed Central

    Podinovskaia, Maria; VanderVen, Brian C.; Yates, Robin M.; Glennie, Sarah; Fullerton, Duncan; Mwandumba, Henry C.; Russell, David G.

    2013-01-01

    Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function, to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enables these activities to be rigorously quantified. This chapter contains the description of several of these assays assessed by different methods of quantitation; including a Fluorescence Resonance Emission Transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometer, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measure by confocal microscope. These assays illustrate both the range parameters that can be quantified as well as the flexibility of instrumentation that can be exploited for their quantitation. PMID:24510516

  12. Paper disk assay for glycosaminoglycan sulfotransferases

    SciTech Connect

    Sugahara, K.; Ishii, T.; Yamashina, I.

    1987-11-01

    A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the (/sup 35/S)sulfate-labeled products are precipitated and then separated from a sulfate donor ((/sup 35/S)PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.

  13. [Cytomegalovirus isolation by conventional cell culture and shell vial assay].

    PubMed

    Galiano, M C; Videla, C M; Sánchez Puch, S; Carballal, G

    2001-01-01

    In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4% of the samples, 11.8% of which were positive by SV and 7.7% by CC. Out of 84 positive samples, concordance between both methods was observed in 36% of the cases. We found that 46% of the samples were positive only by SV, while 18% were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43% were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18% of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity.

  14. Plateletworks: A screening assay for clopidogrel therapy monitoring in healthy cats

    PubMed Central

    Hamel-Jolette, Avril; Dunn, Marilyn; Bédard, Christian

    2009-01-01

    Plateletworks is a screening assay used in human medicine to monitor platelet-inhibiting drugs. As arterial thromboembolism is a common complication in cats suffering from cardiomyopathy, they are often treated with anti-platelet medication. Clopidogrel (Plavix), an anti-platelet aggregation drug, has recently been evaluated in healthy cats. The purpose of this study was to determine if the Plateletworks method can detect a decrease in platelet aggregation in cats receiving clopidogrel. Nine healthy adult cats were used for this study. Platelet aggregation was measured before and after a 3-day clopidogrel treatment (18.75 mg SID). Platelet aggregation after the clopidogrel treatment was significantly lower (P < 0.01). The Plateletworks method appears to be a promising test to monitor clopidogrel therapy in cats. PMID:19337399

  15. Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates.

    PubMed

    Battegay, M; Cooper, S; Althage, A; Bänziger, J; Hengartner, H; Zinkernagel, R M

    1991-06-01

    Titers of lymphocytic choriomeningitis virus (LCMV) were determined on adherent fibroblast cell lines in 24- or 96-well plates. After absorption of virus by cells and 48 h incubation under a methylcellulose overlay, cell monolayers were fixed with 4% formaldehyde in phosphate-buffered saline, permeabilized by incubation in 0.5% Triton X-100 in balanced salt solution and then stained with a monoclonal rat anti-LCMV and a peroxidase-labeled second stage antibody. The sensitivity of the assay is within a factor of 2-4 of conventional plaquing methods. The method also detects poorly or non-plaquing LCMV isolates, and therefore drastically reduces the need for titration of LCMV in mice. The method is quicker (2-3 days), as compared to conventional methods (4-6 days) and less expensive in terms of work and materials.

  16. Performance Evaluation of the Serum Thyroglobulin Assays With Immunochemiluminometric Assay and Immunoradiometric Assay for Differentiated Thyroid Cancer

    PubMed Central

    Cho, Yoon Young; Chun, Sejong; Lee, Soo-Youn; Chung, Jae Hoon

    2016-01-01

    Background Measurement of postoperative serum thyroglobulin (Tg) is important for detecting persistent or recurrent differentiated thyroid cancer. We evaluated the analytic performance of the DxI 800 assay (Beckman Coulter, USA) for serum Tg and anti-thyroglobulin antibodies (TgAbs) in comparison with that of the GAMMA-10 assay (Shinjin Medics Inc., Korea) for serum Tg and RIA-MAT 280 assay (Stratec, Germany) for TgAb. Methods We prospectively collected blood samples from 99 patients thyroidectomized for thyroid cancer. The functional sensitivity was investigated in standards and human serum. Precision and linearity were evaluated according to the guidelines of the Clinical and Laboratory Standards Institute. The correlation between the two assays was assessed in samples with different Tg ranges. Results The functional sensitivity of the DxI 800 assay for serum Tg was between 0.0313 and 0.0625 ng/mL. The total CV was 3.9–5.6% for serum Tg and 5.3–6.9% for serum TgAb. The coefficient of determination (R2) was 1.0 and 0.99 for serum Tg and TgAb, respectively. The cut-offs for serum TgAb were 4.0 IU/mL (DxI 800) and 60.0 IU/mL (RIA-MAT 280), and the overall agreement was 68.7%. The correlation between the two assays was excellent; the correlation coefficient was 0.99 and 0.88 for serum Tg and TgAb, respectively. Conclusions The DxI 800 is a sensitive assay for serum Tg and TgAb, and the results correlated well with those from the immunoradiometric assays (IRMA). This assay has several advantages over the IRMA and could be considered an alternative test for Tg measurement. PMID:27374705

  17. Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals

    PubMed Central

    Wong, Chin Lin; Lam, Ai-Leen; Smith, Maree T.; Ghassabian, Sussan

    2016-01-01

    The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight

  18. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

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

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  19. Nano-immunosafety: issues in assay validation

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

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

  20. DNA Methyltransferase Activity Assays: Advances and Challenges.

    PubMed

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

    2016-01-01

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

  1. Global haemostasis assays, from bench to bedside.

    PubMed

    van Geffen, Mark; van Heerde, Waander L

    2012-06-01

    Bleeding and thrombosis are the ultimate clinical outcomes of aberrations in the haemostatic process. Haemostasis prevents excessive blood loss due to the effort of various compartments like the vasculature, blood cells, coagulation and fibrinolysis. The complexity of all processes involved makes the diagnosis of aberrations difficult, cumbersome and expensive. A single assay to detect any factor disturbing this haemostatic balance with high sensitivity and specificity would be of great value, especially if the outcome of this assay correlates well with clinical outcome. Despite years of research, such an assay is not yet available; however, some interesting candidates are under development and combine the effects of various compartments. This review describes the development of global haemostasis assays and summarizes the current state of the art of these haemostasis assays covering thrombin and plasmin generation, turbidity and thromboelastography/thromboelastometry. Finally, we discuss the applicability of global assays in clinical practice and we provide a future perspective on the ongoing development of automation and miniaturisation as it is our belief that these developments will benefit the standardization of global haemostasis assays.

  2. The root causes of pharmacodynamic assay failure.

    PubMed

    Ferry-Galow, Katherine V; Makhlouf, Hala R; Wilsker, Deborah F; Lawrence, Scott M; Pfister, Thomas D; Marrero, Allison M; Bigelow, Kristina M; Yutzy, William H; Ji, Jiuping J; Butcher, Donna O; Gouker, Brad A; Kummar, Shivaani; Chen, Alice P; Kinders, Robert J; Parchment, Ralph E; Doroshow, James H

    2016-08-01

    Robust pharmacodynamic assay results are valuable for informing go/no-go decisions about continued development of new anti-cancer agents and for identifying combinations of targeted agents, but often pharmacodynamic results are too incomplete or variable to fulfill this role. Our experience suggests that variable reagent and specimen quality are two major contributors to this problem. Minimizing all potential sources of variability in procedures for specimen collection, processing, and assay measurements is essential for meaningful comparison of pharmacodynamic biomarkers across sample time points. This is especially true in the evaluation of pre- and post-dose tumor biopsies, which suffer from high levels of tumor insufficiency due to variations in biopsy collection techniques and significant specimen heterogeneity within and across patients. Developing methods to assess heterogeneous biopsies is necessary in order to evaluate a majority of tumor biopsies collected for pharmacodynamic biomarker studies. Improved collection devices and standardization of methods are being sought in order to improve the tumor content and quality of tumor biopsies. In terms of reagent variability, we have found that stringent initial reagent qualification and quality control of R&D-grade reagents is critical to minimize lot-to-lot variability and prevent assay failures, especially for clinical pharmacodynamic questions, which often demand assay performance that meets or exceeds clinical diagnostic assay standards. Rigorous reagent specifications and use of appropriate assay quality control methodologies help to ensure consistency between assay runs, laboratories and trials to provide much needed pharmacodynamic insights into the activity of investigational agents. PMID:27663480

  3. Inhibitor-neutralisation assay and electro-immuno assay of human factor IX (Christmas factor).

    PubMed

    Bertina, R M; van der Linden, I K

    1977-06-15

    A rabbit antibody specifically precipitating human factor IX has been used in the assay of factor IX antigen. The results obtained with two different methods (inhibitor-neutralisation assay and electro-immunoassay) have been compared in a group of healthy individuals and in a group of hemophilia B patients and carriers. In general, identical results are obtained with both methods, except in some hemophilia B+ carriers and patients, where the electroimmuno assay gives 1.5-2.0 times higher levels. Results obtained by electroimmuno assay are more accurate and reproducible than those obtained by inhibitor-neutralisation assay, which is of importance for its potential use in carrier detection.

  4. Nondestructive assay confirmatory assessment experiments: mixed oxide

    SciTech Connect

    Lemming, J.F.

    1980-04-30

    The confirmatory assessment experiments demonstrate traceable nondestructive assay (NDA) measurements of plutonium in mixed oxide powder using commercially available spontaneous-fission assay systems. The experiments illustrate two major concepts: the production of calibration materials using calorimetric assay, and the use of paired measurements for measurement assurance. Two batches of well-characterized mixed oxide powder were used to establish the random and systematic error components. The major components of an NDA measurement assurance technique to establish and maintain traceability are identified and their functions are demonstrated. 20 refs., 10 figs., 10 tabs.

  5. Methods for threshold determination in multiplexed assays

    SciTech Connect

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2014-06-24

    Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.

  6. Human somatic mutation assays as biomarkers of carcinogenesis.

    PubMed Central

    Compton, P J; Hooper, K; Smith, M T

    1991-01-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutation can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed. PMID:1954924

  7. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  8. Proximity assays for sensitive quantification of proteins.

    PubMed

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S; Bustin, Stephen A

    2015-06-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein-protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  9. Proximity assays for sensitive quantification of proteins

    PubMed Central

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S.; Bustin, Stephen A.

    2015-01-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  10. BIOMARKER ASSAYS IN NIPPLE APIRATE FLUID

    EPA Science Inventory

    ABSTRACT

    The noninvasive technique of nipple aspiration as a potential source of biomarkers of breast cancer risk was evaluated. The feasibility of performing mutagenesis assays, amplifying DNA and performing protein electrophoresis on nipple aspirate fluid was explored. ...

  11. Assessment of the in vivo genotoxicity of cadmium chloride, chloroform, and D,L-menthol as coded test chemicals using the alkaline comet assay.

    PubMed

    Wada, Kunio; Fukuyama, Tomoki; Nakashima, Nobuaki; Matsumoto, Kyomu

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) international validation study of in vivo rat alkaline comet assays, we examined cadmium chloride, chloroform, and D,L-menthol under blind conditions as coded chemicals in the liver and stomach of Sprague-Dawley rats after 3 days of administration. Cadmium chloride showed equivocal responses in the liver and stomach, supporting previous reports of its poor mutagenic potential and non-carcinogenic effects in these organs. Treatment with chloroform, which is a non-genotoxic carcinogen, did not induce DNA damage in the liver or stomach. Some histopathological changes, such as necrosis and degeneration, were observed in the liver; however, they did not affect the comet assay results. D,L-Menthol, a non-genotoxic non-carcinogen, did not induce liver or stomach DNA damage. These results indicate that the comet assay can reflect genotoxic properties under blind conditions.

  12. Electrochemical Assay of Gold-Plating Solutions

    NASA Technical Reports Server (NTRS)

    Chiodo, R.

    1982-01-01

    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  13. The comet assay: a heavenly method!

    PubMed

    Collins, Andrew R

    2015-01-01

    The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey.

  14. The comet assay in marine animals.

    PubMed

    Frenzilli, Giada; Lyons, Brett P

    2013-01-01

    Comet assay is a quick and versatile technique for assessing DNA damage in individual cells. It allows the detection of DNA single- and double-strand breaks, as well as the presence of alkali-labile sites and cross-links. Here we describe the protocols for the single-cell gel electrophoresis (Comet) assay in its alkaline (pH > 13), mild alkaline (pH = 12.1), and neutral (pH = 8) versions, when applied in marine animals.

  15. Automated optical sensing system for biochemical assays

    NASA Astrophysics Data System (ADS)

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

    1994-03-01

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

  16. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  17. Scintillation Proximity Assay of Arginine Methylation

    PubMed Central

    Wu, Jiang; Xie, Nan; Feng, You; Zheng, Y. George

    2011-01-01

    Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein post-translational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors. PMID:21821785

  18. Dot-immunobinding assay (Dot-Iba).

    PubMed

    Surendran, Sumi; Mathai, Annamma; Radhakrishnan, Vishnampet Venkataraman

    2015-01-01

    Dot-immunobinding assay (Dot-Iba) is a simple and highly reproducible immunodiagnostic method. Antibody or antigen is dotted directly onto nitrocellulose membrane (NCM) discs. The diagnostic material to be checked can be incubated on this disc. Presence of antigen-antibody complex in NCM discs can be directly demonstrated with enzyme-conjugated antiglobulins and substrate. Development of a purple-pink colored, insoluble substrate product in the nitrocellulose membrane will be considered a positive result in the assay. This assay allows the processing of multiple specimens at a time and the entire operational procedures required only 4-6 h. Dot-IBA is rapid and the technical steps involved in the assay are much simpler than the other immunoassays such as enzyme-linked immunosorbent assay in detecting circulating antigen and antibody in clinical samples. The Dot-Iba showed an overall sensitivity of 60 % for tuberculous meningitis diagnosis and no false positive results were encountered. Hence this assay is highly specific for the diagnosis of paucibacillary diseases like extrapulmonary tuberculosis. Dot-Iba is best suited to laboratories in developing world where there are constraints in laboratory resources.

  19. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  20. Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality

    PubMed Central

    2012-01-01

    Background There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. Results We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). Conclusion Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality. PMID:22316115

  1. Long-term effects of the rhapontic rhubarb extract ERr 731® on estrogen-regulated targets in the uterus and on the bone in ovariectomized rats.

    PubMed

    Keiler, Annekathrin Martina; Papke, Anja; Kretzschmar, Georg; Zierau, Oliver; Vollmer, Günter

    2012-01-01

    The efficacy of ERr 731(®), a commercially available extract isolated from Rheum rhaponticum, in terms of menopausal complaints like hot flushes, depression, anxiety and vaginal dryness has been proven in a two-year clinical study. Further a recent preclinical study excluded unwanted side effects on the endometrium by showing a lack of stimulation of proliferation marker genes by ERr 731(®) or its constituents in the 3-day uterotrophic assay. The present study aimed at further substantiating the safety of ERr 731(®) in terms of endometrial hyperplasia and at the same time test for potential estrogenic effects in the bone. Therefore, ovariectomized (ovx) rats were treated in a dietary long-term administration for 90 days. Hence, the modulation of proliferation in the uterus was investigated by examining the effects on the mRNA expression of proliferation marker genes (Mki67, Pcna), on the estrogen-responsive gene C3 and on the estrogen receptors ERα and ERβ. We additionally performed densitometry analysis of the proximal tibia metaphysis using peripheral computed tomography (pQCT) and quantified bone homeostasis markers in the serum to examine potential effects on the bone. In this study design, neither an uterotrophic response nor a modulation of proliferation marker genes on mRNA level has been observed as response to the long-term application of the rhapontic extract. Furthermore, no impact of the two administered ERr 731(®) doses on the E2 deprivation-induced bone loss has been evident at the end of the study. In conclusion, the observations from previous trials regarding the endometrial safety of ERr 731(®) have been supported by our experimental findings that exclude a stimulatory activity on proliferation in the uterus in a long-term administration in the young adult rat but no effect on the bone mineral density could be observed.

  2. Controlling variation in the comet assay

    PubMed Central

    Collins, Andrew R.; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation. PMID:25368630

  3. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

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

    2007-07-01

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

  4. Measurement of dabigatran in standardly used clinical assays, whole blood viscoelastic coagulation, and thrombin generation assays.

    PubMed

    van Ryn, Joanne; Grottke, Oliver; Spronk, Henri

    2014-09-01

    Dabigatran, a direct thrombin inhibitor, is increasingly used clinically as one of the new oral anticoagulants. This review summarizes the assays available to measure its activity and includes the relative sensitivity of the different assays for this agent. In addition to plasma-based clotting tests, assays commonly used in surgical/emergency settings, such as activated clotting time and thromboelastometry/thromboelastography, are reviewed. In addition, the thrombin generation assay is discussed as an important method to determine the potential risk of thrombosis or bleeding and its relevance to the measurement of direct thrombin inhibitors. PMID:25168938

  5. Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

    PubMed

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

    2016-01-01

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories. PMID:26867747

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

    PubMed Central

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

    2016-01-01

    Summary The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories. PMID:26867747

  7. Research highlights: digital assays on chip.

    PubMed

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino

    2015-01-01

    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  8. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs

    PubMed Central

    Sewing, Sabine; Boess, Franziska; Moisan, Annie; Bertinetti-Lapatki, Cristina; Minz, Tanja; Hedtjaern, Maj; Tessier, Yann; Schuler, Franz; Singer, Thomas; Roth, Adrian B.

    2016-01-01

    Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development. PMID:27442522

  9. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

    PubMed

    Sewing, Sabine; Boess, Franziska; Moisan, Annie; Bertinetti-Lapatki, Cristina; Minz, Tanja; Hedtjaern, Maj; Tessier, Yann; Schuler, Franz; Singer, Thomas; Roth, Adrian B

    2016-01-01

    Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development. PMID:27442522

  10. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

    PubMed

    Sewing, Sabine; Boess, Franziska; Moisan, Annie; Bertinetti-Lapatki, Cristina; Minz, Tanja; Hedtjaern, Maj; Tessier, Yann; Schuler, Franz; Singer, Thomas; Roth, Adrian B

    2016-01-01

    Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.

  11. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

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

  12. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-05

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

  13. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

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

    2009-06-29

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

  14. Assays for aptamer-based platforms.

    PubMed

    Citartan, Marimuthu; Gopinath, Subash C B; Tominaga, Junji; Tan, Soo-Choon; Tang, Thean-Hock

    2012-04-15

    Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.

  15. Miniaturization of hydrolase assays in thermocyclers.

    PubMed

    Lucena, Severino A; Moraes, Caroline S; Costa, Samara G; de Souza, Wanderley; Azambuja, Patrícia; Garcia, Eloi S; Genta, Fernando A

    2013-03-01

    We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein. PMID:23123426

  16. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  17. Fungicide resistance assays for fungal plant pathogens.

    PubMed

    Secor, Gary A; Rivera, Viviana V

    2012-01-01

    Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

  18. The Soft Agar Colony Formation Assay

    PubMed Central

    Borowicz, Stanley; Van Scoyk, Michelle; Avasarala, Sreedevi; Karuppusamy Rathinam, Manoj Kumar; Tauler, Jordi; Bikkavilli, Rama Kamesh; Winn, Robert A.

    2014-01-01

    Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model. PMID:25408172

  19. Neutron Assay System for Confinement Vessel Disposition

    SciTech Connect

    Frame, Katherine C; Bourne, Mark M; Crooks, William J; Evans, Louise; Mayo, Douglas R; Miko, David K; Salazar, William R; Stange, Sy; Valdez, Jose I; Vigil, Georgiana M

    2012-07-13

    Waste will be removed from confinement vessels remaining from 1970s-era experiments. Los Alamos has 9+ spherical confinement vessels remaining from experiments. Each vessel contains {approx} 500 lbs of radioactive debris such as actinide metals and oxides, metals, powdered silica, graphite, and wires and hardware. In order to dispose of the vessels, debris and contamination must be removed. Neutron assay system was designed to assay vessels before and after cleanout. System requirements are: (1) Modular and moveable; (2) Capable of detecting {approx}100g {sup 239}Pu equivalent in a 2-inch thick steel sphere with 6 foot diameter; and (3) Capable of safeguards-quality assays. Initial design parameters arethe use of 4-atm {sup 3}He tubes with length of 6 feet, and {sup 3}He tubes embedded in polyethelene for moderation. This paper describes the calibration of the Confinement Vessel Assay System (CVAS) and quantification of its uncertainties. Assay uncertainty depends on five factors: (1) Statistical uncertainty in the assay measurement; (2) Statistical uncertainty in the background measurement; (3) Statistical uncertainty in the isotopics determination - This should be much smaller than the other uncertainties; (4) Systematic uncertainty due to position bias; and (5) Systematic uncertainty due to fluctuations in cosmic ray spallation. This one can be virtually eliminated by performing the background measurement with an empty vessel - but that may not be possible. We used modeling and experiments to quantify the systematic uncertainties. The calibration assumes a uniform distribution of material, but reality will be different. MCNPX modeling was used to quantify the positional bias. The model was benchmarked to build confidence in its results. Material at top of vessel is 44% greater than amount assayed, according to singles. Material near 19-tube detector is 38% less than amount assayed, according to singles. Cosmic ray spallation contributes significantly to the

  20. A new fluorescent assay for enalapril maleate.

    PubMed

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

    2005-09-01

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

  1. Pulmonary embolism aggravated by two short flights 3 days apart.

    PubMed

    Grossman, Charles M; White, Jocelyn C; Dunn, Patrick M

    2005-07-01

    An elderly internist experienced aggravation of preexisting pulmonary emboli following each of two short flights of 75 min apiece 3 d apart. A brief discussion of risk factors is included. It is suggested that pertinent reports should separate venous thromboembolic disease (VTE) from uncomplicated deep venous thrombosis (DVT).

