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Sample records for 3-day uterotrophic assay

  1. The mouse uterotrophic assay: a reevaluation of its validity in assessing the estrogenicity of bisphenol A.

    PubMed Central

    Markey, C M; Michaelson, C L; Veson, E C; Sonnenschein, C; Soto, A M

    2001-01-01

    The prevalence of synthetic chemicals in our environment that are capable of mimicking the female hormone estrogen is a growing concern. One such chemical, bisphenol A (BPA), has been shown to leach from a variety of resin-based and plastic products, including dental sealants and food and beverage containers, in concentrations that are sufficient to induce cell proliferation in vitro. The response to BPA in vivo has been varied; thus the aims of this study were to investigate a) whether BPA has an estrogenic effect in CD-1 mice, a strain that is useful for developmental studies; and b) whether the uterotrophic assay is a valid means of determining the estrogenicity of BPA by comparing it with other end points measured in the uterus. Immature female CD-1 mice were exposed to BPA in concentrations ranging from 0.1 to 100 mg/kg body weight for 3 days. Results showed that BPA induced a significant increase in the height of luminal epithelial cells within the uterus at concentrations of 5, 75, and 100 mg/kg and that BPA induced lactoferrin at concentrations of 75 and 100 mg/kg. A uterotrophic response (increase in uterine wet weight) was induced by 100 mg/kg BPA only. Further, the proportion of mice showing vaginal opening was greater after exposure to 0.1 and 100 mg/kg BPA, relative to the control animals and those receiving intermediate doses of BPA. These results demonstrate that BPA induces changes in the mouse uterus that differ depending on the exposure dose and the end point measured, and reveal that certain tissue effects show a nonmonotonic relationship with dose. These data also demonstrate that BPA induces estrogenic changes in the uterus of the CD-1 mouse, and highlight the need to reevaluate the validity of the mouse uterotrophic assay as an end point for determining the estrogenicity of suspected environmental estrogens. PMID:11171525

  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. The estrogenic effects of benzylparaben at low doses based on uterotrophic assay in immature SD rats.

    PubMed

    Hu, Ying; Zhang, Zhaobin; Sun, Libei; Zhu, Desheng; Liu, Qingchun; Jiao, Jian; Li, Jun; Qi, Mingwen

    2013-03-01

    Benzylparaben (BzP), a type of parabens being used as a preservative agent in cosmetics, food, and pharmaceutical products, may be ingested by humans. In this study, we performed an immature uterotrophic assay using Sprague Dawley (SD) rats by intragastric administration to determine the estrogenic effects of BzP and found significant increases in uterine weight with doses of 0.16 mg/kg body weight and higher (P<0.05). The in vivo estrogenicity of BzP was supported by in vitro results from the human estrogen receptor α (hERα)-coactivator recruiting assay and in silico molecular docking analysis performed in this study. The in vitro estrogenic activity of BzP can be observed at concentrations of 1.0×10(-8) M and higher. Molecular docking analysis showed that BzP fits well into the agonist pocket of hERα. The lowest observed effect dose (LOED) (0.16 mg/kg/day) of BzP is much lower than the documented LOEDs of other parabens. Actual risk may exist for people who consume a diet high in BzP or use BzP-laden cosmetics. In addition, we tested the sensitivity of Wistar rats to 17β-estradiol by immature uterotrophic assay, and no obvious uterotrophic response was observed in the rats given doses up to 100 μg/kg body weight. PMID:23220609

  4. Increasing the sensitivity of the rodent uterotrophic assay to estrogens, with particular reference to bisphenol A.

    PubMed Central

    Ashby, J

    2001-01-01

    The gravimetric uterotrophic assay is currently the most well-established, short-term rodent estrogenicity assay. Increasing attention is being paid to the extent to which use of morphometric or molecular changes in the uterus could act as surrogates for the gravimetric end point of the assay, thereby perhaps increasing the sensitivity of the assay. In this paper I discuss the available data, paying particular attention to studies on bisphenol A (BPA) because it offers the largest database for consideration. I conclude that the case has yet to be made for augmenting the gravimetric end point of the uterotrophic assay. To resolve this important question, it will be necessary to conduct detailed dose-response studies where the no-observed-effect level (NOEL) for the proposed surrogate end points are compared with the NOEL for the gravimetric end point. Currently, few such studies exist, and among those that do no clear message emerges. The general trend to increasing use of molecular assays in toxicology (multigene microarrays and real-time polymerase chain reaction) emphasizes the need for clear criteria for comparing the performance of individual markers of toxicity. PMID:11712991

  5. Lack of estrogenic or (anti-)androgenic effects of d-phenothrin in the uterotrophic and Hershberger assays.

    PubMed

    Yamada, Tomoya; Ueda, Shinji; Yoshioka, Kaoru; Kawamura, Satoshi; Seki, Takaki; Okuno, Yasuyoshi; Mikami, Nobuyoshi