  2. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal

    Cancer.gov

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page: https://assays.cancer.gov/support/

  3. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425...

  4. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  5. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  6. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425...

  7. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425...

  8. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  9. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  10. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425...

  11. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425...

  12. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  13. Assay for Angiotensin-Converting Enzyme.

    ERIC Educational Resources Information Center

    Russo, Salvatore F.

    1983-01-01

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

  14. Benzodiazepine Synthesis and Rapid Toxicity Assay

    ERIC Educational Resources Information Center

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

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

  15. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    PubMed

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  16. Micropallets for cell and biological assay applications

    NASA Astrophysics Data System (ADS)

    Jensen-McMullin, Cynthia

    2007-12-01

    Interest in the subjects of microfluidics, nanotechnology and lab-on-a-chip is ever increasing. Several features of microanalysis and biological assays are desired, such as low reagent use and rapid results. These features can be achieved by developing a flexible, encoded technology capable of multiplexing. The work presented in this dissertation introduces microcarriers referred to as 'micropallets' which are encoded structures ranging in size from 25mum to several hundred microns. These small structures are fabricated using photoresist or other polymer materials. Micropallets may be used in static detection systems or for the transportation and manipulation of attached biological or chemical samples through a microfluidic system. Encoding options for micropallets are discussed. Encoding may be accomplished through the use of barcodes or other markings and may be engineered to optimally suit the application. This work presents the encoded micropallet microcarriers and the corresponding microfluidic and static systems used with micropallets. We discuss the importance of encoding towards the development of flexible, multiplexed assays and decoding strategies used or under development. Cell and antibody assays were selected and investigated to assess the utility of micropallets. We conclude from the results of this work, as well as ongoing interests, micropallets achieve the goals of improving biological techniques including cellular and other biological assays through the options of encoding and multiplexing.

  17. Advanced analysis techniques for uranium assay

    SciTech Connect

    Geist, W. H.; Ensslin, Norbert; Carrillo, L. A.; Beard, C. A.

    2001-01-01

    Uranium has a negligible passive neutron emission rate making its assay practicable only with an active interrogation method. The active interrogation uses external neutron sources to induce fission events in the uranium in order to determine the mass. This technique requires careful calibration with standards that are representative of the items to be assayed. The samples to be measured are not always well represented by the available standards which often leads to large biases. A technique of active multiplicity counting is being developed to reduce some of these assay difficulties. Active multiplicity counting uses the measured doubles and triples count rates to determine the neutron multiplication (f4) and the product of the source-sample coupling ( C ) and the 235U mass (m). Since the 35U mass always appears in the multiplicity equations as the product of Cm, the coupling needs to be determined before the mass can be known. A relationship has been developed that relates the coupling to the neutron multiplication. The relationship is based on both an analytical derivation and also on empirical observations. To determine a scaling constant present in this relationship, known standards must be used. Evaluation of experimental data revealed an improvement over the traditional calibration curve analysis method of fitting the doubles count rate to the 235Um ass. Active multiplicity assay appears to relax the requirement that the calibration standards and unknown items have the same chemical form and geometry.

  18. Calcium flux assay in Xenopus oocytes.

    PubMed

    Murphy, P M

    2001-05-01

    Many G protein-coupled receptors of interest to neuroscientists induce transient increases in [Ca(2+)](i), which can be used as a convenient measure of receptor activation in a variety of applications. This unit describes a simple calcium flux assay applied to Xenopus oocytes. PMID:18428482

  19. Three-dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichert, Anke

    2001-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flue virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  20. Homogeneous screening assay for human tankyrase.

    PubMed

    Narwal, Mohit; Fallarero, Adyary; Vuorela, Pia; Lehtiö, Lari

    2012-06-01

    Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors. PMID:22357873

  1. Nondestructive assay of boxed radioactive waste

    SciTech Connect

    Gilles, W.P.; Roberts, R.J.; Jasen, W.G.

    1992-12-01

    This paper describes the problems related to the nondestructive assay (NDA) of boxed radioactive waste at the Hanford Site and how Westinghouse Hanford company (WHC) is solving the problems. The waste form and radionuclide content are described. The characteristics of the combined neutron and gamma-based measurement system are described.

  2. Analysis of Gold Ores by Fire Assay

    ERIC Educational Resources Information Center

    Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

    2004-01-01

    Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

  3. Toxoplasma gondii detection and viability assays in ham legs and shoulders from experimentally infected pigs.

    PubMed

    Gomez-Samblas, M; Vilchez, S; Racero, J C; Fuentes, M V; Osuna, A

    2016-09-01

    Epidemiological studies of toxoplasmosis show that infection in humans is mainly caused by the consumption of raw, undercooked or cured meat. Cured "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. The "Serrano" ham is prepared from pork meat and undergoes a process known as curing and a subsequent fermentation without thermal or smoking treatments. The viability of Toxoplasma gondii in hams and shoulders from experimentally infected pigs that have been subject to different curing processes has been studied in order to evaluate the best method to completely eliminate the viable protozoa. The different treatments include, i) freezing the legs and shoulders below -20 °C for 3 days before salting with marine salt, ii) salting the meat with marine salt and nitrites, iii) salting only with marine salt (traditional process) and iv) salting with marine salt and then freezing at -20 °C for 3 days after the curing period. The ham leg samples were cured for 7 months and the shoulder samples for 5 months. The presence of T. gondii in the different treatments was studied by a "magnetic-capture" method for the isolation of T. gondii DNA and a quantitative real-time PCR to estimate the T. gondii burden in the ham legs and shoulders. The infectivity capacity of T. gondii in positive samples was assayed by bioassays in mice and some physicochemical parameters, such as pH, water activity (aw) and salt content, were evaluated at the end of the curing time. In all the cases where the samples were frozen the T. gondii infectivity was eliminated. In samples in which the meat was salted in marine salt plus nitrites, the parasite viability remained for longer than in the traditional salting process. The methods described here could be useful for producers to guarantee the safety of their products. PMID:27217366

  4. Toxoplasma gondii detection and viability assays in ham legs and shoulders from experimentally infected pigs.

    PubMed

    Gomez-Samblas, M; Vilchez, S; Racero, J C; Fuentes, M V; Osuna, A

    2016-09-01

    Epidemiological studies of toxoplasmosis show that infection in humans is mainly caused by the consumption of raw, undercooked or cured meat. Cured "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. The "Serrano" ham is prepared from pork meat and undergoes a process known as curing and a subsequent fermentation without thermal or smoking treatments. The viability of Toxoplasma gondii in hams and shoulders from experimentally infected pigs that have been subject to different curing processes has been studied in order to evaluate the best method to completely eliminate the viable protozoa. The different treatments include, i) freezing the legs and shoulders below -20 °C for 3 days before salting with marine salt, ii) salting the meat with marine salt and nitrites, iii) salting only with marine salt (traditional process) and iv) salting with marine salt and then freezing at -20 °C for 3 days after the curing period. The ham leg samples were cured for 7 months and the shoulder samples for 5 months. The presence of T. gondii in the different treatments was studied by a "magnetic-capture" method for the isolation of T. gondii DNA and a quantitative real-time PCR to estimate the T. gondii burden in the ham legs and shoulders. The infectivity capacity of T. gondii in positive samples was assayed by bioassays in mice and some physicochemical parameters, such as pH, water activity (aw) and salt content, were evaluated at the end of the curing time. In all the cases where the samples were frozen the T. gondii infectivity was eliminated. In samples in which the meat was salted in marine salt plus nitrites, the parasite viability remained for longer than in the traditional salting process. The methods described here could be useful for producers to guarantee the safety of their products.

  5. Production and assay of forskolin antibodies

    SciTech Connect

    Ho, L.T.; Ho, R.J.

    1986-05-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  6. Assays for mammalian tyrosinase: a comparative study

    SciTech Connect

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.

  7. Comet assay: rapid processing of multiple samples.

    PubMed

    McNamee, J P; McLean, J R; Ferrarotto, C L; Bellier, P V

    2000-03-01

    The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets. PMID:10751727

  8. Non-separation assay for glycohemoglobin.

    PubMed

    Blincko, S; Edwards, R

    1998-06-01

    The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD +/-0.03)%HbA1 +0.6(SD +/-0.3), S(y/x) = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD +/-0.04)%HbA1c + 1.8(SD +/-0.3), S(y/x) = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was <2% (intraassay, range 5-22% GHb) and <4.2% (interassay, range 4-16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 micromol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results. PMID:9625057

  9. Variola Virus-Specific Diagnostic Assays: Characterization, Sensitivity, and Specificity

    PubMed Central

    Kondas, Ashley V.; Olson, Victoria A.; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard

    2015-01-01

    A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified. PMID:25673790

  10. Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity.

    PubMed

    Kondas, Ashley V; Olson, Victoria A; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard; Damon, Inger K

    2015-04-01

    A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

  11. Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

    PubMed

    Vaidya, Sunil R; Kumbhar, Neelakshi S; Bhide, Vandana S

    2014-12-01

    Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies.

  12. Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

    PubMed

    Vaidya, Sunil R; Kumbhar, Neelakshi S; Bhide, Vandana S

    2014-12-01

    Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies. PMID:25244651

  13. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    PubMed

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

  14. Storage and Assay of Tritium in STAR

    SciTech Connect

    Longhurst, Glen R.; Anderl, Robert A.; Pawelko, Robert J.; Stoots, Carl J.

    2005-07-15

    The Safety and Tritium Applied Research (STAR) facility at the Idaho National Engineering and Environmental Laboratory (INEEL) is currently being commissioned to investigate tritium-related safety questions for fusion and other technologies. The tritium inventory for the STAR facility will be maintained below 1.5 g to avoid the need for STAR to be classified as a Category 3 nuclear facility. A key capability in successful operation of the STAR facility is the ability to receive, inventory, and dispense tritium to the various experiments underway there. The system central to that function is the Tritium Storage and Assay System (SAS).The SAS has four major functions: (1) receiving and holding tritium, (2) assaying, (3) dispensing, and (4) purifying hydrogen isotopes from non-hydrogen species.This paper describes the design and operation of the STAR SAS and the procedures used for tritium accountancy in the STAR facility.

  15. Bio-assays for microchemical environmental contaminants

    PubMed Central

    Warner, Richard E.

    1967-01-01

    A solution of the problem of environmental contamination must be based on accurate measurement of the extent of the contamination and of the resulting hazards. This paper reviews the methods for the estimation of microchemical contaminants in water with the aid of living organisms. The methods are grouped according to the nature of the response of the organism to the contaminant—namely, acute response (usually death), behavioural change, physiological change, biochemical and histochemical change, ecological change, embryological and regenerational change, growth change, histological change and perception by man or aquatic organisms. Finally, the following problems are discussed: selection of appropriate tests and standardization, the dangers of sequential concentration and the need for multi-parametric assays (assays involving several responses of a single organism, or responses of several organisms) for complete characterization of the effects of a contaminant on the environment. ImagesFIG. 2FIG. 3FIG. 4FIG. 5FIG. 6 PMID:5299747

  16. Detailed assays conducted on Vietnamese crude oils

    SciTech Connect

    Du, P.Q. )

    1990-07-16

    More oil property data, in the form of recent crude oil assays, have been made available for two Vietnamese crude oils, Bach Ho (White Tiger) and Dai Hung (Big Bear). Crude oil data presented earlier gave limited properties of the crudes,which are from the Miocene formations. Further analyses have been conducted on Bach Ho crude from the Oligocene formations. Production from Oligocene is far more representative of the oils produced from the Bach Ho field and marketed worldwide. Currently, Bach Ho is the only producing field. Dai Hung is expected to be in production during the next few years. Bach Ho is currently producing at the rate of 20,000 b/d. That figure is projected to grow to 100,000 b/d by 1992 and to 120,000 b/d by 1995. Detailed assays of both crude oils are presented.

  17. Expert system technology for nondestructive waste assay

    SciTech Connect

    Becker, G.K.; Determan, J.C.

    1998-07-01

    Nondestructive assay waste characterization data generated for use in the National TRU Program must be of known and demonstrable quality. Each measurement is required to receive an independent technical review by a qualified expert. An expert system prototype has been developed to automate waste NDA data review of a passive/active neutron drum counter system. The expert system is designed to yield a confidence rating regarding measurement validity. Expert system rules are derived from data in a process involving data clustering, fuzzy logic, and genetic algorithms. Expert system performance is assessed against confidence assignments elicited from waste NDA domain experts. Performance levels varied for the active, passive shielded, and passive system assay modes of the drum counter system, ranging from 78% to 94% correct classifications.

  18. A specific endpoint assay for ubiquitin.

    PubMed Central

    Rose, I A; Warms, J V

    1987-01-01

    Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. The method for measuring Ub makes use of the reaction of iodoacetamide-treated Ub-activating enzyme (E): [3H]ATP + Ub + E----E X [3H]AMP-Ub + PPi and PPi----2Pi (in the presence of pyrophosphatase). The Ub is then measured by determining the acid-insoluble radioactivity. The reaction is accompanied by a slow enzyme-catalyzed hydrolysis of the complex to AMP plus Ub. The presence of ubiquitin-activating enzyme in excess of Ub by approximately equal to 0.1 microM assures that the steady state will be close to the endpoint for total Ub. A preparation of the activating enzyme from human erythrocytes that does not depend on affinity chromatography is described. Several applications of the assay are presented. PMID:3031643

  19. Immunonephelometric and immunoturbidimetric assays for proteins.

    PubMed

    Whicher, J T; Price, C P; Spencer, K

    1983-01-01

    Immunonephelometric and immunoturbidimetric techniques for the measurement of proteins have developed and expanded rapidly in recent years and are fast replacing the time-honoured gel precipitation techniques. The reasons for this are primarily an increased awareness of the value of specific protein measurements with the impetus of improved chemical reagents and advances in instrument technology. This review discusses the background to such free fluid phase immunochemical measurement systems and the developments which have occurred in this field. The following topics are reviewed. (1) The theoretical background of light scattering theory as applied to the measurement of antibody-antigen complexes. (2) The nature and kinetics of the antibody-antigen reaction in fluid media and the effect of enhancing polymers. (3) The sample and antibody requirements for nephelometric and turbidimetric assay. (4) Instrumental systems for nephelometry. (5) Instrumental systems for turbidimetry. (6) Methods of establishing, assessing and monitoring nephelometric assays for specific proteins in the laboratory.

  20. Quantum Dot-Based Cell Motility Assay

    SciTech Connect

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

    2005-06-06

    Because of their favorable physical and photochemical properties, colloidal CdSe/ZnS-semiconductor nanocrystals (commonly known as quantum dots) have enormous potential for use in biological imaging. In this report, we present an assay that uses quantum dots as markers to quantify cell motility. Cells that are seeded onto a homogeneous layer of quantum dots engulf and absorb the nanocrystals and, as a consequence, leave behind a fluorescence-free trail. By subsequently determining the ratio of cell area to fluorescence-free track area, we show that it is possible to differentiate between invasive and noninvasive cancer cells. Because this assay uses simple fluorescence detection, requires no significant data processing, and can be used in live-cell studies, it has the potential to be a powerful new tool for discriminating between invasive and noninvasive cancer cell lines or for studying cell signaling events involved in migration.

  1. Delayed gamma technique for fissile material assay

    SciTech Connect

    Mozin, Vladimir; Tobin, Stephen; Vujie, Jasmina; Hunt, Alan

    2010-01-01

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

  2. Taste Preference Assay for Adult Drosophila.

    PubMed

    Bantel, Andrew P; Tessier, Charles R

    2016-01-01

    Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound. PMID:27684591

  3. Depleted uranium waste assay at AWE

    SciTech Connect

    Miller, T.J.

    2007-07-01

    The Atomic Weapons Establishment (AWE) at Aldermaston has recently conducted a Best Practical Means (BPM) study, for solid Depleted Uranium (DU) waste assay, in order to satisfy key stakeholders that AWE is applying best practice. This study has identified portable passive High Resolution Gamma Spectrometry (HRGS), combined with an analytical software package called Spectral Nondestructive Assay Platform (SNAP), as the preferred option with the best balance between performance and costs. HRGS/SNAP performance has been assessed by monitoring 200 l DU waste drum standards and also heterogeneous, high density drums from DU firing trials. Accuracy was usually within 30 % with Detection Limits (DL) in the region of 10 g DU for short count times. Monte Carlo N-Particle (MCNP) calculations have been used to confirm the shape of the calibration curve generated by the SNAP software procured from Eberline Services Inc. (authors)

  4. Neutron Assay System for Confinement Vessel Disposition

    SciTech Connect

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Mayo, Douglas R.; Miko, David K.; Salazar, William R.; Stange, Sy; Valdez, Jose I.; Vigil, Georgiana M.

    2012-07-13

    Los Alamos National Laboratory has a number of spherical confinement vessels (CVs) remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1-inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the CVs. The Confinement Vessel Assay System (CVAS) was developed to measure the amount of special nuclear material (SNM) in CVs before and after cleanout. Prior to cleanout, the system will be used to perform a verification measurement of each vessel. After cleanout, the system will be used to perform safeguards-quality assays of {le}100-g {sup 239}Pu equivalent in a vessel for safeguards termination. The CVAS has been tested and calibrated in preparation for verification and safeguards measurements.

  5. System and method for assaying a radionuclide

    SciTech Connect

    Cadieux, James R; King, III, George S; Fugate, Glenn A

    2014-12-23

    A system for assaying a radionuclide includes a liquid scintillation detector, an analyzer connected to the liquid scintillation detector, and a delay circuit connected to the analyzer. A gamma detector and a multi-channel analyzer are connected to the delay circuit and the gamma detector. The multi-channel analyzer produces a signal reflective of the radionuclide in the sample. A method for assaying a radionuclide includes selecting a sample, detecting alpha or beta emissions from the sample with a liquid scintillation detector, producing a first signal reflective of the alpha or beta emissions, and delaying the first signal a predetermined time. The method further includes detecting gamma emissions from the sample, producing a second signal reflective of the gamma emissions, and combining the delayed first signal with the second signal to produce a third signal reflective of the radionuclide.

  6. DREAM Assay for Studying Microbial Electron Transfer.

    PubMed

    Vishwanathan, A S; Devkota, Ranjan; Siva Sankara Sai, S; Rao, Govind

    2015-12-01

    Methylene blue undergoes reduction with an accompanying colour change reaction, from blue to colourless, enabling its use as a metric for estimating reducing power. A dye reduction-based electron-transfer activity monitoring (DREAM) assay is demonstrated as a tool to study and understand the process of microbes sourcing electrons from organic substrates and transferring them to an electron acceptor. The rate at which electrons can be transferred to the thermodynamically most feasible electron acceptor directly depends on the activity of microbes. Nature of available substrate determines the quantum of electrons available. Dissolved oxygen intercepts electrons from the microbes before they can be taken up by the dye. Sodium sulfite can be used to offset the detrimental effects of the presence of dissolved oxygen. This easy-to-perform assay has been demonstrated as a proof-of-concept having potential to be extended to other practical applications. PMID:26386586

  7. Assay and Inhibition of Diacylglycerol Lipase Activity

    PubMed Central

    Johnston, Meghan; Bhatt, Shachi R.; Sikka, Surina; Mercier, Richard W.; West, Jay M.; Makriyannis, Alexandros; Gatley, S. John; Duclos, Richard I.

    2012-01-01

    A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-14C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-14C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-14C]arachidonic acid. PMID:22738638

  8. [Thrombin generation assays and their clinical application].