    2003-04-22

    Synthetic pyrethroids are among the most common insecticides and pesticides currently in use worldwide. Recently, d-phenothrin, a synthetic pyrethroid, is suspected to have endocrine activities through the estrogen and androgen receptors. However, no study has been conducted to evaluate its potential for hormonal activity using an in vivo test specifically focused on estrogenic and androgenic activities. In this study, we evaluated the interaction of d-phenothrin (0, 100, 300 or 1000 mg/kg per day, p.o.) with estrogen- or androgen-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the uterotrophic and Hershberger assays have not yet been fully developed, both are widely used and are being considered by the OECD as short-term screening assays for hormonal activity. The highest dose level tested for d-phenothrin was a limit dose (1000 mg/kg per day) designated in the current draft protocol by the OECD, and in fact there was no excessive systemic toxicity in both assays; slightly increased liver weight but no change of serum androgen levels in accessing anti-androgenicity. Potential estrogenic effect of d-phenothrin was evaluated by means of 3-day uterotrophic assay using immature Crj:CD(SD)IGS rats (20 days of age). No increase in uterine weight (wet or blotted) was observed following oral exposure to d-phenothrin. Reference control ethynyl estradiol (0.001 mg/kg per day) showed a significant effect in this assay protocol. A 10-day Hershberger assay using castrated peripubertal male rats measures the androgenic or anti-androgenic effects of the test chemicals on several accessory glands/tissues (the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscles, glans penis and Cowper's glands). d-Phenothrin was administered by oral gavage for 10 days to castrated male Crj:CD(SD)IGS rats (7 weeks of age, rats were castrated at 6 weeks of age) with or

  6. D-003 does not possess oestrogenic potential in-vivo: findings of the uterotrophic assay.

    PubMed

    Noa, Miriam; Mendoza, Sarahí; Mas, Rosa; Gámez, Rafael; Valle, Maikel; Pardo, Balia; Gutiérrez, Ariadne; Mendoza, Nilda

    2007-10-01

    D-003 is a mixture of long-chain fatty acids purified from sugarcane wax that inhibits both cholesterol synthesis prior to mevalonate formation, and lipid peroxidation. D-003 has been shown to prevent bone loss and bone resorption in ovariectomized rats, and significantly improves bone resorption markers in postmenopausal women with reduced bone mineral density. As hormone-replacement therapy, D-003 displays cholesterol-lowering and anti-resorptive effects. We have studied its potential oestrogenic activity in-vivo using the uterotrophic assay. Rats were randomly distributed into five groups: a sham-operated group and four groups of ovariectomized rats, one treated with vehicle, one with D-003 (50 mg kg(-1)), one with oestradiol benzoate (30 microg kg(-1)) and one with D-003 (50 mg kg(-1)) plus oestradiol benzoate (30 microg kg(-1)). Treatments were administered for 14 days. Ovariectomy decreased the values of relative uterus weight, epithelium cell height and endometrial thickness compared with sham-operated rats, and these effects were all significantly reduced with oestradiol benzoate, but not with D-003. Concurrent administration of D-003 and oestradiol benzoate had statistically similar effects on all variables as oestradiol benzoate alone. In conclusions, D-003 orally given at 50 mg kg(-1), a dose that prevents bone loss and bone resorption in ovariectomized rats, did not display oestrogenic/anti-oestrogenic activity in-vivo, as assessed in the uterotrophic assay. PMID:17910820

  7. 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

  8. Lack of (anti-) androgenic or estrogenic effects of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the Hershberger and uterotrophic assays.

    PubMed

    Kunimatsu, Takeshi; Yamada, Tomoya; Ose, Keiko; Sunami, Osamu; Kamita, Yusuke; Okuno, Yasuyoshi; Seki, Takaki; Nakatsuka, Iwao

    2002-04-01

    Synthetic pyrethroids are among the most common pesticides and insecticides currently in use worldwide. Recently, chemicals classified as synthetic pyrethroids are suspected as being endocrine disrupting chemicals. However, no study has been conducted to assess their potential hormonal activities using in vivo test specifically focused on endocrine disruption. In the present study, we evaluated the interaction of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) with androgen receptor (AR)- and estrogen receptor (ER)-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the Hershberger and uterotrophic assays have not yet been fully developed, both are widely used and are being considered by OECD as short-term screening assays for hormonal activity. A 5-day Hershberger assay using castrated male rats measures agonistic and androgenic ability of the test chemicals to AR of several accessory glands/tissues (the ventral prostate, dorsolateral prostate, seminal vesicles with coagulating glands, and levator ani plus bulbocavernosus muscles). Esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (25, 50, or 75 mg/kg/day) was administered by oral gavage for 5 days to castrated male Crj:CD(SD)IGS rats (1 week after the castration, 11 weeks of age) with or without coadministration of 0.25 mg/kg/day testosterone propionate (subcutaneous injection on the dorsal surface). The highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. None of esfenvalerate, fenvalerate, and permethrin showed any androgenic or antiandrogenic effects. Reference control of p,p'-DDE and methyltestosterone (100 mg/kg/day) provided significant effects in this assay protocol. Potential effects of these pyrethroids mediated through the ER were evaluated by means of 3-day uterotrophic assay using ovariectomized Crj

  9. 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

  10. 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

  11. Validation of the intact rat weanling uterotrophic assay with notes on the formulation and analysis of the positive control chemical in vehicle.