    PubMed

    Kern, Anita; Várnai, Katalin; Vásárhelyi, Barna

    2014-06-01

    Thrombin is a key enzyme of the coagulation system, having both pro- and anticoagulant functions. Thus, the generation of thrombin is one of the most important steps in coagulation. Global haemostasis assay, the so-called thrombin generation test is appropriate for its assessment. Since thrombin generation is sensible for both pro- and anticoagulant processes it can be applied for the general characterisation of the risk of thrombosis and bleeding, too. Clinical studies confirmed augmented thrombin generation in patients with high risk of venous or arterial thrombosis. Anticoagulant therapy (also novel oral anticoagulant treatment) can be monitored by thrombin generation. In case of haemophilia thrombin generation assays reflect bleeding severity. It is applicable for monitoring of both conventional haemophilia treatment and inhibitor-bypassing therapy, which is needed when inhibitors develop in patients. Standardization of thrombin generation methods and determination of cut off values are required before its application in clinical practice.

  9. System and method for assaying radiation

    SciTech Connect

    DiPrete, David P; Whiteside, Tad; Pak, Donald J; DiPrete, Cecilia C

    2013-11-12

    A system for assaying radiation includes a sample holder configured to hold a liquid scintillation solution. A photomultiplier receives light from the liquid scintillation solution and generates a signal reflective of the light. A control circuit biases the photomultiplier and receives the signal from the photomultiplier reflective of the light. A light impermeable casing surrounds the sample holder, photomultiplier, and control circuit. A method for assaying radiation includes placing a sample in a liquid scintillation solution, placing the liquid scintillation solution in a sample holder, and placing the sample holder inside a light impermeable casing. The method further includes positioning a photomultiplier inside the light impermeable casing and supplying power to a control circuit inside the light impermeable casing.

  10. DREAM Assay for Studying Microbial Electron Transfer.

    PubMed

    Vishwanathan, A S; Devkota, Ranjan; Siva Sankara Sai, S; Rao, Govind

    2015-12-01

    Methylene blue undergoes reduction with an accompanying colour change reaction, from blue to colourless, enabling its use as a metric for estimating reducing power. A dye reduction-based electron-transfer activity monitoring (DREAM) assay is demonstrated as a tool to study and understand the process of microbes sourcing electrons from organic substrates and transferring them to an electron acceptor. The rate at which electrons can be transferred to the thermodynamically most feasible electron acceptor directly depends on the activity of microbes. Nature of available substrate determines the quantum of electrons available. Dissolved oxygen intercepts electrons from the microbes before they can be taken up by the dye. Sodium sulfite can be used to offset the detrimental effects of the presence of dissolved oxygen. This easy-to-perform assay has been demonstrated as a proof-of-concept having potential to be extended to other practical applications.

  11. Transient expression assays in tobacco protoplasts.

    PubMed

    Vanden Bossche, Robin; Demedts, Brecht; Vanderhaeghen, Rudy; Goossens, Alain

    2013-01-01

    The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.

  12. Developments in plutonium waste assay at AWE.

    PubMed

    Miller, T J

    2009-06-01

    In 2002 a paper was presented at the 43rd Annual Meeting of the Institute of Nuclear Materials Management (INMM) on the assay of low level plutonium (Pu) in soft drummed waste (Miller 2002 INMM Ann. Meeting (Orlando, FL, 23-27 July 2002)). The technique described enabled the Atomic Weapons Establishment (AWE), at Aldermaston in the UK, to meet the stringent Low Level Waste Repository at Drigg (LLWRD) conditions for acceptance for the first time. However, it was initially applied to only low density waste streams because it relied on measuring the relatively low energy (60 keV) photon yield from Am-241 during growth. This paper reviews the results achieved when using the technique to assay over 10,000 waste packages and presents the case for extending the range of application to denser waste streams.

  13. Label-free functional selectivity assays.

    PubMed

    Ferrie, Ann M; Goral, Vasiliy; Wang, Chaoming; Fang, Ye

    2015-01-01

    G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the β2-adrenergic receptor (β2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of β2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the β2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map. PMID:25563188

  14. Assay of brines for common radiolysis products

    SciTech Connect

    MacDougall, C.S.

    1981-01-01

    Brines are assayed for four common products of radiolytic reaction. Free chlorine is determined spectrophotometrically after reaction with o-tolidine. The test is specific for chlorine, and quantities of chlorine from 0.1 to 6 ..mu..g in the test aliquot are determined with a precision of about +- 5%. Hydrogen peroxide is reacted with xylenol orange and determined spectrophotometrically with a precision of +- 5% on 2-..mu..g quantities of peroxide. A spectrophotometric method using thiocyanate is employed in the chlorate assay. After subtracting the bias caused by any H/sub 2/O/sub 2/ or Cl/sub 2/, 1-..mu..g quantities of chlorate can be determined with a precision of +- 10%. Perchlorate ion quantities of 1 ppM can be determined directly in brines by ion chromatography with a precision of about +- 15%.

  15. Quantifiable Lateral Flow Assay Test Strips

    NASA Technical Reports Server (NTRS)

    2003-01-01

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

  16. Polymeric assay film for direct colorimetric detection

    DOEpatents

    Charych, Deborah; Nagy, Jon; Spevak, Wayne

    2002-01-01

    A lipid bilayer with affinity to an analyte, which directly signals binding by a changes in the light absorption spectra. This novel assay means and method has special applications in the drug development and medical testing fields. Using a spectrometer, the system is easily automated, and a multiple well embodiment allows inexpensive screening and sequential testing. This invention also has applications in industry for feedstock and effluent monitoring.

  17. Polymeric assay film for direct colorimetric detection

    DOEpatents

    Charych, Deborah; Nagy, Jon; Spevak, Wayne

    1999-01-01

    A lipid bilayer with affinity to an analyte, which directly signals binding by a changes in the light absorption spectra. This novel assay means and method has special applications in the drug development and medical testing fields. Using a spectrometer, the system is easily automated, and a multiple well embodiment allows inexpensive screening and sequential testing. This invention also has applications in industry for feedstock and effluent monitoring.

  18. Methods and devices for protein assays

    DOEpatents

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee

    2009-11-03

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

  19. Lymphocyte chromosomal aberration assay in radiation biodosimetry

    PubMed Central

    Agrawala, Paban K.; Adhikari, J. S.; Chaudhury, N. K.

    2010-01-01

    Exposure to ionizing radiations, whether medical, occupational or accidental, leads to deleterious biological consequences like mortality or carcinogenesis. It is considered that no dose of ionizing radiation exposure is safe. However, once the accurate absorbed dose is estimated, one can be given appropriate medical care and the severe consequences can be minimized. Though several accurate physical dose estimation modalities exist, it is essential to estimate the absorbed dose in biological system taking into account the individual variation in radiation response, so as to plan suitable medical care. Over the last several decades, lots of efforts have been taken to design a rapid and easy biological dosimeter requiring minimum invasive procedures. The metaphase chromosomal aberration assay in human lymphocytes, though is labor intensive and requires skilled individuals, still remains the gold standard for radiation biodosimetry. The current review aims at discussing the human lymphocyte metaphase chromosomal aberration assay and recent developments involving the application of molecular cytogenetic approaches and other technological advancements to make the assay more authentic and simple to use even in the events of mass radiation casualties. PMID:21829315

  20. Assessment of plaque assay methods for alphaviruses.

    PubMed

    Juarez, Diana; Long, Kanya C; Aguilar, Patricia; Kochel, Tadeusz J; Halsey, Eric S

    2013-01-01

    Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, identification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recognition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated. Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method showed consistently greater sensitivity than the semisolid method. Also, a "semisolid-variant method" using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifications in plaque assay conditions affect significantly results and therefore evaluation of the performance of each new assay is needed.

  1. Reporter Immobilization Assay (REIA) for Bioconjugating Reactions.

    PubMed

    Schatte, Martin; Bocola, Marco; Roth, Teresa; Martinez, Ronny; Kopetzki, Erhard; Schwaneberg, Ulrich; Bönitz-Dulat, Mara

    2016-06-15

    Enzymes able to ligate biomolecules are emerging tools to generate site-specific bioconjugates. In this study we present a detection and screening method for bioconjugating enzymes which overcomes limitations of analytical methods such as HPLC or MS. These techniques are experimentally demanding and often limited in sensitivity and throughput compared to enzymatic assays. The principle of this Reporter Immobilization Assay (REIA) is the ligation of a reporter enzyme to a peptide carrying an affinity handle, which can be utilized for its isolation. The REIA system exhibits a high sensitivity with a linear range down to 1 μg/mL (55 nM), a variation coefficient of 6.5%, and can be performed cost-efficiently in 96-well microtiter plate format. The application of this assay allowed the characterization of a thiol transpeptidase sortase from S. aureus which is an important drug target and a biotechnological tool for ligation and modification of proteins. Thereby, yet-undetectable promiscuous activity of sortase could be detected, e.g., the acceptance of alanine as nucleophile. In addition, we were able to provide evidence that the REIA is suitable for high throughput screening of enzyme libraries using crude cellular extract with a throughput of 600 samples per hour. PMID:27182715

  2. Hyperpolarized NMR Probes for Biological Assays

    PubMed Central

    Meier, Sebastian; Jensen, Pernille R.; Karlsson, Magnus; Lerche, Mathilde H.

    2014-01-01

    During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized) molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments. PMID:24441771

  3. Gamma neutron assay method and apparatus

    DOEpatents

    Cole, J.D.; Aryaeinejad, R.; Greenwood, R.C.

    1995-01-03

    The gamma neutron assay technique is an alternative method to standard safeguards techniques for the identification and assaying of special nuclear materials in a field or laboratory environment, as a tool for dismantlement and destruction of nuclear weapons, and to determine the isotopic ratios for a blend-down program on uranium. It is capable of determining the isotopic ratios of fissionable material from the spontaneous or induced fission of a sample to within approximately 0.5%. This is based upon the prompt coincidence relationships that occur in the fission process and the proton conservation and quasi-conservation of nuclear mass (A) that exists between the two fission fragments. The system is used in both passive (without an external neutron source) and active (with an external neutron source) mode. The apparatus consists of an array of neutron and gamma-ray detectors electronically connected to determine coincident events. The method can also be used to assay radioactive waste which contains fissile material, even in the presence of a high background radiation field. 7 figures.

  4. Transformation Assay for Identification of Psychrotrophic Achromobacters

    PubMed Central

    Juni, Elliot; Heym, Gloria A.

    1980-01-01

    The finding that many psychrotrophic, gram-negative, nonmotile, oxidase-positive coccobacilli (achromobacters) are competent for genetic transformation made possible the development of a transformation assay that permits recognition of genetically related strains. It has been demonstrated that 109 independently isolated achromobacters are genetically related since deoxyribonucleic acid samples from all of these organisms were able to transform a single competent auxotrophic strain to prototrophy. Genetically interacting bacteria included strains that lacked one or more of the characteristics typical for most achromobacters. An oxidase-negative mutant of one of these strains reacted positively in the transformation assay, unlike other oxidase-negative bacteria. Achromobacters were derived from fish, poultry, irradiated foods, seawater, and other sources. One strain previously classified as Micrococcus cryophilus has been shown to be related to the achromobacters. Two achromobacters had an optimum growth temperature of 35°C and behaved as typical mesophiles. The moraxellae and Acinetobacter were shown to be unrelated to the achromobacters by using the transformation assay. The ready demonstration of genetic relatedness provides a new basis for taxonomic grouping of the psychrotrophic achromobacters. Images PMID:16345673

  5. Hyperpolarized NMR probes for biological assays.

    PubMed

    Meier, Sebastian; Jensen, Pernille R; Karlsson, Magnus; Lerche, Mathilde H

    2014-01-16

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

  6. The comet assay in human biomonitoring.

    PubMed

    Anderson, Diana; Dhawan, Alok; Laubenthal, Julian

    2013-01-01

    Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects, and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single- and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate towards the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.

  7. Gamma neutron assay method and apparatus

    DOEpatents

    Cole, Jerald D.; Aryaeinejad, Rahmat; Greenwood, Reginald C.

    1995-01-01

    The gamma neutron assay technique is an alternative method to standard safeguards techniques for the identification and assaying of special nuclear materials in a field or laboratory environment, as a tool for dismantlement and destruction of nuclear weapons, and to determine the isotopic ratios for a blend-down program on uranium. It is capable of determining the isotopic ratios of fissionable material from the spontaneous or induced fission of a sample to within approximately 0.5%. This is based upon the prompt coincidence relationships that occur in the fission process and the proton conservation and quasi-conservation of nuclear mass (A) that exists between the two fission fragments. The system is used in both passive (without an external neutron source and active (with an external neutron source) mode. The apparatus consists of an array of neutron and gamma-ray detectors electronically connected to determine coincident events. The method can also be used to assay radioactive waste which contains fissile material, even in the presence of a high background radiation field.

  8. Quantitative comparisons of in vitro assays for estrogenic activities.

    PubMed Central

    Fang, H; Tong, W; Perkins, R; Soto, A M; Prechtl, N V; Sheehan, D M

    2000-01-01

    Substances that may act as estrogens show a broad chemical structural diversity. To thoroughly address the question of possible adverse estrogenic effects, reliable methods are needed to detect and identify the chemicals of these diverse structural classes. We compared three assays--in vitro estrogen receptor competitive binding assays (ER binding assays), yeast-based reporter gene assays (yeast assays), and the MCF-7 cell proliferation assay (E-SCREEN assay)--to determine their quantitative agreement in identifying structurally diverse estrogens. We examined assay performance for relative sensitivity, detection of active/inactive chemicals, and estrogen/antiestrogen activities. In this examination, we combined individual data sets in a specific, quantitative data mining exercise. Data sets for at least 29 chemicals from five laboratories were analyzed pair-wise by X-Y plots. The ER binding assay was a good predictor for the other two assay results when the antiestrogens were excluded (r(2) is 0.78 for the yeast assays and 0.85 for the E-SCREEN assays). Additionally, the examination strongly suggests that biologic information that is not apparent from any of the individual assays can be discovered by quantitative pair-wise comparisons among assays. Antiestrogens are identified as outliers in the ER binding/yeast assay, while complete antagonists are identified in the ER binding and E-SCREEN assays. Furthermore, the presence of outliers may be explained by different mechanisms that induce an endocrine response, different impurities in different batches of chemicals, different species sensitivity, or limitations of the assay techniques. Although these assays involve different levels of biologic complexity, the major conclusion is that they generally provided consistent information in quantitatively determining estrogenic activity for the five data sets examined. The results should provide guidance for expanded data mining examinations and the selection of appropriate

  9. Prospects for cellular mutational assays in human populations

    SciTech Connect

    Mendelsohn, M.L.

    1984-06-29

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

  10. 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.

  11. Evaluation of a new fourth generation enzyme-linked immunosorbent assay, the LG HIV Ag-Ab Plus, with a combined HIV p24 antigen and anti-HIV-1/2/O screening test.

    PubMed

    Yeom, Joon-Sup; Jun, Gyo; Chang, Young; Sohn, Mi-Jin; Yoo, Seungbum; Kim, Eunkyung; Ryu, Seung-Ho; Kang, Hee-Jung; Kim, Young-A; Ahn, Sun-Young; Cha, Je-Eun; Youn, Sung-Tae; Park, Jae-Won

    2006-11-01

    The LG HIV Ag-Ab Plus, a new fourth generation diagnostic assay for HIV infection, was evaluated in comparison to the Enzygnost HIV Integral, an established fourth generation HIV assay. The LG assay showed 100% sensitivity with 109 samples with anti-HIV-1, anti-HIV-2 or anti-HIV-1 group O reactivity. It also detected correctly all 51 positives on three BBI performance panels, slightly outperforming the Enzygnost HIV Integral, which detected 50. The specificity of the LG HIV Ag-Ab Plus was 99.9% with 999 sera from healthy blood donors, which was slightly inferior to the performance of the Enzygnost HIV Integral, which had 100% specificity. The LG assay showed 100% specificity with 81 specimens with underlying diseases including hepatitis B, demonstrating a low risk of cross-reactivity with other infections. The reduction of the diagnostic window by the LG HIV Ag-Ab Plus, compared to a third generation HIV assay, was 6.3 days. The LG assay also showed sufficiently high intra-person and inter-person reproducibility. The overall performance of this new fourth generation HIV assay was adequate for screening and diagnosis of HIV infection.

  12. Nondestructive boxed transuranic (TRU) waste assay systems

    NASA Astrophysics Data System (ADS)

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.

    1999-01-01

    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  13. Nondestructive assay measurements of GNEP related materials

    SciTech Connect

    Santi, Peter A; Crooks, William J.; Geist, William H.; Gonzales, Robert; Helland, Carolyn A.; Jackson, Jay M.; Frame, Katherine C.; Martinez, Michael M.; Scherer, Caroylnn P.; Vo, Duc T.

    2008-06-12

    Because the reprocessing technologies that are currently being considered for the Global Nuclear Energy Partnership (GNEP) will keep various actinides commingled with plutonium at all times throughout the process, the resulting nuclear fuel that is intended for the Advanced Burner Reactor will present unique measurement challenges for the various Nondestructive Assay (NDA) techniques. In order to begin clarifying which types of materials and measurement scenarios that may exist within GNEP require the development of new measurement technologies, an initial series of measurements have been performed on materials with radiation properties that are similar to those being considered within GNEP.

  14. A universal nanoparticle cell secretion capture assay.

    PubMed

    Fitzgerald, Wendy; Grivel, Jean-Charles

    2013-02-01

    Secreted proteins play an important role in intercellular interactions, especially between cells of the immune system. Currently, there is no universal assay that allows a simple noninvasive identification and isolation of cells based on their secretion of various products. We have developed such a method. Our method is based on the targeting, to the cell surface, of heterofunctional nanoparticles coupled to a cell surface-specific antibody and to a secreted protein-specific antibody, which captures the secreted protein on the surface of the producing cell. Importantly, this method does not compromise cellviability and is compatible with further culture and expansion of the secreting cells.

  15. Assay of the Martian Regolith with Neutrons

    NASA Technical Reports Server (NTRS)

    Drake, Darrell M.; Reedy, R.; Jakowsky, B.; Clark, B.; Squyres, S.

    1998-01-01

    Different aspects of assaying Martian regolith using neutrons have been investigated. The epithermal portion of moderated neutrons spectra is dramatically effected by the presence of hydrogen (usually in the form of water). A simple analytic formula has been derived to describe the amplitude of this portion of the neutron spectrum as a function of water concentration. Several demonstration experiments have been performed and modeled with a Monte Carlo code. Results of these experiments generally agreed with the calculations to within 20%. In addition to He-3 detectors, lithium-glass scintillators and U-238 fission ion chambers were investigated to determine their applicability to space experiments.

  16. Test procedure for boxed waste assay system

    SciTech Connect

    Wachter, J.

    1994-12-07

    This document, prepared by Los Alamos National Laboratory`s NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system`s embedded operating and data reduction software.

  17. Protein kinase profiling assays: a technology review.

    PubMed

    Wang, Yuren; Ma, Haiching

    2015-11-01

    Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

  18. Lipase turbidimetric assay and acute pancreatitis.

    PubMed

    Orda, R; Orda, S; Baron, J; Wiznitzer, T

    1984-04-01

    The simplified turbidimetric assay for lipase activity was used for the differential diagnosis of acute pancreatitis. Serum lipase levels were found to be increased in a group of 17 patients in whom acute pancreatitis was clinically suspected and confirmed by a high ACCR and decreased uptake of the radionuclide in the pancreas scan. The lipase levels were within normal limits in a control group of 14 patients suffering from diseases other than acute pancreatitis. The turbidimetric test was helpful for rapid quantitative determination of serum lipase and thus for the early and accurate diagnosis of acute pancreatitis. PMID:6200277

  19. Assaying the Potency of Influenza Vaccines

    PubMed Central

    Minor, Philip D.

    2015-01-01

    The potency of vaccines must be determined to ensure that the appropriate dose is given. The manufacture and assessment of influenza vaccines are complicated by the continuously changing nature of the pathogen, which makes efficacy estimates difficult but also confounds attempts to produce a well-validated, consistent potency assay. Single radial diffusion has been used for decades and provides a relatively simple way to measure the amount of biologically active materials present in the vaccine. It requires reagents, which are updated on a regular, frequently yearly, basis and alternative methods continue to be sought. PMID:26344948

  20. Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus.

    PubMed Central

    Yolken, R H; Stopa, P J

    1979-01-01

    Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen. PMID:226564

  1. Clinical Assay Development Support - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The NCI’s Division of Cancer Treatment and Diagnosis and the Cancer Diagnosis Program announce a request for applications for the Clinical Assay Development Program (CADP) for investigators seeking clinical assay development and validation resources.