    PubMed

    Tyl, Rochelle W; Marr, Melissa C; Brown, Sherri S; Dolbow, Emily A; Myers, Christina B

    2010-10-01

    The intact female weanling version in the Organization for Economic Cooperation and Development (OECD) uterotrophic assay Test Guideline (TG) 440 is proposed as an alternative to the adult ovariectomized female version, because it does not involve surgical intervention (vs the ovariectomized version) and detects direct/indirect-acting estrogenic/anti-estrogenic substances (vs the ovariectomized version which detects only direct-acting estrogenic/anti-estrogenic substances binding to the estrogen receptor). This validation study followed OECD TG 440, with six female weanling rats (postnatal day 21) per dose group and six treatment groups. Females were weighed and dosed once daily by oral gavage for three consecutive days, with one of six doses of 17α-ethinyl estradiol in corn oil at 5 ml kg⁻¹ at 0 and 0.1-10 µg kg⁻¹ per day. On postnatal day 24, the juvenile females were euthanized by CO₂ asphyxiation, weighed, livers weighed and uteri weighed wet and blotted. The presence or absence of vaginal patency was recorded. Absolute and relative (to terminal body weight) uterine wet and blotted weights and uterine luminal fluid weights were significantly increased at 3.0 and 10.0 (both P < 0.01) µg kg⁻¹ per day, and increased to ~140% of control values at 1.0 µg kg⁻¹ per day (not statistically significantly). In vivo body weights, weight changes, feed consumption, liver weights and terminal body weights were unaffected. Vaginal patency was not acquired in any female at any dose, although vaginal puckering was observed in one female at 10.0 µg kg⁻¹ per day. Therefore, this intact weanling uterotrophic assay is validated in our laboratory for use under US and European endocrine toxicity testing programs/legislation. PMID:20981862

  12. Lack of binding to isolated estrogen or androgen receptors, and inactivity in the immature rat uterotrophic assay, of the ultraviolet sunscreen filters Tinosorb M-active and Tinosorb S.

    PubMed

    Ashby, J; Tinwell, H; Plautz, J; Twomey, K; Lefevre, P A

    2001-12-01

    The presence of structurally diverse chemicals as contaminants in the environment has led to concerns regarding their possible endocrine disturbing effects. Recently, some ultraviolet absorbing components of sunscreen preparations have given positive responses in assays monitoring estrogen-like activity both in vitro and in vivo. Consequently, two recently developed sunscreen components, Tinosorb M-active and Tinosorb S, were evaluated using the in vitro estrogen and androgen receptor competitive binding assays. Neither compound gave a positive response in either of the assays, consistent with the large molecular dimensions of each chemical disfavoring binding to the hormone receptors. Both of the chemicals were inactive in immature rat uterotrophic assays conducted using the subcutaneous route of administration. It is concluded that neither of these agents possess intrinsic estrogenic/antiestrogenic or androgenic/antiandrogenic activity. The several positive control chemicals evaluated gave the expected positive responses in the assays used. PMID:11754532

  13. 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

  14. 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...

  15. 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...

  16. 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...

  17. 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

  18. Comparative uterotrophic effects of endoxifen and tamoxifen in ovariectomized Sprague-Dawley rats.

    PubMed

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

    2014-12-01

    Endoxifen (4-hydroxy-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 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-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

  19. 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

  20. The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

    PubMed

    Kanno, J; Onyon, L; Haseman, J; Fenner-Crisp, P; Ashby, J; Owens, W

    2001-08-01

    The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists. PMID:11564613

  1. The OECD program to validate the rat uterotrophic bioassay. Phase 2: dose-response studies.

    PubMed Central

    Kanno, Jun; Onyon, Lesley; Peddada, Shyamal; Ashby, John; Jacob, Elard; Owens, William

    2003-01-01

    The Organisation for Economic Co-operation and Development has completed phase 2 of an international validation program for the rodent uterotrophic bioassay. The purpose of the validation program was to demonstrate the performance of two versions of the uterotrophic bioassay, the immature female rat and the adult ovariectomized rat, in four standardized protocols. This article reports the dose-response studies of the validation program; the coded single-dose studies are reported in an accompanying paper. The dose-response study design used five selected weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT. These weak agonists were administered in a prescribed series of doses to measure the performance and reproducibility of the protocols among the participating laboratories. All protocols successfully detected increases in uterine weights when the weak agonists were administered. Within each protocol, there was good agreement and reproducibility of the dose response among laboratories with each substance. Substance-specific variations were observed in the influence of the route of administration on the uterine response, the potency as related to the dose producing the first statistically significant increase in uterine weights, and the maximum increase in uterine weight. Substantive performance differences were not observed between the uterotrophic bioassay versions or among the standardized protocols, and these were judged to be qualitatively equivalent. It is noteworthy that these results were reproducible under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age), indicating that the bioassay's performance as a screen is robust. In conclusion, both the intact, immature, and adult OVX versions, and all protocols appear to be reproducible and transferable across laboratories and are able to detect weak estrogen agonists. PMID:12948896

  2. 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

  3. Triclosan exposure modulates estrogen-dependent responses in the rat uterotrophic assay.

    EPA Science Inventory

    Our previous studies in the juvenile rat indicated that the biocide triclosan may alter steroid hormone levels. Here, we hypothesize that triclosan possesses estrogenic activity. In the first study, we evaluated the potential estrogenicity of triclosan using the immature rat uter...

  4. Direction of estradiol metabolism as a control of its hormonal action--uterotrophic activity of estradiol metabolites.

    PubMed

    Martucci, C; Fishman, J

    1977-12-01

    The uterotrophic activities of the catechol metabolites of estradiol 2-hydroxyestrone, 2-methoxyestrone and 2-hydroxyestradiol were measured under conditions of continuous administration of sc implanted paraffin pellets. The activity of these estrogens was compared to that of estradiol-17beta and its other principal metabolites estrone, estriol and 15alpha-hydroxyestriol (estetrol). The major catechol estrogens, 2-hydroxyestrone and 2-methoxyestrone, and the pregnancy metabolite, 15alpha-hydroxyestriol, exhibited no uterotrophic activity. The minor catecholestrogen, 2-hydroxyestradiol, showed some activity whose character was different from that exhibited by implants of estradiol, estrone and estriol all of which were equipotent uterotrophic agents. Implants of 2-hydroxyestrone in the presence of estradiol or estriol pellets did not diminish the response to the latter indicating that the 2-hydroxyestrone is not antiestrogenic under these conditions. It is concluded that the direction of estradiol metabolism can have a profound influence on the expression of peripheral hormonal activity with hydroxylation at C-2 terminating and hydroxylation at C-16 extending it. PMID:590186

  5. No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats.

    PubMed

    Kim, Hyung Sik; Han, Soon Young; Kim, Tae Sung; Kwack, Seung Jun; Lee, Rhee Da; Kim, In Young; Seok, Ji-Hyun; Lee, Byung Mu; Yoo, Sun Dong; Park, Kui Lea

    2002-09-01

    Several studies have demonstrated that bisphenol A (BPA) exhibited weak estrogenic activity in the 3-day uterotrophic assay using ovariectomized (OVX) and immature rats (Toxicol. Lett. 115 (2000) 231; Regul. Toxicol. Pharmacol. 32 (2000) 118; J. Toxicol. Sci. 26 (2001) 111) and BPA also possessed anti-androgenic activity in in vitro yeast based assays (J. Endocrinol. 158 (1998) 327). To investigate anti-androgenic effects of BPA. a rodent Hershberger assay was carried out using immature Sprague-Dawley male rats. An androgen agonist, testosterone (0.4 mg/kg per day), was administered for 7 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, a pure androgen antagonist, flutamide (1, 5. 10 mg/kg per day. oral) was co-administered with testosterone (0.4 mg/kg per day s.c.). BPA was also administered orally with or without testosterone (0.4 mg/kg per day, s.c.) for 7 consecutive days. In the testosterone treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles (LABC) weights were significantly increased compared with control. However. flulamide dose-dependently inhibited the testosterone-induced re-growth of seminal vesicles, ventral prostate, and LABC, with a significant decrease at flutamide 1.0 mg/kg and above (P<0.05). Serum LH levels were also significantly increased (5 mg/kg and above, P<0.05), but no changes in serum testosterone levels. In contrast, BPA had no effects on the re-growth of seminal vesicles, ventral prostate and LABC induced by testosterone, and no significant differences were observed in serum LH and testosterone levels. In summary, the Hershberger assay could be a sensitive method for detecting androgenic or anti-androgenic chemicals, but BPA did not exhibit any androgenic or anti-androgenic activities in Hershberger assay. PMID:12243870

  6. 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

  7. Environmental stress and 3-day eventing: effects of altitude.

    PubMed

    Foreman, J H; Waldsmith, J K; Lalum, R B

    1999-07-01

    Three-day event horses are subject to various external environmental stresses including changes in ambient temperature, humidity, altitude, and test severity. Considerable research on the adverse effects of increased heat and humidity preceded the 1996 Olympic Summer Games in Atlanta, Georgia USA, but no research has been done previously on the effects of altitude on 3-day eventing. Physical and venous blood gas data were collected on horses (n = 24) competing in the High Prairie Preliminary (CCN*) and Intermediate (CCN**) 3-day events and Preliminary Horse Trials in Parker, Colorado (1900 m above sea level). Despite the increased altitude, only post exercise rectal temperature and pH were higher (P < 0.05) whereas heart rate (HR), [K+], and ionized calcium (ICa++) were lower (P < 0.05) in 3-day event horses compared to horse trial horses. All other variables (respiratory rate [RR], PCV, [Hb], PCO2, [tCO2], [HCO3-], BE, and [Na+]) were not different between groups (P > 0.05). When these preliminary horse trial horses in Colorado were compared to those previously studied at preliminary horse trials at sea level in Arizona, post exercise HR and RR were higher (P < 0.05) and pH, PCO2, [tCO2], [HCO3-], BE and [iCa++] were lower (P < 0.05) at altitude. These data show that increased altitude (1900 m above sea level) was more stressful for 3-day event horses, but did not result in the severe physiological changes and inability to complete prescribed exercise tests seen in previous studies with increased heat and humidity. It is clear from these and previous data that increased heat and humidity are the more important environmental stressors in 3-day eventing. PMID:10659288

  8. Metabolism of ifosfamide during a 3 day infusion.

    PubMed Central

    Hartley, J. M.; Hansen, L.; Harland, S. J.; Nicholson, P. W.; Pasini, F.; Souhami, R. L.

    1994-01-01

    Urinary drug metabolites were measured in 21 patients receiving ifosfamide by continuous infusion over 3 days. Mean values for the proportion of drug excreted as parent compound, 2-dechloroethylifosfamide (2-DC), 3-dechloroethylifosfamide (3-DC), carboxyifosfamide (CX) and ifosforamide mustard (IPM) were 19, 6, 10, 7 and 8% of dose respectively. The proportion of urinary drug products in the form of ifosfamide fell considerably over the course of the 3 days. This was mirrored by an increase in the proportion of 2-DC, 3-DC and CX. The proportion in the form of IPM, however, remained unchanged. With successive cycles the amount of 2-DC and IPM increased by about 10% per course. A very wide variation in the amount of each metabolite was reproducibly seen between patients, but no evidence for a genetic polymorphism was found. Urinary dechloroethyl metabolites correlated positively with each other and negatively with CX. Although autoinduction increases 'activation' of ifosfamide when given over 3 days, our evidence suggests that competing metabolic pathways prevent an increase in the amount of active metabolite formed. PMID:8180026

  9. 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

  10. 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

  11. 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...