  2. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    ERIC Educational Resources Information Center

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

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

  3. Storage and Assay of Tritium in STAR

    SciTech Connect

    Glen R. Longhurst; Robert A. Anderl; Robert J. Pawelko

    2004-09-01

    The Safety and Tritium Applied Research (STAR) facility has recently been commissioned to investigate tritium-related safety questions for fusion and other technologies. The authorized inventory of tritium is 1.6 grams, the threshold quantity for nuclear facility classification. A key capability in successful operation of the STAR facility is the ability to receive, inventory, and dispense tritium to the various experiments underway there. The system central to that function is the Tritium Storage and Assay System (SAS). The SAS has four major functions: (1) receiving and holding tritium from shipping containers brought into the STAR facility, (2) assaying the amount of tritium in the SAS, (3) dispensing tritium to secondary beds or containers used for transferring it to the experimental systems in the STAR facility, and (4) purifying hydrogen isotopes from non-hydrogen species. To that may be added a fifth, optional function, isotopic separation of hydrogen isotopes using bed-to-bed transfer techniques. This paper documents the design and operation of the STAR SAS and the procedures used for tritium accountancy in the STAR facility.

  4. The clinical value of tissue factor assays.

    PubMed

    Francis, J L; Carvalho, M; Francis, D A

    1995-06-01

    Tissue factor (TF) is now considered to be the primary physiologic activator of the blood coagulation system. Coupled with recent advances in our understanding of the biochemistry of TF this has heightened interest in measuring aspects of TF activity in disease states. Expression of TF by blood monocytes in various diseases is an established trigger for intravascular coagulation and there is now a considerable body of experience with its measurement. This has considerable clinical potential although more widespread application awaits a consensus on the most appropriate methodologic approach to its measurement. TF can be detected in urine and may reflect the activation state of renal macrophages. Urinary TF is increased in cancer and could have diagnostic and prognostic value in a variety of malignant diseases. Finally, it is now possible to measure soluble TF in plasma. One such assay is commercially available and is technically simple to perform. The clinical value of such assays, however, must await better understanding of the source and function of soluble TF in plasma.

  5. Application of nondestructive assay techniques in Kazakstan

    SciTech Connect

    Sprinkle, J.K. Jr.; Butler, G.; Collins, M.

    1997-11-01

    As Kazakstan has transitioned from being part of the Soviet Union to a nonweapons state (Treaty of Nonproliferation of Nuclear Weapons [NPT] signatory) under International Atomic Energy Agency (IAEA) inspections, significant changes have been required. Some of these changes have occurred in nuclear material protection, control, and accounting at the four nuclear facility sites in the Republic of Kazakstan. Specifically, the Republic of Kazakstan has changed from relying primarily on a subset of physical protection methods to a graded safeguards approach using a balance of material control, material accounting, and physical protection. Once more intensive material control and accounting procedures and systems are in place, a necessary step is to supply the accounting systems with measured values of high quality. This need can be met with destructive and nondestructive methods. Material control systems can also use qualitative nondestructive assay information as input. This paper will discuss the nondestructive assay techniques and systems the US Department of Energy (DOE) is providing to Kazakstan under both DOE programs and the Cooperative Threat Reduction Act as part of the nuclear material control and accounting upgrades at four facilities in Kazakstan. 4 refs., 6 figs.

  6. The clinical value of tissue factor assays.

    PubMed

    Francis, J L; Carvalho, M; Francis, D A

    1995-06-01

    Tissue factor (TF) is now considered to be the primary physiologic activator of the blood coagulation system. Coupled with recent advances in our understanding of the biochemistry of TF this has heightened interest in measuring aspects of TF activity in disease states. Expression of TF by blood monocytes in various diseases is an established trigger for intravascular coagulation and there is now a considerable body of experience with its measurement. This has considerable clinical potential although more widespread application awaits a consensus on the most appropriate methodologic approach to its measurement. TF can be detected in urine and may reflect the activation state of renal macrophages. Urinary TF is increased in cancer and could have diagnostic and prognostic value in a variety of malignant diseases. Finally, it is now possible to measure soluble TF in plasma. One such assay is commercially available and is technically simple to perform. The clinical value of such assays, however, must await better understanding of the source and function of soluble TF in plasma. PMID:7647219

  7. Standardization of anti-DNA antibody assays.

    PubMed

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  8. Assaying Environmental Nickel Toxicity Using Model Nematodes

    PubMed Central

    Rudel, David; Douglas, Chandler D.; Huffnagle, Ian M.; Besser, John M.; Ingersoll, Christopher G.

    2013-01-01

    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species. PMID:24116204

  9. Rapid Multiple Immunoenzyme Assay of Mycotoxins

    PubMed Central

    Urusov, Alexandr E.; Zherdev, Anatoly V.; Petrakova, Alina V.; Sadykhov, Elchin G.; Koroleva, Olga V.; Dzantiev, Boris B.

    2015-01-01

    Mycotoxins are low molecular weight fungal metabolites that pose a threat as toxic contaminants of food products, thereby necessitating their effective monitoring and control. Microplate ELISA can be used for this purpose, but this method is characteristically time consuming, with a duration extending to several hours. This report proposes a variant of the ELISA method for the detection and quantification of three mycotoxins, ochratoxin A, aflatoxin B1 and zearalenone, in the kinetic regime. The main requirement for the proposed kinetic protocol was to provide a rapid method that combined sensitivity and accuracy. The use of biotin with an extended spacer together with a streptavidin–polyperoxidase conjugate provided high signal levels, despite these interactions occurring under non-equilibrium conditions. Duration of the individual mycotoxin assays was 20 min, whereas the analysis of all three mycotoxins in parallel reached a maximum duration of 25 min. Recovery of at least 95% mycotoxins in water-organic extracts was shown. The developed assays were successfully validated using poultry processing products and corn samples spiked with known quantities of mycotoxins. The detection limits for aflatoxin B1, ochratoxin A and zearalenone in these substances were 0.24, 1.2 and 3 ng/g, respectively. PMID:25633750

  10. Improved flow cytometer measurement of binding assays

    NASA Astrophysics Data System (ADS)

    Saunders, G. C.

    1984-05-01

    A method of measuring binding assays is carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also known quantity of smaller particles with a coating of binder reactant. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating.

  11. Preliminary assay reveals quality of Mars blend

    SciTech Connect

    Rhodes, A.K.

    1996-06-10

    Shell Oil Co. has assayed a preproduction well sample of Mars blend, from the Louisiana Gulf of Mexico. The assay shows the crude to have an API gravity of 31{degree} and a sulfur content of 2.0 wt%. Distillation data for the crude are shown in this paper. When partners Shell (71.5%) and BP (28.5%) begin commercial production in July, several producing zones will be tapped through a number of wells. Shell says the final sales quality of Mars Blend may vary from the qualities of this representative sample. The crude will be produced from a tension-leg platform (TLP) at a water depth of 2,940 ft (OGJ, Apr. 8, p. 23). The TLP is anchored in Mississippi Canyon block 807, about 130 miles southwest of New Orleans (map). Mars field will be the principal source of the crude initially. Shell says production will escalate to about 100,000 b/d crude and 110 MMcfd natural gas in 1997. Ultimate recovery is estimated to be 700 million BOE.

  12. Standardisation of the factor H autoantibody assay.

    PubMed

    Watson, Rachael; Lindner, Susanne; Bordereau, Pauline; Hunze, Eva-Maria; Tak, Federico; Ngo, Stéphanie; Zipfel, Peter F; Skerka, Christine; Dragon-Durey, Marie-Agnes; Marchbank, Kevin J

    2014-01-01

    The screening of all atypical haemolytic uraemic syndrome (aHUS) patients for factor H autoantibodies is best practice. However, there is no consensus assay for the reporting of factor H autoantibody titres. In this study, three European complement laboratories with expertise in the field of autoantibody testing address this by systematically evaluating several ELISA methods used for the detection of factor H autoantibodies. All methods tested adequately detect high titre samples. However, this study recommends the Paris method for the detection and reporting of factor H autoantibodies to be used when setting up a factor H autoantibody screen. The importance of individual sample background subtraction in these ELISA tests was established. The use of a relative or arbitrary unit index with a common positive and negative serum allowed for consistent comparison of findings from different test centres. Therefore, it is recommended that a standard arbitrary unit scale based on a titration curve from a common positive anti-serum be adopted to allow future establishment of the relative importance of particular titres of factor H autoantibodies in aHUS. Systematic assay for the presence of factor H autoantibodies in patients using the Paris method will provide the longitudinal analysis needed to fully establish the importance of factor H autoantibodies in disease. This will feed into additional research to clarify whether additional factors have a bearing on the phenotype/outcome of autoimmune aHUS. PMID:23891327

  13. Improved flow cytometer measurement of binding assays

    DOEpatents

    Saunders, G.C.

    1984-05-30

    The invention relates to a method of measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant. The binding reactant is the same as the binding reactant present in the sample. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number and strength of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating. (LEW)

  14. Survey of assay methods of antivenins

    PubMed Central

    Grasset, E.

    1957-01-01

    In view of the multiplicity of methods used at present for the preparation and assay of antivenins and as a first step towards the international standardization of antivenins, it seemed advisable to make a comparative study of the methods used in the institutes specializing in the production of these sera. With this end in view, the author circulated to the serologists of institutes concerned a detailed questionnaire on the assay methods used for the determination of the neutralization potency of the various types of antivenins prepared under their direction. The information supplied by these institutes is reproduced, in condensed form, in this report and is analysed by the author. The author emphasizes that the great variety in the constitution of venoms necessitates: (1) the use of monovalent standard sera against homologous “test” venoms of high activity and stability; and (2) the establishment, on a regional basis, of standard antivenins corresponding to groups of snakes characterized by venoms of common or closely related antigenic constitution. PMID:13413648

  15. Synthesis and Assays of Inhibitors of Methyltransferases.

    PubMed

    Cai, X-C; Kapilashrami, K; Luo, M

    2016-01-01

    Epigenetic regulation requires site-specific modification of the genome and is involved in multiple physiological processes and disease etiology. Methyltransferases, which catalyze the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to various substrates, are critical components of the epigenetic machinery. This group of enzymes can methylate diverse substrates including DNA, RNA, proteins, and small-molecule metabolites. Their dysregulation has also been implicated in multiple disease states such as cancer, neurological, and cardiovascular disorders. Developing potent and selective small-molecule inhibitors of methyltransferases is valuable not only for therapeutic intervention but also for investigating the roles of these enzymes in disease progression. In this chapter, we will discuss the strategies of designing and synthesizing methyltransferases inhibitors based on the SAM scaffold. Following the section of inhibitor design, we will briefly review representative assays that are available to evaluate the potency of these inhibitors along with a detailed description of the most commonly used radiometric assay. PMID:27423865

  16. Assaying environmental nickel toxicity using model nematodes

    USGS Publications Warehouse

    Rudel, David; Douglas, Chandler; Huffnagle, Ian; Besser, John M.; Ingersoll, Christopher G.

    2013-01-01

    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegansand P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  17. Two light, sweet Indonesian crudes assayed

    SciTech Connect

    Rhodes, A.K.

    1994-10-31

    Two crudes from Southeast Asia have been assayed. Belida, from the South Natuna Sea, is a light, sweet crude, with an API gravity of 45.1 [degree] and almost no sulfur (0.02 wt %). Hydra, from the Zone of Cooperation between Indonesia and Australia in the Timor Sea, has a gravity of 37.5 API and a sulfur content of 0.08 wt%. Belida operator Conoco Indonesia Ltd. began phase-two oil flow from a directional development well last October. Production from the field was 84,665 b/d on Oct. 21, 1993, and was expected to reach 90,000 b/d by the end of that month. The operator expected a peak of 100,00 b/d some time this year. The second crude, Hydra, is from the first well drilled in the Zone of Cooperation between Indonesia and Australia. Marathon Oil Co. spudded the first Hydra well in block ZOCA 9-11 in late 1992. As of early last year, five operating groups were expected to drill seven wells in second-half 1993. And a total of 26 wells has been committed for the Zone of Cooperation between 1995 and 1997, making the area a hotbed of exploration. Although the Journal has acquired no additional information on the Hydra program since that time, the assay may provided an idea of the quality of the crudes from that area.

  18. Assay optimization: a statistical design of experiments approach.

    PubMed

    Altekar, Maneesha; Homon, Carol A; Kashem, Mohammed A; Mason, Steven W; Nelson, Richard M; Patnaude, Lori A; Yingling, Jeffrey; Taylor, Paul B

    2007-03-01

    With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses. This article focuses on the use of statistically designed experiments in assay optimization.

  19. Performance Characteristics of Xpert Flu/RSV XC Assay.

    PubMed

    Popowitch, Elena B; Miller, Melissa B

    2015-08-01

    The Xpert Flu/RSV XC assay was compared to laboratory-developed tests (LDTs) (n = 207) and the Xpert Flu assay (n = 147) using archived nasopharyngeal swabs. The percentages of positive agreements with LDTs were 97.8% for influenza A, 97.2% for influenza B, and 89.3% for RSV. The sensitivity of influenza detection was improved with the Xpert Flu/RSV XC assay compared to the Xpert Flu assay.

  20. Development of an immunofluorescence focus assay for Ebola virus.

    PubMed Central

    Truant, A L; Regnery, R L; Kiley, M P

    1983-01-01

    A 48-h indirect immunofluorescence focus assay for the quantitation of Ebola virus was developed, utilizing HeLa-229 cell monolayers. The dose dependency and the sensitivity of this assay as compared with conventional assays are reported. This indirect immunofluorescence focus assay can be used as a rapid, quantitative test for the detection of Ebola virus, an agent from Africa known to cause hemorrhagic fever. Images PMID:6352735

  1. Hormone assays: some aspects that endocrinologists should know.

    PubMed

    Alfayate, Rocío; Mauri, Montserrat

    2008-02-01

    Since the pioneering works of Yalow and Berson that introduced radioimmunoassays (RIA), hormone assays have been developed gradually, with improvements in all aspects of their design, from immunoradiometric assays to automatization. Examples of this evolution are the thyrotropin (TSH) and parathyroid (PTH) assays. Despite the strong accuracy and reliability of currently used hormone assays, some limitations should be reviewed, such as interference by autoantibodies, heterophile antibodies or macroprolactin and the hook effect.

  2. Prediction of Non-Genotoxic Carcinogenicity Based on Genetic Profiles of Short Term Exposure Assays

    PubMed Central

    Pérez, Luis Orlando; González-José, Rolando; García, Pilar Peral

    2016-01-01

    Non-genotoxic carcinogens are substances that induce tumorigenesis by non-mutagenic mechanisms and long term rodent bioassays are required to identify them. Recent studies have shown that transcription profiling can be applied to develop early identifiers for long term phenotypes. In this study, we used rat liver expression profiles from the NTP (National Toxicology Program, Research Triangle Park, USA) DrugMatrix Database to construct a gene classifier that can distinguish between non-genotoxic carcinogens and other chemicals. The model was based on short term exposure assays (3 days) and the training was limited to oxidative stressors, peroxisome proliferators and hormone modulators. Validation of the predictor was performed on independent toxicogenomic data (TG-GATEs, Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System, Osaka, Japan). To build our model we performed Random Forests together with a recursive elimination algorithm (VarSelRF). Gene set enrichment analysis was employed for functional interpretation. A total of 770 microarrays comprising 96 different compounds were analyzed and a predictor of 54 genes was built. Prediction accuracy was 0.85 in the training set, 0.87 in the test set and increased with increasing concentration in the validation set: 0.6 at low dose, 0.7 at medium doses and 0.81 at high doses. Pathway analysis revealed gene prominence of cellular respiration, energy production and lipoprotein metabolism. The biggest target of toxicogenomics is accurately predict the toxicity of unknown drugs. In this analysis, we presented a classifier that can predict non-genotoxic carcinogenicity by using short term exposure assays. In this approach, dose level is critical when evaluating chemicals at early time points.

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

    PubMed

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

    2010-01-01

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

  4. A Luciferase-Based Quick Potency Assay to Predict Chondrogenic Differentiation.

    PubMed

    Oberbauer, Eleni; Steffenhagen, Carolin; Feichtinger, Georg; Hildner, Florian; Hacobian, Ara; Danzer, Martin; Gabriel, Christian; Redl, Heinz; Wolbank, Susanne

    2016-05-01

    Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs. PMID:27019357

  5. Characterization of a method for quantitating food consumption for mutation assays in Drosophila

    SciTech Connect

    Thompson, E.D.; Reeder, B.A.; Bruce, R.D. )

    1991-01-01

    Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period. Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.

  6. 21 CFR 866.2350 - Microbiological assay culture medium.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from...

  7. 21 CFR 866.2350 - Microbiological assay culture medium.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from...

  8. 21 CFR 866.2350 - Microbiological assay culture medium.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from...

  9. 21 CFR 866.2350 - Microbiological assay culture medium.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from...

  10. 21 CFR 866.2350 - Microbiological assay culture medium.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from...

  11. Multiplexing a high-throughput liability assay to leverage efficiencies.

    PubMed

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  12. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    DOEpatents

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

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

  13. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  14. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  15. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  16. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  17. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  18. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  19. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  20. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  1. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  3. A Different Approach to Validating Screening Assays for Developmental Toxicity

    EPA Science Inventory

    BACKGROUND: There continues to be many efforts around the world to develop assays that are shorter than the traditional embryofetal developmental toxicity assay, or use fewer or no mammals, or use less compound, or have all three attributes. Each assay developer needs to test th...

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

    DOEpatents

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

    2016-01-26

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

  5. Antibody secreting cell assay for influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  6. Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

    PubMed

    Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

    2015-02-21

    This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings. PMID:25474561

  7. Antiviral activities of 15 dengue NS2B-NS3 protease inhibitors using a human cell-based viral quantification assay.

    PubMed

    Chu, Justin Jang Hann; Lee, Regina Ching Hua; Ang, Melgious Jin Yan; Wang, Wei-Ling; Lim, Huichang Annie; Wee, John Liang Kuan; Joy, Joma; Hill, Jeffrey; Brian Chia, C S

    2015-06-01

    The dengue virus is a mosquito-borne pathogen responsible for an estimated 50-100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor's ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 μM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 μM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay.

  8. Accuracy of the endpoint assay for virus titration.

    PubMed

    Nielsen, L K; Smyth, G K; Greenfield, P F

    1992-01-01

    The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was found to be inconsistent. A superior estimator based on the maximum likelihood principle was developed. The results are discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.

  9. Antioxidant assay using genetically engineered bioluminescent Escherichia coli

    NASA Astrophysics Data System (ADS)

    Bartolome, Amelita; Macalino, Bernadette; Pastoral, Ian Lemuel; Sevilla, Fortunato, III

    2006-02-01

    A new antioxidant activity assay based on the reactive oxygen species (ROS)-inducible bacterial strain (E. coli DPD2511) is described. The strain harbors the plasmid pKatG::luxCDABE and responds to hydrogen peroxide treatment by increasing light emission at 490 nm. Antioxidant capacity is evaluated through the ability of an agent to inhibit the hydrogen peroxide-induced bioluminescence of E. coli DPD2511. Applicability of the developed assay in detecting levels of antioxidants in various aqueous plant extracts is demonstrated. The assay was validated against 2,2-diphenylpicrylhydrazyl (DPPH) assay, a known antioxidant assay.

  10. Universal fieldable assay with unassisted visual detection

    NASA Technical Reports Server (NTRS)

    Chelyapov, Nicolas (Inventor)

    2012-01-01

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

  11. Rapid Automated Sample Preparation for Biological Assays

    SciTech Connect

    Shusteff, M

    2011-03-04

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

  12. Nondestructive Assay Options for Spent Fuel Encapsulation

    SciTech Connect

    Tobin, Stephen J.; Jansson, Peter

    2014-10-02

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

  13. A miniaturized fibrinolytic assay for plasminogen activators

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Nachtwey, D. S.; Damron, K. L.

    1991-01-01

    This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.

  14. Analyte detection using an active assay

    DOEpatents

    Morozov, Victor; Bailey, Charles L.; Evanskey, Melissa R.

    2010-11-02

    Analytes using an active assay may be detected by introducing an analyte solution containing a plurality of analytes to a lacquered membrane. The lacquered membrane may be a membrane having at least one surface treated with a layer of polymers. The lacquered membrane may be semi-permeable to nonanalytes. The layer of polymers may include cross-linked polymers. A plurality of probe molecules may be arrayed and immobilized on the lacquered membrane. An external force may be applied to the analyte solution to move the analytes towards the lacquered membrane. Movement may cause some or all of the analytes to bind to the lacquered membrane. In cases where probe molecules are presented, some or all of the analytes may bind to probe molecules. The direction of the external force may be reversed to remove unbound or weakly bound analytes. Bound analytes may be detected using known detection types.