  12. “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

  13. Erythropoietin improves mood and modulates the cognitive and neural processing of emotion 3 days post administration.

    PubMed

    Miskowiak, Kamilla; Inkster, Becky; Selvaraj, Sudhakar; Wise, Richard; Goodwin, Guy M; Harmer, Catherine J

    2008-02-01

    Erythropoietin (Epo) has neuroprotective and neurotrophic effects and is a promising candidate for treatment of neurodegenerative and psychiatric disorder. Recently, we demonstrated that Epo modulates memory-relevant hippocampal response and fear processing in human models of antidepressant drug action 1 week post-administration, and improves self-reported mood for 3 days immediately following administration. The present study explored the effects of Epo (40 000 IU) vs saline on self-reported mood and on neural and cognitive function in healthy volunteers 3 days post-administration to test the reliability of the rapid mood improvement and its neuropsychological basis. Neuronal responses during the processing of happy and fearful faces were investigated using functional magnetic resonance imaging (fMRI); facial expression recognition performance was assessed after the fMRI scan. Daily ratings of mood were obtained for 3 days after Epo/saline administration. During faces processing Epo enhanced activation in the left amygdala and right precuneus to happy and fearful expressions. This was paired with improved recognition of all facial expressions, in particular of low intensity happiness and fear. This is similar to behavioral effects observed with acute administration of serotonergic antidepressants. Consistent with our previous finding, Epo improved self-reported mood for all 3 days post-administration. Together, these results suggest that characterization of the effects of Epo in a clinically depressed group is warranted. PMID:17473836

  14. 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.…

  15. 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…

  16. Efficacy of essential oil mouthwash with and without alcohol: a 3-Day plaque accumulation model

    PubMed Central

    2011-01-01

    Background The aim of this study was to evaluate the antiplaque effect of a new alcohol free essential oil mouthwash with respect to a control of an essential oil with alcohol mouthwash, using an in vivo plaque regrowth model of 3-days. Methods The study was designed as a double-masked, randomized, crossover clinical trial, involving 30 volunteers to compare two different essential oil containing mouthwashes, during a 3-day plaque accumulation model. After receiving a thorough professional prophylaxis at the baseline, over the next 3-days each volunteer refrained from all oral hygiene measures and had two daily rinses with 20 ml of the test mouthwash (alcohol free essential oil) or the control mouthwash (essential oil with alcohol). At the end of the each experimental period, plaque was assessed and the panelists filled out a questionnaire. Each subject underwent a 14 days washout period and there was a second allocation. Results The essential oil mouthwash with ethanol shows a better inhibitory effect of plaque regrowth in 3-days than the mouthwash test with only essential oil in the whole mouth (plaque index = 2.18 against 2.46, respectively, p < 0.05); for the lower jaw (plaque index = 2.28 against 2.57, respectively, p < 0.05); for the upper jaw (plaque index = 2.08 against 2.35, respectively, p < 0.05); for the incisors (plaque index = 1.93 against 2.27, respectively, p < 0.05); and the canines (plaque index = 1.99 against 2.47, respectively, p < 0.05). Conclusion The essential oil containing mouthwash without alcohol seems to have a less inhibiting effect on the plaque regrowth than the traditional alcoholic solution. Trial Registration ClinicalTrials.gov NCT01411618 PMID:22171999

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

    PubMed

    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 (282 mg/day) stimulates fat oxidation and lipolysis in 24 overweight subjects (age = 30 ± 2 yrs, BMI = 27.7 ± 0.3 kg/m(2)). 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

  18. 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

  19. Preflight Adjustment to Eastward Travel: 3 Days of Advancing Sleep with and without Morning Bright Light

    PubMed Central

    Burgess, Helen J.; Crowley, Stephanie J.; Gazda, Clifford J.; Fogg, Louis F.; Eastman, Charmane I.

    2005-01-01

    Jet lag is caused by a misalignment between circadian rhythms and local destination time. As humans typically take longer to re-entrain after a phase advance than a phase delay, eastward travel is often more difficult than westward travel. Previous strategies to reduce jet lag have focused on shaping the perceived light-dark cycle after arrival, in order to facilitate a phase shift in the appropriate direction. Here we tested treatments that travelers could use to phase advance their circadian rhythms prior to eastward flight. Thus, travelers would arrive with their circadian rhythms already partially re-entrained to local time. We determined how far the circadian rhythms phase advanced, and the associated side effects related to sleep and mood. Twenty-eight healthy young subjects participated in 1 of 3 different treatments, which all phase advanced each subject’s habitual sleep schedule by 1 h/day for 3 days. The 3 treatments differed in morning light exposure for the 1st 3.5 h after waking on each of the 3 days: continuous bright light (> 3000 lux), intermittent bright light (> 3000 lux, 0.5 h on, 0.5 off, etc.), or ordinary dim indoor light (< 60 lux). Aphase assessment in dim light (< 10 lux) was conducted before and after the treatments to determine the endogenous salivary dim light melatonin onset (DLMO). The mean DLMO phase advances in the dim, intermittent, and continuous light groups were 0.6, 1.5, and 2.1 h, respectively. The intermittent and continuous light groups advanced significantly more than the dim light group (p < 0.01) but were not significantly different from each other. The side effects as assessed with actigraphy and logs were small. A 2-h phase advance may seem small compared to a 6- to 9-h time zone change, as occurs with eastward travel from the USA to Europe. However, a small phase advance will not only reduce the degree of re-entrainment required after arrival, but may also increase postflight exposure to phase-advancing light relative