  15. Trace Technology for Assays of Novel Biomarkers

    PubMed Central

    2011-01-01

    The availability of an easily measurable specific marker for diagnosis of a disease is an important but not always reachable objective. Emergency area is an area where decisions must be taken as quick as possible, and sometimes it is not clear if the patient is well enough to be treated as an outpatient or must be hospitalized. During last few years many new biomarkers and techniques for their measurement have been developed, and surely some others will appear. The aim of this report was to present TRACE technology for specific assays such as: copeptin, proadrenomedullin and proatrial natriuretic peptide. Ongoing research is being done to decide in which diseases they might be useful and if they will be needed for diagnosis, prognosis or treatment monitoring. Results are not conclusive yet, but in the future some of these markers could be used in routine laboratory work if their utility is documented by new data.

  16. Mutagenesis assays of human amniotic fluid

    SciTech Connect

    Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.

    1985-01-01

    Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.

  17. Assays for investigating deSUMOylation enzymes.

    PubMed

    Madu, Ikenna G; Chen, Yuan

    2012-07-01

    Post-translational modifications by the SUMO (Small Ubiquitin-like MOdifier) family of proteins are recently discovered essential regulatory mechanisms. All SUMO proteins are synthesized as larger precursors that are matured by SUMO-specific proteases, known as SENPs, which remove several C-terminal amino acids of SUMO to expose the Gly-Gly motif. SENPs also remove SUMO modifications from target proteins, making this modification highly dynamic. At least six deSUMOylation enzymes, all of which are encoded by essential genes, have been identified in mammals. SENP1 has been shown to play an important role in the development of prostate cancer and in angiogenesis. This unit describes and discusses methods for characterizing the deSUMOylation enzymes. These assays enable the identification of inhibitors of these enzymes and investigation of their mechanism of inhibition in order to develop research tools and future therapeutics.

  18. Assay of DAGLα/β Activity.

    PubMed

    Bisogno, Tiziana

    2016-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Since 2-AG biosynthetic enzymes have been identified only recently, little information on methodological approaches for measuring DAGL activity is as yet available. Here, a highly sensitive radiometric assay to measure DAGL activity by using 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as the substrate is reported. All the steps needed to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [(14)C]-oleic acid via scintillation counting are described in detail. PMID:27245901

  19. Nondestructive assay (NDA) techniques and procedures

    SciTech Connect

    Not Available

    1994-05-01

    Report No. 4 is precursory to Report No. 5 {open_quotes}Determination of the Quantity and Locations of the Pu Currently Retained in the Cimarron Fuel Plant Systems{close_quotes} which will be presented upon completion of the decontamination of the Cimarron Plutonium Fuel Fabrication Facility. This report presents the Non-Destructive Assay (NDA) procedures which were developed and used by Sequoyah Fuels Corporation (successor to Kerr-McGee Nuclear Corporation) to measure equipment hold-up of plutonium materials for inventory purposes during operation of the plant. These procedures are also used to measure plutonium contamination on the equipment removed from the Material Balance Areas (MBA`s) during final decontamination. Report No. 5 will compare the measurements taken during this final decontamination period to previous inventory hold-up measurements, the date will be statistically analyzed, and a long-term assessment of the performance of the NDA equipment will be described.

  20. Low Background Assay Results for LZ

    NASA Astrophysics Data System (ADS)

    Oliver-Mallory, Kelsey; Thomas, Keenan; Lux-Zeplin Collaboration; Berkeley Low Background Facility Team

    2016-03-01

    The next generation dark matter experiment LUX-ZEPLIN (LZ) requires careful control of intrinsic radioactivity in all critical detector components in order to reach its unprecedented target sensitivity to Weakly Interacting Massive Particles (WIMPs): 2 ×10-48 cm2 at 50 GeV/c2. Appropriate material selection is essential to meeting this goal, and an extensive campaign of low background screening is currently being carried out using assay devices at the Sanford Underground Research Facility and the Boulby Underground Laboratory. We will present results from this work, including measurements for the Ti cryostat, PMT bases, PMT raw materials, PTFE, and other components. This work was partially supported by the U.S. Department of Energy (DOE) under Award Number DE-AC02-05CH11231, and is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1106400.

  1. High throughput assays for analyzing transcription factors.

    PubMed

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed. PMID:16834538

  2. Quality Assurance Specifications for Planetary Protection Assays

    NASA Astrophysics Data System (ADS)

    Baker, Amy

    As the European Space Agency planetary protection (PP) activities move forward to support the ExoMars and other planetary missions, it will become necessary to increase staffing of labo-ratories that provide analyses for these programs. Standardization of procedures, a comprehen-sive quality assurance program, and unilateral training of personnel will be necessary to ensure that the planetary protection goals and schedules are met. The PP Quality Assurance/Quality Control (QAQC) program is designed to regulate and monitor procedures performed by labora-tory personnel to ensure that all work meets data quality objectives through the assembly and launch process. Because personnel time is at a premium and sampling schedules are often de-pendent on engineering schedules, it is necessary to have flexible staffing to support all sampling requirements. The most productive approach to having a competent and flexible work force is to establish well defined laboratory procedures and training programs that clearly address the needs of the program and the work force. The quality assurance specification for planetary protection assays has to ensure that labora-tories and associated personnel can demonstrate the competence to perform assays according to the applicable standard AD4. Detailed subjects included in the presentation are as follows: • field and laboratory control criteria • data reporting • personnel training requirements and certification • laboratory audit criteria. Based upon RD2 for primary and secondary validation and RD3 for data quality objectives, the QAQC will provide traceable quality assurance safeguards by providing structured laboratory requirements for guidelines and oversight including training and technical updates, standardized documentation, standardized QA/QC checks, data review and data archiving.

  3. Improved internal control for molecular diagnosis assays.

    PubMed

    Vinayagamoorthy, T; Maryanski, Danielle; Vinayagamoorthy, Dilanthi; Hay, Katie S L; Yo, Jacob; Carter, Mark; Wiegel, Joseph

    2015-01-01

    The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1-4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. •The analyte and internal control have the same PCR and sequencing annealing sequences.•This method assures for little or no false negatives and false positives due to the method's design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.•This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay.

  4. Performance of MycAssay Aspergillus DNA real-time PCR assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples.

    PubMed

    Danylo, Alexis; Courtemanche, Chantal; Pelletier, René; Boudreault, Alexandre A

    2014-08-01

    Invasive aspergillosis (IA) is a major problem in the immunocompromised population, and its diagnosis is difficult due to the low sensitivity of available tests. Detection of Aspergillus nucleic acid by polymerase chain reaction (PCR) in serum samples is a promising diagnostic tool; however, use of multiple "in-house" methods precludes standardization. The first commercial PCR assay, MycAssay Aspergillus (Myconostica, Ltd), became available recently, and its performance in the diagnosis of IA was evaluated and compared with the galactomannan (GM) assay. Serum samples obtained from patients with hematological cancer were tested retrospectively with MycAssay Aspergillus PCR. Per-episode and per-test analyses were undertaken with 146 sera from 35 hematological patients. Sixteen patients had proven or probable IA and 19 had possible or no IA. In per-episode analysis, MycAssay Aspergillus had a sensitivity of 43.8% (95% confidence interval [CI], 19.8%-70.1%) and a specificity of 63.2% (95% CI, 38.4%-83.7%) for IA diagnosis. In per-test analyses, MycAssay Aspergillus had a lower specificity than the GM assay (83.3% vs. 93.1%, P = 0.04). The addition of PCR to routine clinical practice would have permitted the diagnosis of one additional probable IA in our cohort. Use of PCR instead of GM assay would have delayed the diagnosis in two cases. Aspergillus DNA detection by PCR with serum specimens using MycAssay showed a lower specificity than the GM assay and was associated with a low sensitivity for IA diagnosis. More studies are needed to determine the exact role of MycAssay in IA diagnosis in patients with hematological malignancy.

  5. Lateral-Flow Assay for Rapid Serodiagnosis of Human Leptospirosis

    PubMed Central

    Smits, Henk L.; Eapen, C. K.; Sugathan, Sheela; Kuriakose, Mariamma; Gasem, M. Hussein; Yersin, Claude; Sasaki, David; Pujianto, Bambang; Vestering, Marc; Abdoel, Theresia H.; Gussenhoven, George C.

    2001-01-01

    An assay device for the rapid detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera is presented. The sensitivity (85.8%) and specificity (93.6%) of the assay compared well (91.9% agreement) with those of an IgM enzyme-linked immunosorbent assay routinely used in the serodiagnosis of leptospirosis. The sensitivity of the assay varied with the stage of the disease. The assay uses stabilized components and is simply performed by the addition of serum and sample fluid to the sample well of the assay device. The assay is read after 10 min, and a positive result is obtained when staining of the test line is observed. PMID:11139212

  6. Membrane Assays to Characterize Interaction of Drugs with ABCB1.

    PubMed

    Fekete, Zsolt; Rajnai, Zsuzsanna; Nagy, Tünde; Jakab, Katalin Tauberné; Kurunczi, Anita; Gémes, Katalin; Herédi-Szabó, Krisztina; Fülöp, Ferenc; Tóth, Gábor K; Czerwinski, Maciej; Loewen, Greg; Krajcsi, Peter

    2015-12-01

    ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions. PMID:25926125

  7. Infectivity assay of bovine rotavirus: evaluation of plaque and end-point methods in comparison with immunofluorescent cell assay.

    PubMed

    Butchaiah, G

    1988-01-01

    Three different methods, namely plaque assay, immunofluorescent cell (IFC) count and end-point dilution (TCID50) were evaluated for quantitative infectivity assay of the cell culture adapted UK strain of bovine rotavirus in secondary calf kidney (CK) cells and BGM cell line. Plaque and IFC count techniques were found equally efficient for infectivity titration of bovine rotavirus. Addition of trypsin into maintenance medium enhanced the sensitivity of the TCID50 method. Both CK and BGM cells served as efficient assay cells for infectivity assay of bovine rotavirus by IFC count and TCID50 methods, whereas, for plaque assay, only CK cells were found suitable.

  8. Health care industries' perspective of viral load assays: the VERSANT HIV-1 RNA 3.0 assay.

    PubMed

    Elbeik, Tarek; Loftus, Richard A; Beringer, Scott

    2002-05-01

    This article will compare the VERSANT HIV-1 RNA 3.0 (bDNA 3.0) assay with other HIV-1 viral load assays, particularly the AMPLICOR HIV-1 MONITOR (Amplicor 1.5), the industry standard. The discussion will cover the history of viral load assay development and challenges to the field. It will finish with a description of the evolving markets for viral load assays in the developing world and the impact of variations in the different assays on their ability to reach those markets and patient populations.

  9. Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase activity in vitro.

    PubMed Central

    Juliette, L Y; Hyman, M R; Arp, D J

    1995-01-01

    We investigated the effects of bovine serum albumin (BSA) on both the assay and the stability of ammonia-oxidizing activity in cell extracts of Nitrosomonas europaea. Ammonia-dependent O2 uptake activity of freshly prepared extracts did not require BSA. However, a dependence on BSA developed in extracts within a short time. The role of BSA in the assay of ammonia-oxidizing activity apparently is to absorb endogenous free fatty acids which are present in the extracts, because (i) only proteins which bind fatty acids, e.g., BSA or beta-lactoglobulin, supported ammonia-oxidizing activity; (ii) exogenous palmitoleic acid completely inhibited ammonia-dependent O2 uptake activity; (iii) the inhibition caused by palmitoleic acid was reversed only by proteins which bind fatty acids; and (iv) the concentration of endogenous free palmitoleic acid increased during aging of cell extracts. Additionally, the presence of BSA (10 mg/ml) or CuCl2 (500 microM) stabilized ammonia-dependent O2 uptake activity for 2 to 3 days at 4 degrees C. The stabilizing effect of BSA or CuCl2 was apparently due to an inhibition of lipolysis, because both additives inhibited the increase in concentrations of free palmitoleic acid in aging extracts. Other additives which are known to modify lipase activity were also found to stabilize ammonia-oxidizing activity. These additives included HgCl2, lecithin, and phenylmethylsulfonyl fluoride. PMID:7665467

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

    PubMed

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

    2014-07-01

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

  11. The ADVIA Centaur infectious disease assays: a technical review.

    PubMed

    Dwyer, Robert

    2004-04-01

    Bayer Healthcare, Diagnostics Division, has developed several new several new assays for hepatitis B, hepatitis C and HIV 1/O/2 on the ADVIA Centaur Immunoassay system. The panel for detection of markers for hepatitis B infection includes assays for hepatitis B surface antigen (HBsAg) and HBsAg. Confirmatory, antibodies to hepatitis B surface antigen (anti-HBs), IgM and IgG antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). The hepatitis B panel is complemented by third generation assays for anti-HCV and anti-HIV that concomitantly detects HIV-1 (including group O) and HIV-2. The features of the ADVIA Centaur and the design of the assays combine to deliver state-of-the-art performance. Features, such as disposable sample pipette tips to prevent sample carryover, clot detection and management to verify sample addition, reagent integrity checks to assure accurate reagent addition, auto repeat capabilities and the capacity to maintain up to 30 assays onboard at 2-8 degrees C all contribute to the systems's utility for infectious disease testing. Furthermore, the flexibility of the ADVIA Centaur has allowed for selection of formats to provide optimal assay performance. The anti-HBs, anti-HBc Total and anti-HIV 1/O/2 assays are antigen-bridging assays. The anti-HBc IgM assay is a class-capture two step assay configured for detection of IgM anti-HBc antibodies during acute HBV infection. The anti-HCV assay is an indirect assay utilizing streptavidin-coated microparticles preformed with a combination of recombinant and synthetic peptide antigens from the NS2, NS4, NS5 and core regions of the HCV genome. The HBsAg assay is a sandwich assay with an incubation time of 28 minutes and the HBsAg Confirmatory assay is a fully automated neutralization assay. The availability of these assays on the ADVIA Centaur Immunoassay system enables laboratories to consolidate other immunoassays along with the infectious

  12. Simple Bulk Readout of Digital Nucleic Acid Quantification Assays.

    PubMed

    Morinishi, Leanna S; Blainey, Paul

    2015-01-01

    Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays. We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout. Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology. PMID:26436576

  13. A quantitative comet infection assay for influenza virus.

    PubMed

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

    2012-02-01

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

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

    PubMed

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

    2006-03-01

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

  15. Emergency gas-chromatographic assay of phenobarbital and phenytoin and liquid-chromatographic assay of theophylline.

    PubMed

    Shihabi, Z K

    1978-09-01

    Phenobarbital and phenytoin are extracted and concentrated in a single step, without solvent evaporation, followed by on-column methylation and gas-chromatographic quanitation. A similar extraction step for the assay of theophylline by high-pressure liquid chromatography is described. The extraction step can be completed in less than 2 min, the chromatographic step in less than 8 min. This extraction method yields clean chromatograms, avoids evaporating health-hazardous solvents, and is applicable to other drugs.

  16. Pi overlapping ring systems contained in a homogeneous assay: a novel homogeneous assay for antigens

    NASA Astrophysics Data System (ADS)

    Kidwell, David A.

    1993-05-01

    A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

  17. WASTE CRATE ASSAY SYSTEM (WCAS) : ASSAY SOLUTIONS FOR VERY LARGE REMOTE HANDLED CRATES

    SciTech Connect

    Menlove, Howard O.; Rinard, Phillip M.; Li, T. K.; Romero, M.; Hiruta, K.; Nasuno, S.

    2001-01-01

    An advanced passive neutron counter has been designed and fabricated to measure the plutonium content in large remote handled (RH) waste crates. The waste crate assay system (WCAS) was developed under an agreement between Los Alamos National Laboratory, Japan Nuclear Fuel Limited (JNFL), and BNFL Instruments Inc. (BII) to measure the plutonium content in the waste generated in the Rokkasho reprocessing facility. The primary goal of the design was to produce an assay system for large waste containers. The system also includes 200-L drum pallet assay capability. The measurements are based on neutron-time correlation counting of the passive neutron emissions from the 240Pu, and the plutonium isotopic ratios are used to calculate the total plutonium. The system is designed for both RH waste and low-activity plutonium waste. The system permits the measurement of the singles, doubles, and triples rates and the multiplicity mode analysis is used together with the 'add-a-source' method to correct for the matrix materials in the crates. In the multiplicity analysis, the efficiency for counting the neutrons emitted from the crate is directly calculated from the three measured rates. For improved detectability limits, advanced methods have been incorporated in the WCAS-A to reduce the cosmic-ray neutron backgrounds. These methods include statistical filters and truncation of high-multiplicity events. The paper describes the WCAS-A design, performance, and calibration.

  18. Adaptive measurement control for calorimetric assay

    SciTech Connect

    Glosup, J.G.; Axelrod, M.C.

    1994-10-01

    The performance of a calorimeter is usually evaluated by constructing a Shewhart control chart of its measurement errors for a collection of reference standards. However, Shewhart control charts were developed in a manufacturing setting where observations occur in batches. Additionally, the Shewhart control chart expects the variance of the charted variable to be known or at least well estimated from previous experimentation. For calorimetric assay, observations are collected singly in a time sequence with a (possibly) changing mean, and extensive experimentation to calculate the variance of the measurement errors is seldom feasible. These facts pose problems in constructing a control chart. In this paper, the authors propose using the mean squared successive difference to estimate the variance of measurement errors based solely on prior observations. This procedure reduces or eliminates estimation bias due to a changing mean. However, the use of this estimator requires an adjustment to the definition of the alarm and warning limits for the Shewhart control chart. The authors propose adjusted limits based on an approximate Student`s t-distribution for the measurement errors and discuss the limitations of this approximation. Suggestions for the practical implementation of this method are provided also.

  19. Fluorescence Polarization Assays in Small Molecule Screening

    PubMed Central

    Lea, Wendy A.; Simeonov, Anton

    2011-01-01

    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  20. Assay of carbon nanoparticles in liquids.

    PubMed

    Nawi, Yehuda; Sasson, Yoel; Dolgin, Bella

    2016-04-01

    The critical assay of carbon black concentration suffers from the lack of available methods, especially in-situ methods suitable for nanoparticles. We propose a useful tool for monitoring carbon nanoparticles concentration in liquids by means of RGB imaging, fluorescence and conductivity measurements. In this study carbon black particles of 25-75nm size were dispersed within two types of "green" liquids (1-butyl-3-methyl imidazolium based ionic liquids and glycerol) and the effect of carbon nanoparticles concentration on the liquids properties was measured. The conductivity of all the liquids increased with carbon concentration, while the slope of the curve was liquid dependent. The fluorescence intensity of ionic liquids decreased dramatically even when a small amount of carbon was added, while water-containing ionic liquids had a more moderate behavior. Glycerol has no native fluorescence, therefore, a known tracer present in soot (dibenzothiophene), having a characteristic fluorescence monitored by synchronous scan mode, was used. The carbon black effect on RGB imaging shows a linear dependence, while the red counts decreases with contamination. The proposed methods are simple and low-cost but nonetheless sensitive. PMID:26780588

  1. The platelet serotonin-release assay.

    PubMed

    Warkentin, Theodore E; Arnold, Donald M; Nazi, Ishac; Kelton, John G

    2015-06-01

    Few laboratory tests are as clinically useful as The platelet serotonin-release assay (SRA): a positive SRA in the appropriate clinical context is virtually diagnostic of heparin-induced thrombocytopenia (HIT), a life- and limb-threatening prothrombotic disorder caused by anti-platelet factor 4 (PF4)/heparin antibodies that activate platelets, thereby triggering serotonin-release. The SRA's performance characteristics include high sensitivity and specificity, although caveats include indeterminate reaction profiles (observed in ∼4% of test sera) and potential for false-positive reactions. As only a subset of anti-PF4/heparin antibodies detectable by enzyme-immunoassay (EIA) are additionally platelet-activating, the SRA has far greater diagnostic specificity than the EIA. However, requiring a positive EIA, either as an initial screening test or as an SRA adjunct, will reduce risk of a false-positive SRA (since a negative EIA in a patient with a "positive" SRA should prompt critical evaluation of the SRA reaction profile). The SRA also provides useful information on whether a HIT serum produces strong platelet activation even in the absence of heparin: such heparin-"independent" platelet activation is a marker of unusually severe HIT, including delayed-onset HIT and severe HIT complicated by consumptive coagulopathy with risk for microvascular thrombosis. PMID:25775976

  2. Virus Characterization by FFF-MALS Assay

    NASA Astrophysics Data System (ADS)

    Razinkov, Vladimer

    2009-03-01

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

  3. Establishment of indirect immunofluorescence assay for rotavirus.

    PubMed

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y

    2016-03-01

    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production.

  4. Rho family and Rap GTPase activation assays.

    PubMed

    Jennings, Richard T; Knaus, Ulla G

    2014-01-01

    The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione S-transferase (GST). Such domains bind preferentially to the GTP-bound form of the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.

  5. Assaying Visual Memory in the Desert Locust.

    PubMed

    Dillen, Senne; Chen, Ziwei; Broeck, Jozef Vanden

    2015-01-01

    The involvement of associative learning cues has been demonstrated in several stages of feeding and food selection. Short neuropeptide F (sNPF), an insect neuropeptide whose effects on feeding behavior have previously been well established, may be one of the factors bridging feeding and learning behavior. Recently, it was shown in Drosophila melanogaster that the targeted reduction of Drome-sNPF transcript levels significantly reduced sugar-rewarded olfactory memory. While Drosophila mainly relies on olfactory perception in its food searching behavior, locust foraging behavior is likely to be more visually orientated. Furthermore, a feeding-dependent regulation of Schgr-sNPF transcript levels has previously been observed in the optic lobes of the locust brain, suggesting a possible involvement in visual perception of food and visual associative memory in this insect species. In this study, we describe the development of a robust and reproducible assay allowing visual associative memory to be studied in the desert locust, Schistocerca gregaria. Furthermore, we performed an exploratory series of experiments, studying the role of Schgr-sNPF in this complex process. PMID:26463192

  6. Establishment of indirect immunofluorescence assay for rotavirus.

    PubMed

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y

    2016-03-01

    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production. PMID:26982471

  7. Evaluation of Consistency in Spheroid Invasion Assays

    PubMed Central

    Cisneros Castillo, Liliana R.; Oancea, Andrei-Dumitru; Stüllein, Christian; Régnier-Vigouroux, Anne

    2016-01-01

    Multicellular tumor spheroids embedded in a matrix represent invaluable tools to analyze cell invasion. Spheroid sizes and invasiveness are the main observables easily measurable to evaluate effects of biological or pharmaceutical manipulations on invasion. They largely account for these 3-D platforms variability, leading to flaws in data interpretation. No method has been established yet that characterizes this variability and guarantees a reliable use of 3-D platforms. Spheroid initial/end sizes and invasiveness were systematically analyzed and compared in spheroids of U87MG cells generated by three different methods and embedded at different times in a collagen matrix. A normality test was used to characterize size distribution. We introduced the linearity-over-yield analysis as a novel mathematical tool to assess end sizes and invasion reproducibility. We further provide a proof of concept by applying these tools to the analysis of a treatment known to be effective beforehand. We demonstrate that implementation of these statistical and mathematical tools warranted a confident quantification and interpretation of in 3-D conducted assays. We propose these tools could be incorporated in a guideline for generation and use of 3-D platforms. PMID:27334575

  8. Clickable enzyme-linked immunosorbent assay.

    PubMed

    Canalle, Luiz A; Vong, TuHa; Adams, P Hans H M; van Delft, Floris L; Raats, Jos M H; Chirivi, Renato G S; van Hest, Jan C M

    2011-10-10

    Click chemistry is explored as a potential cost-effective and selective immobilization method for the production of an enzyme-linked immunosorbent assay (ELISA). Coatings were formulated containing either a terminal alkyne or a bicyclo[6.1.0]non-4-yne (BCN) chemical handle, and a diagnostic peptide was subsequently immobilized onto these coatings by the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) or copper-free strain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC), respectively. The terminal alkyne-containing coating showed high background levels in subsequent ELISA's due to the copper catalyst used in the immobilization step. The BCN-containing coating, however, was successfully employed and presents a cost-effective alternative to existing (strept)avidin-biotin immobilization methods. This technology was illustrated with an ELISA used for the diagnosis of rheumatoid arthritis (RA) but could be easily applied to a wide range of diagnostic tests. PMID:21866934

  9. Introduction to Antigen and Antibody Assays.

    PubMed

    Day, Michael J

    2015-12-01

    Serological tests are used widely in veterinary practice; most often in the diagnosis of infectious disease. Such tests may be used to detect antigen from an infectious agent within a biological sample or to detect the presence of serum antibody specific for the pathogen as evidence of immunological exposure. These tests are all based on the fundamental principles of interaction between antigenic epitopes and antibodies of either the immunoglobulin (Ig) G, IgM, IgA, or IgE classes. The relative concentration of specific antibody within a sample is traditionally determined by calculation of the titer of antibody. With few exceptions, the primary interaction between an antigen and antibody in vitro cannot be visualized and so serological tests generally employ a secondary indicator system based on the use of a polyclonal antiserum or monoclonal antibody. A range of such tests has been developed, but many in veterinary medicine are based on the principle of the enzyme-linked immunosorbent assay, which is described in detail in this article. The interpretation of serological tests must be made carefully, taking into consideration the sensitivity and specificity of the test and the possible reasons for false-positive and false-negative outcomes. PMID:27154595

  10. Interpreting clinical assays for histone deacetylase inhibitors

    PubMed Central

    Martinet, Nadine; Bertrand, Philippe

    2011-01-01

    As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When “imprinted” early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient’s bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients. PMID:21625397

  11. Biochemical assays on plasminogen activators and hormones from kidney sources

    NASA Technical Reports Server (NTRS)

    Barlow, Grant H.; Lewis, Marian L.; Morrison, Dennis R.

    1988-01-01

    Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

  12. A comprehensive company database analysis of biological assay variability.

    PubMed

    Kramer, Christian; Dahl, Göran; Tyrchan, Christian; Ulander, Johan

    2016-08-01

    Analysis of data from various compounds measured in diverse biological assays is a central part of drug discovery research projects. However, no systematic overview of the variability in biological assays has been published and judgments on assay quality and robustness of data are often based on personal belief and experience within the drug discovery community. To address this we performed a reproducibility analysis of all biological assays at AstraZeneca between 2005 and 2014. We found an average experimental uncertainty of less than a twofold difference and no technologies or assay types had higher variability than others. This work suggests that robust data can be obtained from the most commonly applied biological assays.

  13. Disagreement between Human Papillomavirus Assays: An Unexpected Challenge for the Choice of an Assay in Primary Cervical Screening

    PubMed Central

    Ejegod, Ditte Møller; Rygaard, Carsten; Lynge, Elsebeth; Bonde, Jesper

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41% tested positive on all four. Agreement was lower in women aged ≥30 years (30%, vs. 49% at <30 years), in primary screening samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30–65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings of considerable disagreement between human papillomavirus assays. This suggested that the extent of disagreement in primary screening is neither population- nor storage media-specific, leaving assay design differences as the most probable cause. The substantially different selection of women testing positive on the various human papillomavirus assays represents an unexpected challenge for the choice of an assay in primary cervical screening, and for follow up of in particular HPV positive/cytology normal women. PMID:24466262

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

    SciTech Connect

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

    2015-12-01

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

  15. Assay of nitrogenase activity in intact plant systems.

    PubMed

    Jain, M K; Vlassak, K

    1975-01-01

    Nitrogenase activity was assayed in intact system of Cichorium intybus, a non-leguminous commercially cultivated crop, Dahlia pinnata and Helianthus annus, and Taraxacum officinale, a common weed plant. The assay was made in fabricated cylinders which could accomodate pot with plants. In such kind of assay along with rhizosphere microflora, the nitrogen fixed by phyllosphere nitrogen fixing microflora could also be accounted, which otherwise was difficult to be accounted for. PMID:1211718

  16. In vitro radiolabel uptake viability assay for Onchocerca microfilariae

    SciTech Connect

    Callahan, H.L.; Wakeman, J.M.; Crouch, R.K.; James, E.R.

    1989-02-01

    A radiolabel uptake viability assay for Onchocerca cervicalis using (/sup 3/H)2-deoxy-D-glucose in Hanks' balanced salt solution, pH 7.5, at 30 C is described and compared to the traditional visual motility assay. A correlation of r = 0.92 between the assays was found, with the radiolabel uptake method apparently a more sensitive indicator of microfilarial viability.

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

    DOEpatents

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

    2012-06-05

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

  18. Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards.

    PubMed

    Lionel, Faivre; Lucie, Lecouflet; Wang-Qing, Liu; Isabelle, Khadher; Camille, Lahaie; Michel, Vidal; Sabine, Legouvello; Jean-Louis, Beaumont; Philippe, Bierling; Hélène, Rouard; Brigitte, Birebent

    2014-09-01

    Background: For the last 40 years, the technique of extracorporeal photopheresis has constantly developed. Among irradiation systems, those called 'off-line' allow the validation of the quality of the cell therapy product. The inhibition of the proliferation of lymphocytes after UVA irradiation is usually verified by the tritiated thymidine assay as in vitro proliferation assay. The document presented here describes the results obtained while performing the setting up of an alternative proliferation assay using flow cytometry according to ISO 15189:2007 Standard. Methods: Cells samples taken before and after UVA irradiation were labeled with CFSE and then cultured with phytohemagglutinin. After 3 days, an analysis of the CFSE staining was realized by flow cytometry. In order to validate the shift in the method used according to Standard, the following tests were performed: 1) comparison with the reference method, 2) robustness test, 3) reagents stability. Results: Comparison method demonstrated that the sensitivity of the CFSE test is 100%, the specificity is 89% and the concordance is almost complete. The CFSE test is robust regarding parameters like cell concentration or PHA concentration. PHA and CFSE are stable for 6 months and one year, respectively. Conclusion: Validation of this alternative test, according to the ISO 15189:2007 Standard, has demonstrated good concordance with reference method. The results of the robustness and stability of reagents are appropriate for its routine use. Thus, the benefits of alternative technique make it a wise choice for the quality control of ECP in a cell therapy laboratory. © 2014 Clinical Cytometry Society.

  19. Cost of gentamicin assays carried out by microbiology laboratories.

    PubMed Central

    Vacani, P F; Malek, M M; Davey, P G

    1993-01-01

    AIMS--To assess the current range of prices charged for gentamicin assays in United Kingdom laboratories; and to examine the laboratories' likely response to increases or decreases in the demand for the service. METHODS--A postal survey of the 420 members of the Association of Medical Microbiologists was used to establish the range of prices charged for aminoglycoside assays. Additionally, eight private institutions were contacted to determine what the private sector was charging for aminoglycoside assays. Reagent costs in the NHS laboratories were calculated by dividing the total cost of all aminoglycoside assay kits by the number of samples analysed. RESULTS--The NHS and the private institutions both showed a wide price variation. Prices charged to an in-hospital requester for a peak and trough assay ranged from 5.00 pounds to 68.20 pounds (n = 44), and to an external private hospital, under a bulk service contract, from 5.00 pounds to 96.00 pounds (n = 47). Prices charged by private laboratories ranged from 49.00 pounds to 84.00 pounds (n = 8). There was a log linear correlation in the NHS laboratories between the reagent costs per assay and the number of assays performed per year, and most laboratories thought that their price per assay would be sensitive to increases or decreases in demand. Laboratories which had purchased their assay machines had lower reagent costs per assay but higher repair and maintenance costs. Overall, number of assays performed and method of payment for assay machinery only accounted for 44.8% of the observed variation in assay kit costs. CONCLUSIONS--The price range for gentamicin assays in the United Kingdom is wide and is only partially explained by the number of assays performed. Most laboratories believe that they would experience a reduction in unit cost as output increases. The currently offered range of prices is, in part, due to variation in the laboratories' approach to costing the service provided and some laboratories

  20. The value of translational biomarkers to phenotypic assays

    PubMed Central

    Swinney, David C.

    2014-01-01

    Phenotypic assays are tools essential for drug discovery. Phenotypic assays have different types of endpoints depending on the goals; (1) empirical endpoints for basic research to understand the underlying biology that will lead to identification of translation biomarkers, (2) empirical endpoints to identify undesired effects related to toxicity of drug candidates, and (3) knowledge-based endpoints (biomarkers) for drug discovery which ideally are translational biomarkers that will be used to identify new drug candidates and their corresponding molecular mechanisms of action. The value of phenotypic assays is increased through effective alignment of phenotypic assay endpoints with the objectives of the relevant stage in the drug discovery and development cycle. PMID:25076910

  1. Electronic microarray assays for avian influenza and Newcastle disease virus.

    PubMed

    Lung, Oliver; Beeston, Anne; Ohene-Adjei, Samuel; Pasick, John; Hodko, Dalibor; Hughes, Kimberley Burton; Furukawa-Stoffer, Tara; Fisher, Mathew; Deregt, Dirk

    2012-11-01

    Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.

  2. Detection of radiation-induced apoptosis using the comet assay.

    PubMed

    Wada, Seiichi; Khoa, Tran Van; Kobayashi, Yasuhiko; Funayama, Tomoo; Yamamoto, Kazuo; Natsuhori, Masahiro; Ito, Nobuhiko

    2003-11-01

    The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. PMID:14665742

  3. Assaying Wnt5A-mediated invasion in melanoma cells.

    PubMed

    O'Connell, Michael P; French, Amanda D; Leotlela, Poloko D; Weeraratna, Ashani T

    2008-01-01

    Wnt5A has been implicated in melanoma metastasis, and the progression of other cancers including pancreatic, gastric, prostate, and lung cancers. Assays to test motility and invasion include both in vivo assays and in vitro assays. The in vivo assays include the use of tail vein or footpad injections of metastatic cells, and are often laborious and expensive. In vitro invasion assays provide quick readouts that can help to establish conditions that either activate or inhibit melanoma cell motility, and to assess whether the conditions in question are worth translating into an in vivo model. Here we describe two standard methods for assaying motility and invasion in vitro including wound healing assays and Matrigel invasion assays (Boyden chamber assays). In addition, we and several other laboratories have previously shown that melanoma cells require matrix metalloproteinase (MMP)-2 for their invasion, and have recently shown that Wnt5A treatment can increase the levels of this enzyme in melanoma cells, as demonstrated by gelatin zymography. The use of these techniques can help to assess the migratory capacity of melanoma cells in response to Wnt treatment.

  4. A multi-detection assay for malaria transmitting mosquitoes.

    PubMed

    Lee, Yoosook; Weakley, Allison M; Nieman, Catelyn C; Malvick, Julia; Lanzaro, Gregory C

    2015-01-01

    The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays. PMID:25867057

  5. Robust versatile tyrosine kinase assay for HTS in drug discovery

    NASA Astrophysics Data System (ADS)

    Deshpande, Sudhir S.; Mineyev, I.; Owicki, John C.

    1999-04-01

    A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

  6. Scintillation proximity assay using polymeric membranes

    SciTech Connect

    Mansfield, R.K.

    1992-01-01

    Liquid scintillation counting (LSC) is typically used to quantify electron emitting isotopes. In LSC, radioactive samples are dissolved in an organic fluor solution (scintillation cocktail) to ensure that the label is close enough to the fluor molecules to be detected. Although efficient, scintillation cocktail is neither specific or selective for samples labeled with the same radioisotope. Scintillation cocktail is flammable posing significant health risks to the user and is expensive to purchase and discard. Scintillation Proximity Assay (SPA) is a radioanalytical technique where only those radiochemical entities (RCE's) bound to fluor containing matrices are detected. Only bound RCE's are in close enough proximity the entrapped fluor molecules to induce scintillations. Unbound radioligands are too far removed from the fluor molecules to be detected. The research in this dissertation focused on the development and evaluation of fluor-containing membranes (scintillation proximity membranes, SP membranes) to be used for specific radioanalytical techniques without using scintillation cocktail. Polysulfone and PVC SP membranes prepared in our laboratory were investigated for radioimmunossay (RIA) where only bound radioligand is detected, thereby eliminating the separation step impeding the automation of RIA. These SP membranes performed RIA where the results were nearly identical to commercial SP microbeads. SP membranes functionalized with quaternary ammonium hydroxide moieties were able to trap and quantify [sup 14]CO[sub 2] without using liquid scintillation cocktail. RCE's bound in the pore structure of SP membranes are intimate with the entrapped fluor providing the geometry needed for high detection efficiencies. Absorbent SP membranes were used in radiation surveys and were shown to be as effective as conventional survey techniques using filter paper and scintillation cocktail.

  7. Inferring Protein Associations Using Protein Pulldown Assays

    SciTech Connect

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  8. Comparative investigation of various cellulase assay procedures

    SciTech Connect

    Canevascini, G.; Gattlen, C.

    1981-07-01

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

  9. Neutron Generators for Spent Fuel Assay

    SciTech Connect

    Ludewigt, Bernhard A

    2010-12-30

    The Next Generation Safeguards Initiative (NGSI) of the U.S. DOE has initiated a multi-lab/university collaboration to quantify the plutonium (Pu) mass in, and detect the diversion of pins from, spent nuclear fuel (SNF) assemblies with non-destructive assay (NDA). The 14 NDA techniques being studied include several that require an external neutron source: Delayed Neutrons (DN), Differential Die-Away (DDA), Delayed Gammas (DG), and Lead Slowing-Down Spectroscopy (LSDS). This report provides a survey of currently available neutron sources and their underlying technology that may be suitable for NDA of SNF assemblies. The neutron sources considered here fall into two broad categories. The term 'neutron generator' is commonly used for sealed devices that operate at relatively low acceleration voltages of less than 150 kV. Systems that employ an acceleration structure to produce ion beam energies from hundreds of keV to several MeV, and that are pumped down to vacuum during operation, rather than being sealed units, are usually referred to as 'accelerator-driven neutron sources.' Currently available neutron sources and future options are evaluated within the parameter space of the neutron generator/source requirements as currently understood and summarized in section 2. Applicable neutron source technologies are described in section 3. Commercially available neutron generators and other source options that could be made available in the near future with some further development and customization are discussed in sections 4 and 5, respectively. The pros and cons of the various options and possible ways forward are discussed in section 6. Selection of the best approach must take a number of parameters into account including cost, size, lifetime, and power consumption, as well as neutron flux, neutron energy spectrum, and pulse structure that satisfy the requirements of the NDA instrument to be built.

  10. Modeling error in experimental assays using the bootstrap principle: understanding discrepancies between assays using different dispensing technologies.

    PubMed

    Hanson, Sonya M; Ekins, Sean; Chodera, John D

    2015-12-01

    All experimental assay data contains error, but the magnitude, type, and primary origin of this error is often not obvious. Here, we describe a simple set of assay modeling techniques based on the bootstrap principle that allow sources of error and bias to be simulated and propagated into assay results. We demonstrate how deceptively simple operations--such as the creation of a dilution series with a robotic liquid handler--can significantly amplify imprecision and even contribute substantially to bias. To illustrate these techniques, we review an example of how the choice of dispensing technology can impact assay measurements, and show how large contributions to discrepancies between assays can be easily understood and potentially corrected for. These simple modeling techniques--illustrated with an accompanying IPython notebook--can allow modelers to understand the expected error and bias in experimental datasets, and even help experimentalists design assays to more effectively reach accuracy and imprecision goals.

  11. Recovery of spiked troponin I in four routine assays

    PubMed Central

    Loh, Tze Ping; Lim, Xiong Chang; Kieu, Karize; Sajiir, Haressh; Neo, Siew Fong; Cheng, Wan Ling; Sethi, Sunil Kumar

    2016-01-01

    Introduction This study aimed to examine the recovery of spiked human cardiac troponin I (cTnI) results measured by four routine assays, and investigate possible interference from microclots. Materials and methods 457 consecutive samples with cTnI concentration below limit of quantitation (12 ng/L), declared by the Vitros TnI ES assay (reference assay), were measured on Beckman Coulter Accu TnI+3, Siemens TnI-Ultra and Roche TnI STAT assays. These samples were enriched with native full-length cTnI to a concentration of 100 ng/L and retested. A post-spiking result that exceeded the critical difference at a predefined probability of 0.0005 of the target concentration (the median post-spiking result for each individual assay) was considered as outlier. To determine whether microclots were a significant cause of critically discrepant outlier results, a separate 50 samples were centrifuged twice between two post-spiking measurements using the Vitros TnI ES assay. Results The median recovery of the enriched cTnI was highest with the Roche assay (271 ng/L) and lowest with the Vitros assay (29 ng/L). The Vitros assay had the highest percentage of results that exceeded the critical difference (49%), followed by the Siemens (38%), Roche (18%) and Beckman Coulter (7%) assays. None of the 50 additional samples produced a critically lower cTnI result after re-centrifugation. Conclusions Our findings underscored the variability of cTnI assays in measuring native cTnI. The lack of cTnI results that became significantly lower after re-centrifugation suggested that microclots are unlikely to be a major cause of the outlier results. PMID:27346968

  12. New flow cytometric assays for monitoring cell-mediated cytotoxicity.

    PubMed

    Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

    2010-06-01

    The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the (51)Cr-release assay and IFN-gamma ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the (51)Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

  13. Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays.

    PubMed

    Zhang, Wei; Zhu, Min; Wang, Feng; Cao, Danhui; Ruan, Jennifer Jin; Su, Weike; Ruan, Benfang Helen

    2016-09-15

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS). PMID:27387057

  14. A Simple Endpoint Assay for Starch-Degrading Enzymes.

    ERIC Educational Resources Information Center

    Kroen, William K.