  20. Localization of Ascaridia galli larvae in the jejunum of chickens 3 days post infection.

    PubMed

    Luna-Olivares, Luz Adilia; Ferdushy, Tania; Kyvsgaard, Niels Christian; Nejsum, Peter; Thamsborg, Stig Milan; Roepstorff, Allan; Iburg, Tine Moesgaard

    2012-04-30

    The normal habitat of the parasitic stages of Ascaridia galli is in the small intestine of poultry but the exact localization is poorly understood. Therefore, a histological study was conducted in order to localize the larvae during the early phase of infection. Six layer pullets seven-week old were infected orally with 20,000 embryonated A. galli eggs each, whereas four chickens were left as un-infected controls. At necropsy 3 days after infection the first half of jejunum/ileum was divided into two equally sized sections (J1 and J2). After taking samples for histology from the middle of J1 and J2 and the junction between these determined JX, the two sections were subjected to parasitological examination. A higher number of A. galli larvae were recovered from section J2 than J1 and the majority of larvae were recovered from the most profound layers. Based on histology 144 larvae were identified and their location was noted. The highest number of larvae was observed in the JX sample as compared to J1 and J2 (P<0.001). Most of them were located in the profound crypt zone of the mucosa (51%) as compared to the other zones (P<0.05). The number of larvae was higher in the lumen (63%) compared to the epithelium (32%) and lamina propria (5%) (P<0.001). A significantly higher number of eosinophils were found in lamina propria of the infected group compared to the control group (P<0.001). This experiment clearly showed that only few larvae had penetrated the epithelium and were positioned in the lamina propria at 3 days post infection. It was far more common that the larvae were localized within the epithelium or in the lumen of the crypts. It is therefore suggested that at least in this early phase "mucosal phase" is a more appropriate term to be used for the A. galli larval localization as compared to the term "histotrophic phase" currently used in many textbooks. PMID:22133491

  1. 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

  2. Human muscle signaling responses to 3-day head-out dry immersion.

    PubMed

    Vilchinskaya, N A; Mirzoev, T M; Lomonosova, Y N; Kozlovskaya, I B; Shenkman, B S

    2015-09-01

    To date little is known about catabolic NO-dependent signaling systems in human skeletal muscle during early stages of gravitational unloading. The goal of the study was to analyze signaling pathways that determine the initial development of proteolytic events in human soleus muscle during short-term gravitational unloading (simulated microgravity). Gravitational unloading was simulated by 3-day head-out dry immersion. Before and after the immersion the samples of soleus muscle were taken under local anesthesia, using biopsy technique. The content of desmin, IRS-1, phospho-AMPK, total and phospho-nNOS in soleus of 6 healthy men was determined using Western-blotting before and after the dry-immersion. Three days of the dry immersion resulted in a significant decrease in desmin, phospho-nNOS and phospho-AMPK as compared to the pre-immersion values. The results of the study suggest that proteolytic processes in human soleus at the early stage of gravitational unloading are associated with inactivation of nNOS. Reduction in AMPK phosphorylation could serve as a trigger event for the development of primary atrophic changes in skeletal muscle. PMID:26350948

  3. 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.

  4. 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...

  5. Y-STR profiling in extended interval (> or = 3 days) postcoital cervicovaginal samples.

    PubMed

    Mayntz-Press, Kathleen A; Sims, Lynn M; Hall, Ashley; Ballantyne, Jack

    2008-03-01

    Depending upon specific situations, some victims of sexual assault provide vaginal samples more than 36-48 h after the incident. We have tested the ability of commercial and in-house Y-STR systems to provide DNA profiles from extended interval (> or =3 days) postcoital samples. The commercial Y-STR systems tested included the AmpFlSTR Yfiler (Applied Biosystems), PowerPlex Y (Promega) and Y-PLEX 12 (Reliagene) products whereas the in-house systems comprised Multiplex I (MPI) and Multiplex B (MPB). Three donor couples were recruited for the study. Postcoital cervicovaginal swabs (x2) were recovered by each of the three females at specified intervals after sexual intercourse (3-7 days). Each time point sample was collected after a separate act of sexual intercourse and was preceded by a 7-day abstention period. As a negative control, a precoital swab was also recovered prior to coitus for each sampling and only data from postcoital samples that demonstrated a lack of male DNA in the associated precoital sample was used. A number of DNA profile enhancement strategies were employed including sampling by cervical brushing, nondifferential DNA extraction methodology, and post-PCR purification. Full Y-STR profiles from cervicovaginal samples recovered 3-4 days after intercourse were routinely obtained. Profiles were also obtainable 5-6 days postcoitus although by this stage partial profiles rather than full profiles were a more likely outcome. The DNA profiles from the sperm fraction of a differential lysis were superior to that obtained when a nondifferential method was employed in that the allelic signal intensities were generally higher and more balanced and exhibited less baseline noise. The incorporation of a simple post-PCR purification process significantly increased the ability to obtain Y-STR profiles, particularly from 5- to 6-day postcoital samples. Remarkably an 8 locus Y-STR profile was obtained from a 7-day postcoital sample, which is approaching the

  6. Effect of 3-day dry immersion on ventilatory response to hypercapnic hypoxia

    NASA Astrophysics Data System (ADS)

    Goncharov, Alexander; Dyachenko, Alexander; Shulagin, Yury; Ermolaev, Evgeny

    . This increase was 34% and 23% respectively. Tidal volume sensitivity in tests 2 and 3 increased in comparison with test 1 (P<0.05). Points of apnea in the 2-nd test changed to increased P _{CO2} values compared with 1-st test (P<0.05} and in the tests 2 and 3 compared with the test 4 (P<0.02). Thus, the VR - P _{CO2} relationship shifted to the right and its slope increased. Conclusion: Ventilation reaction to hypercapnia combined with hypoxia was changed during 3-day dry immersion. The VR - P _{CO2} relationship shifted to the right and its slope increased.