    1998-01-01

    Since many of the important energy-transferring pathways involve synthesis or degradation of biological macromolecules, observations of the enzymes responsible for starch breakdown provide a useful case study. Provides a short, one-step assay for the enzymes amylase and amyloglucosidase. Topics covered include goals, preparation, assay procedure,…

  15. Overview of mutation assays in transgenic mice for routine testing.

    PubMed

    Gorelick, N J

    1995-01-01

    There is scientific and regulatory interest in using mutation assays in transgenic mice in safety assessments for new chemicals and drugs. Currently these assays are in the process of being validated, and protocols for routine testing are being defined. Some of the issues and results to date with regard to assay validation include reproducibility of the assay results (they are qualitatively reproducible), relevance of the test system (the transgene closely approximates an endogenous mammalian gene as a mutational target for the limited number of compounds tested), and the predictivity of the assay for heritable effects (unknown at this time) or carcinogenicity (the assays show good positive predictivity for carcinogenicity; the negative predictivity of the assay requires further investigation). Definition of appropriate study protocols for routine testing requires that applicable statistical methods are available and that the experimental parameters that affect the detection of mutations are known. Progress made in identifying these parameters is discussed. A proposal is made for the custom design of routine safety studies, which is based on the anticipated use of each individual test agent. A working group has been formed to conduct some of the studies still required for validation of these assays.

  16. Student-Designed Enzyme-Linked Metabolite Assay Kits

    ERIC Educational Resources Information Center

    Hancock, D.; Johnston, J.; Dimauro, J.; Denyer, G.

    2004-01-01

    The extensive use of commercial kits in molecular biology and biochemistry has prompted us to design a series of practical sessions to help students become familiar with the uses and limitations of pre-packaged assay systems. To facilitate an understanding of these assay systems and to promote reflection on their appropriate use, students…

  17. AN OVERVIEW OF VIRUS EXTRACTION AND ASSAY METHODS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enteric viruses, particularly norovirus and hepatitis A virus, are major contaminants of molluscan shellfish and can cause illness. A host of procedures have been developed for the extraction of viruse from shellfish and for their assay. Some extraction and assay methods focused on detection of vacc...

  18. Infectious Viral Quantification of Chikungunya Virus-Virus Plaque Assay.

    PubMed

    Kaur, Parveen; Lee, Regina Ching Hua; Chu, Justin Jang Hann

    2016-01-01

    The plaque assay is an essential method for quantification of infectious virus titer. Cells infected with virus particles are overlaid with a viscous substrate. A suitable incubation period results in the formation of plaques, which can be fixed and stained for visualization. Here, we describe a method for measuring Chikungunya virus (CHIKV) titers via virus plaque assays.

  19. A colorimetric assay for viral agents that produce cytopathic effects.

    PubMed

    Heldt, Caryn L; Hernandez, Raquel; Mudiganti, Usharani; Gurgel, Patrick V; Brown, Dennis T; Carbonell, Ruben G

    2006-07-01

    Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.

  20. Methylcellulose media for plaque assay of murine leukemia virus.

    PubMed

    Watanabe, T; Horikawa, Y; Sato, K; Saito, H

    1982-09-01

    When ecotropic murine leukemia virus was assayed by a methylcellulose-XC cell procedure, plaque titers showed less test-to-test variation, more uniform dose-response curves, and larger plaque sizes, as compared with results of the conventional liquid overlay-XC cell test system. This assay therefore seems to be reliable and useful for the titration of ecotropic murine leukemia virus.

  1. Validation of a Fish Short-term Reproduction Assay

    EPA Science Inventory

    The Fish Short-term Reproduction Assay is an in vivo assay conducted with fathead minnows and is designed to detect changes in spawning, gross morphology, histopathology, and specific biochemical endpoints that reflect disturbances in the hypothalamic-pituitary-gonadal (HPG) axis...

  2. The Hornworm Assay: Useful in Mathematically-Based Biological Investigations

    ERIC Educational Resources Information Center

    Rice, Stanley A.; Griffin, Jennifer R.

    2004-01-01

    Hornworms are good assay organisms for leaf toxins, and can be raised on an artificial medium ("chow"), consisting of corn meal, soy flour, dry milk, yeast and other additives and preservatives. The hornworm assay is less useful in ecological and toxicological research, but is very useful in learning about experimental design and hypothesis…

  3. Nanochannel-based electrochemical assay for transglutaminase activity.

    PubMed

    Fernández, Iñigo; Sánchez, Alfredo; Díez, Paula; Martínez-Ruiz, Paloma; Di Pierro, Prospero; Porta, Raffaele; Villalonga, Reynaldo; Pingarrón, José M

    2014-11-11

    A novel electrochemical assay to quantify transglutaminase activity is reported. The assay is based on the enzyme-controlled diffusion of Fe(CN)6(3-/4-) through amino-functionalized nanochannels of a mesoporous silica thin film on a Au surface in the presence of N-benzyloxycarbonyl-L-glutaminylglycine.

  4. A phytoplankton growth assay for routine in situ environmental assessments.

    PubMed

    Moreira-Santos, Matilde; Soares, Amadeu M V M; Ribeiro, Rui

    2004-06-01

    This study proposes an ecologically relevant and cost-effective phytoplankton growth assay for routine in situ toxicity assessments. Assay procedures were developed applying, to the extent possible, the rationale behind the design of standard algal assays. Chlorella vulgaris was selected as test species because it grows well immobilized in alginate beads and has a wide geographic distribution. The performance of the assay in a freshwater system impacted by acid mine drainage demonstrated the suitability of assay chambers and procedures. The test system, made of inexpensive materials, allowed the rapid and easy deployment of the assay. The deployment of extra chambers at reference sites provided the ability to periodically check whether algal growth had already reached recommended growth criteria (time at which the assay should end). By deploying chambers filled with control medium at all sites, temperature was identified to explain 95% of the variation in growth. By using an artificial nutrient source shown capable of promoting algal growth according to recommended standards, toxicity from the mine effluent was distinguish from in situ nutrient limitation effects. The very good agreement (r2 = 90%) between mean in situ growth rates estimated by microscopy and by spectrophotometry and their similar coefficient of variation showed the latter to be a suitable straightforward methodology for assay endpoint estimation.

  5. Engineering luciferase enzymes and substrates for novel assay capabilities

    NASA Astrophysics Data System (ADS)

    Wood, Keith V.

    2004-06-01

    In the development of HTS as a central paradigm of drug discovery, fluorescent reporter molecules have generally been adopted as the favored signal transducer. Nevertheless, luminescence has maintained a prominent position among certain methodologies, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a broader range of applications due to their sensitivity, extensive linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by development several new assay designs for diverse targets such as kinases, cytochrome p450's, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for drug discovery, highlighting new detection capabilities brought about by engineering luciferase enzymes and substrates. In reporter gene applications, modified luciferases have provided greatly improved expression efficiency in mammalian cells, improved responsiveness to changes of transcriptional rate, and increased the magnitude of the reporter response. Highly stabilized luciferase mutants have enabled new assays strategies for high-throughput screening based on detection of ATP and luciferin. Assays based on ATP support rapid analysis of cell metabolism and enzymatic processes coupled to ATP hydrolysis. Although luciferin is found natively only in luminous beetles, coupled assays have been designed using modified forms of luciferin requiring the action of second enzyme to yield luminescence. Due to the very low inherent background and protection of the photon-emitter afforded by the enzyme, bioluminescent assays often outperform the analogous fluorescent assays for analyses performed in multiwell plates.

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

    EPA Science Inventory

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

  7. Microfluidic assay without blocking for rapid HIV screening and confirmation.

    PubMed

    Song, Lusheng; Zhang, Yi; Wang, Wenjun; Ma, Liying; Liu, Yong; Hao, Yanlin; Shao, Yiming; Zhang, Wei; Jiang, Xingyu

    2012-08-01

    The essential step for HIV spreading limitation is the screening tests. However, there are multiple disadvantages in current screening assays which need further confirmation test. Herein we developed a rapid HIV assay combining screening and confirmation test by using the microfluidic network assay. Meanwhile, the assay is accelerated by bypassing the step of blocking. We call this method as microfluidic assay without blocking (MAWB). Both the limit of detection and reagent incubation time of MAWB are determined by screening of one model protein pair: ovalbumin and its antibody. The assay time is accelerated about 25% while the limit of detection (LOD) is well kept. Formatting the method in for both HIV screening (testing 8 HIV-related samples) and confirmation (assaying 6 kinds of HIV antibodies of each sample) within 30 min was successful. Fast HIV screening and confirmation of 20 plasma samples were also demonstrated by this method. MAWB improved the assay speed while keeping the LOD of conventional ELISA. Meanwhile, both the accuracy and throughput of MAWB were well improved, which made it an excellent candidate for a quick HIV test for both screening and confirmation. Methods like this one will find wide applications in clinical diagnosis and biochemical analysis based on the interactions between pairs of molecules. PMID:22374476

  8. Microneutralization assay for swine influenza virus in swine serum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The microneutralization (MN) assay is a modification of the serum virus neutralization assay and is a serological test to detect the presence of functional systemic antibodies that prevent infectivity of virus. When infectious virus is mixed with serum antibody, the virus infectivity can be "neutral...

  9. AN OVERVIEW OF ENTERIC VIRUS EXTRACTION AND ASSAY METHODS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enteric viruses, particularly norovirus and hepatitis A virus, are major contaminants of molluscan shellfish, leading to outbreaks of viral illness. A host of procedures have been developed for the extraction and assay of viruses from shellfish. Early extraction and assay methods focused on the de...

  10. Reconsidering pluripotency tests: do we still need teratoma assays?

    PubMed

    Buta, Christiane; David, Robert; Dressel, Ralf; Emgård, Mia; Fuchs, Christiane; Gross, Ulrike; Healy, Lyn; Hescheler, Jürgen; Kolar, Roman; Martin, Ulrich; Mikkers, Harald; Müller, Franz-Josef; Schneider, Rebekka K; Seiler, Andrea E M; Spielmann, Horst; Weitzer, Georg

    2013-07-01

    The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.

  11. Confocal detection of planar homogeneous and heterogeneous immunosorbent assays

    NASA Astrophysics Data System (ADS)

    Ghafari, Homanaz; Zhou, Yanzhou; Ali, Selman; Hanley, Quentin S.

    2009-11-01

    Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

  12. Confocal detection of planar homogeneous and heterogeneous immunosorbent assays.

    PubMed

    Ghafari, Homanaz; Zhou, Yanzhou; Ali, Selman; Hanley, Quentin S

    2009-01-01

    Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

  13. Two Types of Assays for Detecting Frog Sperm Chemoattraction

    PubMed Central

    Burnett, Lindsey A.; Tholl, Nathan; Chandler, Douglas E.

    2011-01-01

    Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 μm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed

  14. Waste Examination Assay Facility operations: TRU waste certification

    SciTech Connect

    Schultz, F.J.; Caylor, B.A.; Coffey, D.E.; Phoenix, L.B.

    1987-01-01

    The ORNL Waste Examination Assay Facility (WEAF) was established to nondestructively assay (NDA) transuranic (TRU) waste generated at Oak Ridge National Laboratory (ORNL). The present facility charter encompasses the NDA and nondestructive examination (NDE) of both TRU and low-level wastes (LLW). Presently, equipment includes a Neutron Assay System (NAS), a Segmented Gamma Scanner (SGS), a drum-sized Real-Time Radiography (RTR) system, and a Neutron Slab Detector (NSD). The first three instruments are computer interfaced. Approximately 2300 TRU waste drums have been assayed with the NAS and the SGS. Another 3000 TRU and LLW drums have been examined with the RTR unit. Computer data bases have been developed to collate the large amount of data generated during the assays and examinations. 6 refs., 1 tab.

  15. Misuse of PCR assay for diagnosis of mollusc protistan infections.

    PubMed

    Burreson, Eugene M

    2008-06-19

    Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

  16. 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.

  17. Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

    PubMed

    Reeves, Will K; Szymczak, Mitchell Scott; Burkhalter, Kristen L; Miller, Myrna M

    2015-12-01

    Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses. PMID:26675463

  18. Solid-Phase Biological Assays for Drug Discovery

    NASA Astrophysics Data System (ADS)

    Forsberg, Erica M.; Sicard, Clémence; Brennan, John D.

    2014-06-01

    In the past 30 years, there has been a significant growth in the use of solid-phase assays in the area of drug discovery, with a range of new assays being used for both soluble and membrane-bound targets. In this review, we provide some basic background to typical drug targets and immobilization protocols used in solid-phase biological assays (SPBAs) for drug discovery, with emphasis on particularly labile biomolecular targets such as kinases and membrane-bound receptors, and highlight some of the more recent approaches for producing protein microarrays, bioaffinity columns, and other devices that are central to small molecule screening by SPBA. We then discuss key applications of such assays to identify drug leads, with an emphasis on the screening of mixtures. We conclude by highlighting specific advantages and potential disadvantages of SPBAs, particularly as they relate to particular assay formats.

  19. Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

    PubMed

    Reeves, Will K; Szymczak, Mitchell Scott; Burkhalter, Kristen L; Miller, Myrna M

    2015-12-01

    Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

  20. A more sensitive and specific radioenzymatic assay for catecholamines

    SciTech Connect

    Kennedy, B.; Ziegler, M.G. )

    1990-01-01

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

  1. Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity.

    PubMed

    Zingue, Stéphane; Magne Nde, Chantal Beatrice; Clyne, Colin; Njamen, Dieudonné

    2014-01-01

    Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs. PMID:27433539

  2. Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity

    PubMed Central

    Zingue, Stéphane; Magne Nde, Chantal Beatrice; Njamen, Dieudonné

    2014-01-01

    Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs. PMID:27433539

  3. DNA damage in Pakistani pesticide-manufacturing workers assayed using the Comet assay.

    PubMed

    Bhalli, Javed A; Khan, Q M; Nasim, A

    2006-10-01

    The production and use of chemical pesticides has increased in recent years. Although the increased use of pesticides may benefit agriculture, they are also the potential source of environmental pollution, and exposure to pesticides can have negative consequences for human health. In the present study, we have assessed DNA damage in blood leukocytes from 29 Pakistani pesticide-factory workers and 35 controls of similar age and smoking history. The workers were exposed to various mixtures of organophosphates, carbamates, and pyrethroids. DNA damage was measured with the single cell gel electrophoresis (SCGE) assay or Comet assay, using the mean comet tail length (microm) as the DNA damage metric. Exposed workers had significantly longer comet tail lengths than the controls (mean +/- SD 19.98 +/- 2.87 vs. 7.38 +/- 1.48, P < 0.001). Of the possible confounding factors, smokers had significantly longer mean comet tail lengths than nonsmokers and exsmokers for both the workers (21.48 +/- 2.58 vs.18.37 +/- 2.28, P < 0.001) and the controls (8.86 +/- 0.56 vs. 6.79 +/- 1.31, P < 0.001), while age had a minimal effect on DNA damage (P > 0.05 and P < 0.05 for workers and controls, respectively). The results of this study indicate that occupational exposure to pesticides causes DNA damage. PMID:16917935

  4. An immunoperoxidase plaque assay for reticuloendotheliosis virus and its application to a sensitive serum neutralization assay.

    PubMed

    Calvert, J G; Nazerian, K

    1994-01-01

    A rapid assay for the enumeration of reticuloendotheliosis virus (REV) is described. Chicken embryo fibroblast monolayer cultures were infected with REV and incubated 6 days under an agar overlay. After removal of the overlay, cells were fixed with acetone/ethanol. Foci of infection (hereafter referred to as plaques) were detected using either an anti-REV envelope monoclonal antibody or convalescent chicken serum as the primary antibody; the secondary antibody was either horseradish peroxidase-conjugated goat anti-mouse IgG (for use with monoclonals) or goat anti-chicken IgG (for use with chicken serum). Staining with a substrate solution containing diaminobenzidine, CoCl2, and H2O2 revealed individual dark plaques on a light gray background. This method worked equally well for the SNV, CSV, and REV-T strains of REV; further, it detected all six field isolates tested. Results indicate that this immunoperoxidase technique is a rapid and reliable method for detection and titration of REV as well as for the assay of neutralizing antibody in chicken serum.

  5. Pantothenic acid quantification: method comparison of a stable isotope dilution assay and a microbiological assay.

    PubMed

    Rychlik, Michael; Roth-Maier, Dora

    2005-05-01

    Different foods and feedstuffs were analyzed for pantothenic acid (PA) by the recently developed stable isotope dilution assay (SIDA) and by the standard method, a microbiological assay (MA). The SIDA involved the use of [13C3, 15N]-pantothenic acid as the internal standard and detection by liquid chromatography-tandem mass spectrometry. The analysis of identical extracts minimized systematic bias due to equal extraction yields and enabled an ideal comparison between both methods. For the samples derived from plants a good accordance between the MA and the SIDA of total PA was found, whereas for the products of animal origin, higher contents were measured by MA than by SIDA. From the results of treatments by pantetheinase and phosphatase on the one hand and papain and diastase on the other, it was concluded that MA is able to measure a significant amount of bound PA. Furthermore, the data imply that microbial enzymes were able to cleave PA conjugates more effectively than pantetheinase and phosphatase treatment. PMID:16028638

  6. AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

    PubMed Central

    Thiel, William H.; Giangrande, Paloma H.

    2016-01-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  7. DNA damage in Pakistani pesticide-manufacturing workers assayed using the Comet assay.

    PubMed

    Bhalli, Javed A; Khan, Q M; Nasim, A

    2006-10-01

    The production and use of chemical pesticides has increased in recent years. Although the increased use of pesticides may benefit agriculture, they are also the potential source of environmental pollution, and exposure to pesticides can have negative consequences for human health. In the present study, we have assessed DNA damage in blood leukocytes from 29 Pakistani pesticide-factory workers and 35 controls of similar age and smoking history. The workers were exposed to various mixtures of organophosphates, carbamates, and pyrethroids. DNA damage was measured with the single cell gel electrophoresis (SCGE) assay or Comet assay, using the mean comet tail length (microm) as the DNA damage metric. Exposed workers had significantly longer comet tail lengths than the controls (mean +/- SD 19.98 +/- 2.87 vs. 7.38 +/- 1.48, P < 0.001). Of the possible confounding factors, smokers had significantly longer mean comet tail lengths than nonsmokers and exsmokers for both the workers (21.48 +/- 2.58 vs.18.37 +/- 2.28, P < 0.001) and the controls (8.86 +/- 0.56 vs. 6.79 +/- 1.31, P < 0.001), while age had a minimal effect on DNA damage (P > 0.05 and P < 0.05 for workers and controls, respectively). The results of this study indicate that occupational exposure to pesticides causes DNA damage.

  8. [Individual response to ionising radiation: What predictive assay(s) to choose?].

    PubMed

    Granzotto, Adeline; Joubert, Aurélie; Viau, Muriel; Devic, Clément; Maalouf, Mira; Thomas, Charles; Vogin, Guillaume; Malek, Karim; Colin, Catherine; Balosso, Jacques; Foray, Nicolas

    2011-02-01

    Individual response to ionizing radiation is an important information required to apply an efficient radiotherapy treatment against tumour and to avoid any adverse effects in normal tissues. In 1981, Fertil and Malaise have demonstrated that the post-irradiation local tumor control determined in vivo is correlated with clonogenic cell survival assessed in vitro. Furthermore, these authors have reminded the relevance of the concept of intrinsic radiosensitivity that is specific to each individual organ (Fertil and Malaise, 1981) [1]. To date, since clonogenicity assays are too time-consuming and do not provide any other molecular information, a plethora of research groups have attempted to determine the molecular bases of intrinsic radiosensitivity in order to propose reliable and faster predictive assays. To this aim, several approaches have been developed. Notably, the recent revolution in genomic and proteomic technologies is providing a considerable number of data but their link with radiosensitivity still remains to be elucidated. On another hand, the systematic screening of some candidate genes potentially involved in the radiation response is highlighting the complexity of the molecular and cellular mechanisms of DNA damage sensoring and signalling and shows that an abnormal radiation response is not necessarily due to the impairment of one single protein. Finally, more modest approaches consisting in focusing some specific functions of DNA repair seem to provide more reliable clues to predict over-acute reactions caused by radiotherapy. In this review, we endeavoured to analyse the contributions of these major approaches to predict human radiosensitivity. PMID:21333944

  9. An adaptation of the human HepaRG cells to the in vitro micronucleus assay.

    PubMed

    Jossé, Rozenn; Rogue, Alexandra; Lorge, Elisabeth; Guillouzo, André

    2012-05-01

    The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing >80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent >1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other

  10. An ethanolic extract of black cohosh causes hematological changes but not estrogenic effects in female rodents

    SciTech Connect

    Mercado-Feliciano, Minerva; Cora, Michelle C.; Witt, Kristine L.; Granville, Courtney A.; Hejtmancik, Milton R.; Fomby, Laurene; Knostman, Katherine A.; Ryan, Michael J.; Newbold, Retha; Smith, Cynthia; Foster, Paul M.; Vallant, Molly K.; Stout, Matthew D.

    2012-09-01

    Black cohosh rhizome (Actaea racemosa) is used as a remedy for pain and gynecological ailments; modern preparations are commonly sold as ethanolic extracts available as dietary supplements. Black cohosh was nominated to the National Toxicology Program (NTP) for toxicity testing due to its widespread use and lack of safety data. Several commercially available black cohosh extracts (BCE) were characterized by the NTP, and one with chemical composition closest to formulations available to consumers was used for all studies. Female B6C3F1/N mice and Wistar Han rats were given 0, 15 (rats only), 62.5 (mice only), 125, 250, 500, or 1000 mg/kg/day BCE by gavage for 90 days starting at weaning. BCE induced dose-dependent hematological changes consistent with a non-regenerative macrocytic anemia and increased frequencies of peripheral micronucleated red blood cells (RBC) in both species. Effects were more severe in mice, which had decreased RBC counts in all treatment groups and increased micronucleated RBC at doses above 125 mg/kg. Dose-dependent thymus and liver toxicity was observed in rats but not mice. No biologically significant effects were observed in other organs. Puberty was delayed 2.9 days at the highest treatment dose in rats; a similar magnitude delay in mice occurred in the 125 and 250 mg/kg groups but not at the higher doses. An additional uterotrophic assay conducted in mice exposed for 3 days to 0.001, 0.01, 0.1, 1, 10, 100 and 500 mg/kg found no estrogenic or anti-estrogenic activity. These are the first studies to observe adverse effects of BCE in rodents. -- Highlights: ► Mice and rats were dosed with black cohosh extract for 90 days starting at weaning. ► Hematological changes were consistent with a non-regenerative macrocytic anemia. ► Peripheral micronucleated red blood cell frequencies increased. ► Puberty was delayed 2.9 days in rats. ► No estrogenic/anti-estrogenic activity was seen in the uterotrophic assay.

  11. The analytical validation of the Oncotype DX Recurrence Score assay

    PubMed Central

    Baehner, Frederick L

    2016-01-01

    In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score® result (scale: 0–100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time. PMID:27729940

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

    PubMed Central

    Herrel, Nicholas R; Johnson, Nadia L; Cameron, Jennifer E; Leigh, Janet; Hagensee, Michael E

    2009-01-01

    Background Human Papillomavirus-32 (HPV-32) has traditionally been associated with focal-epithelial-hyperplasia (FEH). It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR) assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization) using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects). Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts. PMID:19558697

  13. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies.

    PubMed

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite(®) has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines.

  14. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies

    PubMed Central

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite® has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

  15. ToxCast Assay Network (TCAN) Viewer: A Visualization Tool for High-throughput Assay Chemical Data (SOT)

    EPA Science Inventory

    USEPA’s ToxCast program has generated high-throughput bioactivity screening (HTS) data on thousands of chemicals. The ToxCast program has described and annotated the HTS assay battery with respect to assay design and target information (e.g., gene target). Recent stakeholder and ...

  16. Portrait Toxigenic Clostridium difficile assay, an isothermal amplification assay detects toxigenic C. difficile in clinical stool specimens.

    PubMed

    Denys, Gerald A

    2014-01-01

    The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8-97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.

  17. Evaluation of the IMMULITE® 2000 CMV IgM assay

    PubMed Central

    2012-01-01

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

  18. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto

    1998-01-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular

  19. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Oehring, H.; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl O.

    1997-12-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular

  20. Plaque assay for titration of bovine enteric coronavirus.

    PubMed

    Vautherot, J F

    1981-10-01

    The plaquing ability of two isolates of bovine enteric coronavirus (BECV) was studied in HRT18 (human rectal adenocarcinoma) cell monolayers. Both isolates were able to induce plaque formation within 2 to 3 days; plaques appeared as round opalescent areas which remained colourless after neutral red or crystal violet staining. A good correlation was found between the titres as determined either by counting the plaques that were visible to the naked eye before and after neutral red staining, or by enumerating fluorescence or haemadsorption foci.

  1. Comparison of the Abbott Architect i2000 assay, the Roche Modular Analytics E170 assay, and an immunoradiometric assay for serum hepatitis B virus markers.

    PubMed

    Kim, Hyunjung; Oh, Eun-Jee; Kang, Mi-Sook; Kim, Sung Hoon; Park, Yeon-Joon

    2007-01-01

    Serum hepatitis B virus (HBV) markers are the most important data for epidemiological screening and clinical diagnosis of HBV infection, especially in endemic areas. We compared the results of the Roche Modular Analytics E170 assay, the Abbott Architect i2000 assay, and an immunoradiometric assay (IRMA) for HBV surface antigen (HBsAg), anti-HBV surface antigen (anti-HBs), HBV e antigen (HBeAg), and anti-HBV e antigen (anti-HBe). A number of serum samples (264, 263, 224, and 202 for HBsAg, anti-HBs, HBeAg, and anti-HBe, respectively) were studied. For samples giving discrepant results for HBeAg between methods, real-time PCR assays were performed. The concordance rates among the three methods were high for HBsAg (100%) and HBeAg (94.6), but low for anti-HBs (91.6%) and anti-HBe (82.2%). For anti-HBs, which could be measured quantitatively by the Modular E170 and Architect i2000 procedures, discrepant results were observed at low levels of anti-HBs. For anti-HBe, the positive rate was highest with Modular E170 (60.9%) followed by the IRMA kit (54.1%) and Architect i2000 (51.0%). This study shows substantial differences between the assay results by the three methods, which should be taken into account in determinations of serum HBV markers.

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

    PubMed

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

    2013-09-01

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

  3. Growth hormone assays: current methodologies and their limitations.

    PubMed

    Bidlingmaier, Martin; Strasburger, Christian J

    2007-01-01

    Measurement of circulating growth hormone (GH) concentrations is essential in diagnosis of either GH deficiency or GH excess. The invention of immunoassays for the measurement of peptide hormones was a major breakthrough, enabling the routine analysis of GH concentrations in larger series of samples. Over the last few decades, measurement technology has evolved from less sensitive, mainly radioactive assays based on polyclonal antisera to the latest generations of highly sensitive chemiluminescence methods employing monoclonal antibodies. Unfortunately, the development of newer assays did not lead to better agreement among the results obtained by different assay methods. On the contrary, the differences tended to increase when monoclonal antibody based assays became more popular. The actual value reported for the GH concentration in a specific patient's sample still mainly depends on the method used by the respective laboratory, limiting the applicability of international consensus guidelines in clinical practice. The heterogeneity of the analyte itself, the availability of different reference preparations for calibration and the interference from matrix components such as GH binding protein are among the reasons why standardizing GH assays is difficult. An additional challenge arose from the availability of a GH receptor antagonist for the treatment of acromegaly, which is basically a mutated form of GH and therefore interferes in many GH assays. This review provides an overview on GH assays used in clinical practice, their limitations and the potential next steps towards standardization.

  4. Applications Where Snap is BPM for Radioactive Waste Assay

    SciTech Connect

    Miller, T.J.

    2008-07-01

    Historically, the Atomic Weapons Establishment (AWE) at Aldermaston in the United Kingdom (UK), has used a variety of assay techniques to measure the radioactive content of a diverse range of waste packages from decommissioning, operational and legacy sources. The regulator, the Environment Agency in the UK, places conditions and limits on AWE through an authorisation within the Radioactive Substances Act (RSA93). The conditions and limits require Best Practical Means (BPM) measurements to be used to demonstrate compliance with the authorisation. Hence, the assay technique employed needs to achieve a balance between risk of exposure, environmental considerations, technological considerations, health and safety considerations and cost effectiveness, without being grossly disproportionate in terms of money, time or trouble. Recently published work has concluded that the Spectral Non-destructive Assay Platform (SNAP) assay system is BPM for Depleted Uranium (DU) waste assay at AWE (1) and low level plutonium in soft drummed waste, HEPA filters and soils (2-4). The purpose of this paper is to highlight other applications where SNAP represents BPM for radioactive waste assay. This has been done by intercomparison studies of SNAP with other assay techniques, such as Segmented Gamma Scanner (SGS) and Passive Neutron Coincidence Counter (PNCC). It has been concluded that, for a large range of waste packages encountered at AWE, SNAP is BPM. (author)

  5. Validation of an LDH Assay for Assessing Nanoparticle Toxicity

    PubMed Central

    Han, Xianglu; Gelein, Robert; Corson, Nancy; Wade-Mercer, Pamela; Jiang, Jingkun; Biswas, Pratim; Finkelstein, Jacob N.; Elder, Alison; Oberdörster, Günter

    2014-01-01

    Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40 nm), silver (Ag-35, 35 nm; Ag-40, 40 nm), and titanium dioxide (TiO2-25, 25 nm) NPs by examining their potential to inactivate LDH and interference with β-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO2-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case. PMID:21722700

  6. Nested PCR Assay for Detection of Granulocytic Ehrlichiae

    PubMed Central

    Massung, Robert F.; Slater, Kim; Owens, Jessica H.; Nicholson, William L.; Mather, Thomas N.; Solberg, Victoria B.; Olson, James G.

    1998-01-01

    A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens. PMID:9542943

  7. Preparation, imaging, and quantification of bacterial surface motility assays.

    PubMed

    Morales-Soto, Nydia; Anyan, Morgen E; Mattingly, Anne E; Madukoma, Chinedu S; Harvey, Cameron W; Alber, Mark; Déziel, Eric; Kearns, Daniel B; Shrout, Joshua D

    2015-01-01

    Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more "temperate swarmers" that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. "Wettability", or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment. PMID:25938934

  8. Molecular genotyping and quantitation assay for rotavirus surveillance.

    PubMed

    Liu, Jie; Lurain, Kate; Sobuz, Shihab U; Begum, Sharmin; Kumburu, Happiness; Gratz, Jean; Kibiki, Gibson; Toney, Denise; Gautam, Rashi; Bowen, Michael D; Petri, William A; Haque, Rashidul; Houpt, Eric R

    2015-03-01

    Rotavirus genotyping is useful for surveillance purposes especially in areas where rotavirus vaccination has been or will be implemented. RT-PCR based molecular methods have been applied widely, but quantitative assays targeting a broad spectrum of genotypes have not been developed. Three real time RT-PCR panels were designed to identify G1, G2, G9, G12 (panel GI), G3, G4, G8, G10 (panel GII), and P[4], P[6], P[8], P[10], P[11] (panel P), respectively. An assay targeting NSP3 was included in both G panels as an internal control. The cognate assays were also formulated as one RT-PCR-Luminex panel for simultaneous detection of all the genotypes listed above plus P[9]. The assays were evaluated with various rotavirus isolates and 89 clinical samples from Virginia, Bangladesh and Tanzania, and exhibited 95% (81/85) sensitivity compared with the conventional RT-PCR-Gel-electrophoresis method, and 100% concordance with sequencing. Real time assays identified a significantly higher rate of mixed genotypes in Bangladeshi samples than the conventional gel-electrophoresis-based RT-PCR assay (32.5% versus 12.5%, P<0.05). In these mixed infections, the relative abundance of the rotavirus types could be estimated by Cq values. These typing assays detect and discriminate a broad range of G/P types circulating in different geographic regions with high sensitivity and specificity and can be used for rotavirus surveillance.

  9. Biological detection of low radiation doses with integrated photothermal assay

    NASA Astrophysics Data System (ADS)

    Zharov, Vladimir P.; Viegas, Mark; Soderberg, Lee S. F.

    2005-04-01

    The goal of this paper was to evaluate the diagnostic value of integrated photothermal (PT) assay with additional fluorescent and photoacoustic (PA) modules to assess both the "safety limit" of exposure to ionizing γ-radiation and optimal therapeutic doses for cancer treatment. With this assay, the influences of γ irradiation on cancer cells (pancreatic-AR42J and hepatocytes-hepG2) and healthy cells (mouse lymphocytes and erythrocytes) was examined as a function of exposure dose (0.6-5 Gy) and time after irradiation, in vitro and in vivo. Independent verification of data obtained with conventional assays revealed that integrated PT assay allowed us to detect the different stages of radiation impact, including changes in cell metabolism at low dose, or stages related to cell death (apoptosis and necrosis) at high doses with a threshold sensitivity of at least three orders of magnitude better than existing assays. Also, PT assay was capable of quantitatively differentiating the biological action of γ irradiation alone and in combination with drug and nicotine impact. Finally, we demonstrated on an animal model that IPT assay has the potential for use in routine rapid evaluation of biological consequences of low-dose exposure a few days after irradiation.

  10. Field evaluation of endotoxin air sampling assay methods.

    PubMed

    Thorne, P S; Reynolds, S J; Milton, D K; Bloebaum, P D; Zhang, X; Whitten, P; Burmeister, L F

    1997-11-01

    This study tested the importance of filter media, extraction and assay protocol, and bioaerosol source on the determination of endotoxin under field conditions in swine and poultry confinement buildings. Multiple simultaneous air samples were collected using glass fiber (GF) and polycarbonate (PC) filters, and these were assayed using two methods in two separate laboratories: an endpoint chromogenic Limulus amebocyte lysate (LAL) assay (QCL) performed in water and a kinetic chromogenic LAL assay (KQCL) performed in buffer with resistant-parallel line estimation analysis (KLARE). In addition, two aqueous filter extraction methods were compared in the QCL assay: 120 min extraction at 22 degrees C with vigorous shaking and 30 min extraction at 68 degrees C with gentle rocking. These extraction methods yielded endotoxin activities that were not significantly different and were very highly correlated. Reproducibility of endotoxin determinations from duplicate air sampling filters was very high (Cronbach alpha all > 0.94). When analyzed by the QCL method GF filters yielded significantly higher endotoxin activity than PC filters. QCL and KLARE methods gave similar estimates for endotoxin activity from PC filters; however, GF filters analyzed by the QCL method yielded significantly higher endotoxin activity estimates, suggesting enhancement of the QCL assay or inhibition of the KLARE asay with GF filters. Correlation between QCL-GF and QCL-PC was high (r = 0.98) while that between KLARE-GF and KLARE-PC was moderate (r = 0.68). Analysis of variance demonstrated that assay methodology, filter-type, barn-type, and interactions between assay and filter-type and between assay and barn-type were important factors influencing endotoxin exposure assessment.

  11. Comparison of the new Mycofast Revolution assay with a molecular assay for the detection of genital mycoplasmas from clinical specimens

    PubMed Central

    2013-01-01

    Background Genital mycoplasmas are opportunistic bacteria that are associated with undesirable gynaecologic and reproductive events. Mycoplasmas are fastidious bacteria with increasing resistance to routine antimicrobials and often fail to grow on conventional culture methods. The commercial Mycofast Revolution assay permits the phenotypic detection and identification of genital mycoplasmas. Antimicrobial susceptibility testing against five antimicrobial agents with MICs corresponding to the CLSI guidelines can also be performed. This study aimed to compare the new commercially available Mycofast Revolution assay with a multiplex PCR assay. Methods Self-collected swabs were obtained from pregnant women attending the antenatal clinic of a tertiary academic hospital in Pretoria, South Africa from October 2012 to November 2012. These swabs were used to seed UMMt and modified Amies transport media. The seeded UMMt transported medium was used to inoculate the Mycofast Revolution assay for the identification, enumeration and antimicrobial susceptibility testing of genital mycoplasmas. Following DNA extraction from the modified Amies transport medium, specimens were subjected to a multiplex PCR assay for the detection of genital mycoplasmas. Results The Mycofast Revolution kit had a sensitivity and specificity of 77.3% (95% CI: 62.15% to 88.51%) and 80% (95% CI: 28.81% to 96.70%), respectively, against the PCR assay. The positive and negative predictive values were 97.1% (95% CI: 85.03% to 99.52%) and 28.6% (95% CI: 8.57% to 58.08%). Genital mycoplasmas were detected in 71.4% (35/49) of samples with the Mycofast Revolution assay with 49% (24/49) being Ureaplasma spp. and 22.4% (11/49) mixed strains. The multiplex PCR assay had a positivity rate of 89.8% (44/49) for genital mycoplasmas; mixed strains were present in 51% (25/49) of samples, Ureaplasma spp. in 16.3% (8/49) and M. hominis in 22.4% (11/49) of samples. Conclusions There was a fair agreement (κ = 0

  12. Effect of dilution on carboxymethylcellulase and xylanase assays

    SciTech Connect

    Cauchon, N.; LeDuy, A.

    1984-08-01

    It has been demonstrated that the dilution of samples prior to the carboxymethylcellulase and xylanase assays causes serious discrepancies in the numerical values obtained for the enzyme activities. Even when the sample is assayed with the identical procedure, one could obtain different numerical values of the enzyme activity U depending on how much this sample has been diluted before the enzyme assay. Two crude commercial cellulase samples of Aspergillus niger and Trichoderma viride as well as the culture filtrate of our newly isolated acidophilic fungus have been used for the demonstration. An empirical method for reporting the cellulolytic activity by taking into account this dilution effect is proposed.

  13. Advances in Assays and Analytical Approaches for Botulinum Toxin Detection

    SciTech Connect

    Grate, Jay W.; Ozanich, Richard M.; Warner, Marvin G.; Bruckner-Lea, Cindy J.; Marks, James D.

    2010-08-04

    Methods to detect botulinum toxin, the most poisonous substance known, are reviewed. Current assays are being developed with two main objectives in mind: 1) to obtain sufficiently low detection limits to replace the mouse bioassay with an in vitro assay, and 2) to develop rapid assays for screening purposes that are as sensitive as possible while requiring an hour or less to process the sample an obtain the result. This review emphasizes the diverse analytical approaches and devices that have been developed over the last decade, while also briefly reviewing representative older immunoassays to provide background and context.

  14. Luciferase reporter assay in Drosophila and mammalian tissue culture cells

    PubMed Central

    Yun, Chi

    2014-01-01

    Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

  15. Electrical lysis of cells for detergent-free droplet assays

    PubMed Central

    Tran, T. M.; Abate, A. R.

    2016-01-01

    Efficient lysis is critical when analyzing single cells in microfluidic droplets, but existing methods utilize detergents that can interfere with the assays to be performed. We demonstrate robust cell lysis without the use of detergents or other chemicals. In our method, cells are exposed to electric field immediately before encapsulation in droplets, resulting in cell lysis. We characterize lysis efficiency as a function of control parameters and demonstrate compatibility with enzymatic assays by measuring the catalysis of β-glucosidase, an important cellulase used in the conversion of biomass to biofuel. Our method enables assays in microfluidic droplets that are incompatible with detergents. PMID:27051471

  16. Ethanal assay, using an enzymo-conductimetric method

    SciTech Connect

    Saad, I.; Wallach, J.M. )

    1992-01-01

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

  17. A Functional Assay for GPR55: Envision Protocol.

    PubMed

    Anavi-Goffer, Sharon; Ross, Ruth A

    2016-01-01

    AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids. PMID:27245893

  18. Electrical lysis of cells for detergent-free droplet assays.

    PubMed

    de Lange, N; Tran, T M; Abate, A R

    2016-03-01

    Efficient lysis is critical when analyzing single cells in microfluidic droplets, but existing methods utilize detergents that can interfere with the assays to be performed. We demonstrate robust cell lysis without the use of detergents or other chemicals. In our method, cells are exposed to electric field immediately before encapsulation in droplets, resulting in cell lysis. We characterize lysis efficiency as a function of control parameters and demonstrate compatibility with enzymatic assays by measuring the catalysis of β-glucosidase, an important cellulase used in the conversion of biomass to biofuel. Our method enables assays in microfluidic droplets that are incompatible with detergents. PMID:27051471

  19. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  20. Independency of Fe ions in hemoglobin on immunomagnetic reduction assay

    NASA Astrophysics Data System (ADS)

    Yang, S. Y.; Lan, C. B.; Chen, C. H.; Horng, H. E.; Hong, Chin-Yih; Yang, H. C.; Lai, Y. K.; Lin, Y. H.; Teng, K. S.

    2009-10-01

    Immunomagnetic reduction (IMR), which involves measuring the reduction in the ac magnetic susceptibility of magnetic reagents, is due to the association between bio-functionalized magnetic nanoparticles and target bio-molecules. This has been demonstrated for assaying proteins in solutions free of Fe ions, such as serum. In this work, the validity of IMR assay for samples rich in Fe ions like hemoglobin (Hb) is investigated. According to the results, there is no magnetic signal contributed by Fe-ion-rich Hb. Furthermore, the results show a high sensitivity in assaying hemoglobin A1c (HbA1c) by using IMR.