  7. Association between Hemoglobin Levels in the First 3 Days of Life and Bronchopulmonary Dysplasia in Preterm Infants.

    PubMed

    Duan, Jun; Kong, Xiangyong; Li, Qiuping; Hua, Shaodong; Zhang, Sheng; Feng, Zhichun; Zhang, Xiaoying

    2016-08-01

    Objective The objective of this study was to determine the association between hemoglobin (Hb) levels in the first 3 days of life and bronchopulmonary dysplasia (BPD) in preterm infants. Study Design The study population comprises 147 neonates with a gestational age (GA) of less than 32 weeks who were admitted to BaYi Children's Hospital Affiliated to Beijing Military General Hospital from January 2014 to May 2015. Hb levels in the first 3 days of life, maternal and infant characteristics, were recorded and then analyzed. Results BPD patients had a lower GA and birth weight than non-BPD patients. Rates of surfactant use, use of early inhalation hormone, days of mechanical ventilation > 2 weeks, and patent ductus arteriosus in BPD patients were higher and have a significant difference. Number of transfusions was higher in BPD patients. Lower Hb levels in the first 3 days of life were also observed in BPD patients. A cutoff value of Hb levels was determined as 155.5 g/L. Hb ≤ 155 g/L in the first 3 days of life was a significant risk factor for BPD. Conclusion Our study demonstrated that lower Hb levels in the first 3 days of life may increase the risk of developing BPD in preterm infants. PMID:27120476

  8. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  9. 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

  10. An open evaluation of a 3-day course of pivmecillinam (ten 200 mg tablets) in women with acute uncomplicated cystitis.

    PubMed

    Donald, J F; Rimmer, D M

    1980-01-01

    One hundred and eighty-four women, with acute uncomplicated cystitis, received a 3-day course of pivmecillinam comprising an initial 400 mg (two tablets) dose, followed by 200 mg every 8 hours; a total of ten tablets. A satisfactory clinical response was achieved in 91% of patients. Bacteriological success was observed in 94% of sixty-eight patients with a proven infection. Side-effects were reported in sixteen patients (8.4%). Two patients ceased therapy prematurely. Pivmecillinam did not select out resistant Gram-negative organisms. A trend towards reinfection with Gram-positive cocci was observed. The possible significance of reinfection with different organisms is discussed. PMID:6245977

  11. Objective Assessment of Fall Risk in Parkinson's Disease Using a Body-Fixed Sensor Worn for 3 Days

    PubMed Central

    Weiss, Aner; Herman, Talia; Giladi, Nir; Hausdorff, Jeffrey M.

    2014-01-01

    Background Patients with Parkinson's disease (PD) suffer from a high fall risk. Previous approaches for evaluating fall risk are based on self-report or testing at a given time point and may, therefore, be insufficient to optimally capture fall risk. We tested, for the first time, whether metrics derived from 3 day continuous recordings are associated with fall risk in PD. Methods and Materials 107 patients (Hoehn & Yahr Stage: 2.6±0.7) wore a small, body-fixed sensor (3D accelerometer) on lower back for 3 days. Walking quantity (e.g., steps per 3-days) and quality (e.g., frequency-derived measures of gait variability) were determined. Subjects were classified as fallers or non-fallers based on fall history. Subjects were also followed for one year to evaluate predictors of the transition from non-faller to faller. Results The 3 day acceleration derived measures were significantly different in fallers and non-fallers and were significantly correlated with previously validated measures of fall risk. Walking quantity was similar in the two groups. In contrast, the fallers walked with higher step-to-step variability, e.g., anterior-posterior width of the dominant frequency was larger (p = 0.012) in the fallers (0.78±0.17 Hz) compared to the non-fallers (0.71±0.07 Hz). Among subjects who reported no falls in the year prior to testing, sensor-derived measures predicted the time to first fall (p = 0.0034), whereas many traditional measures did not. Cox regression analysis showed that anterior-posterior width was significantly (p = 0.0039) associated with time to fall during the follow-up period, even after adjusting for traditional measures. Conclusions/Significance These findings indicate that a body-fixed sensor worn continuously can evaluate fall risk in PD. This sensor-based approach was able to identify transition from non-faller to faller, whereas many traditional metrics were not successful. This approach may facilitate earlier detection of fall

  12. 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

  13. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  14. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  15. 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

  16. 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.

  17. Comparative efficacy of 3-day and 7-day chemotherapy with twice-daily pivmecillinam in urinary tract infections seen in general practice.

    PubMed

    Richards, H H

    1984-01-01

    In a multi-centre general practice study, 183 females suffering from symptoms of acute urinary tract infection were randomly assigned to receive 400 mg pivmecillinam twice-daily for either 3 or 7 days. The clinical response was equally good in both treatment groups with a mean reduction in symptom scores of 88%. Positive pre-treatment bacteriological cultures were obtained from 48 (36%) of the 134 patients for whom data were complete. Bacteriological cure was achieved in all these patients except for 1 in the 3-day treatment group. Pivmecillinam was well tolerated, with side-effects reported by 7 (7%) patients in the 3-day group and 12 (13%) patients in the 7-day group. One patient in the 3-day group and 2 patients in the 7-day group stopped treatment prematurely due to side-effects. PMID:6499513

  18. 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. PMID:26634419

  19. Comparative efficacy and safety of 3-day azithromycin and 10-day penicillin V treatment of group A beta-hemolytic streptococcal pharyngitis in children.

    PubMed Central

    Pacifico, L; Scopetti, F; Ranucci, A; Pataracchia, M; Savignoni, F; Chiesa, C

    1996-01-01

    The efficacy and safety of a 3-day course of azithromycin oral suspension (10 mg/kg of body weight once daily) were compared with those of penicillin V (50,000 U/kg/day in two divided doses) in children aged 3 to 12 years for the treatment of symptomatic pharyngitis caused by the group A beta-hemolytic streptococcus (GABHS). For the 154 evaluable patients, the original infecting strain of GABHS was eliminated at the end of follow-up (34 to 36 days after treatment started) from 67 (85.8%) of 78 penicillin-treated patients and 41 (53.9%) of 76 azithromycin-treated patients (P < 0.0001). Overall clinical success was achieved in 71 (91.0%) of 78 penicillin V-treated patients and 57 (75.0%) of 76 azithromycin-treated patients (P < 0.05). Potential drug-related adverse events were reported for 5.5 and 8.6% of the penicillin V- and azithromycin-treated patients, respectively (P = 0.6). In the present study, a once-daily (10 mg/kg), 3-day oral regimen of azithromycin was as safe as a 10-day course of penicillin but did not represent an effective alternative to penicillin for the treatment of GABHS pharyngitis, even for those children with azithromycin-susceptible strains. PMID:8849215

  20. 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.

  1. 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.

  2. Immunochromatographic assay on thread.

    PubMed

    Zhou, Gina; Mao, Xun; Juncker, David

    2012-09-18

    Lateral-flow immunochromatographic assays are low-cost, simple-to-use, rapid tests for point-of-care screening of infectious diseases, drugs of abuse, and pregnancy. However, lateral flow assays are generally not quantitative, give a yes/no answer, and lack multiplexing. Threads have recently been proposed as a support for transporting and mixing liquids in lateral-flow immunochromatographic assays, but their use for quantitative high-sensitivity immunoassays has yet to be demonstrated. Here, we introduce the immunochromatographic assay on thread (ICAT) in a cartridge format that is suitable for multiplexing. The ICAT is a sandwich assay performed on a cotton thread knotted to a nylon fiber bundle, both of which are precoated with recognition antibodies against one target analyte. Upon sample application, the assay results become visible to the eye within a few minutes and are quantified using a flatbed scanner. Assay conditions were optimized, the binding curves for C-reactive protein (CRP) in buffer and diluted serum were established and a limit of detection of 377 pM was obtained. The possibility of multiplexing was demonstrated using three knotted threads coated with antibodies against CRP, osteopontin, and leptin proteins. The performance of the ICAT was compared with that of the paper-based and conventional assays. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests. PMID:22889381

  3. 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.

  4. Rapid mercury assays

    SciTech Connect

    Szurdoki, S.; Kido, H.; Hammock, B.D.

    1996-10-01

    We have developed rapid assays with the potential of detecting mercury in environmental samples. our methods combine the simple ELISA-format with the selective, high affinity complexation of mercuric ions by sulfur-containing ligands. The first assay is based on a sandwich chelate formed by a protein-bound ligand immobilized on the wells of a microliter plate, mercuric ion of the analyzed sample, and another ligand conjugated to a reporter enzyme. The second assay involves competition between mercuric ions and an organomercury-conjugate to bind to a chelating conjugate. Several sulfur containing chelators (e.g., dithiocarbamates) and organomercurials linked to macromolecular carriers have been investigated in these assay formats. The assays detect mercuric ions in ppb/high ppt concentrations with high selectivity.

  5. 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.

  6. A comprehensive survey of atmospheric quasi 3 day planetary-scale waves and their impacts on the day-to-day variations of the equatorial ionosphere

    NASA Astrophysics Data System (ADS)

    Liu, Guiping; England, Scott L.; Immel, Thomas J.; Frey, Harald U.; Mannucci, Anthony J.; Mitchell, Nicholas J.

    2015-04-01

    This study reports a comprehensive survey of quasi 3 day (2.5-4.5 day period) planetary-scale waves in the low-latitude mesosphere and lower thermosphere using the temperature observations from Thermosphere Ionosphere and Mesosphere Electric Dynamics/Sounding of the Atmosphere using Broadband Emission Radiometry throughout 2002-2012. Occurrences and properties of the waves, including the eastward propagating zonal wave numbers of 1-3 (E1-E3) and vertical wavelengths, are determined for each case. The impacts of these waves on the equatorial ionosphere are investigated by searching for the corresponding variations with the same periods and wave numbers in total electron content (TEC) from the concurrent observations of the ground-based GPS network. For a threshold amplitude of 4 K in temperature, a total of 300 waves are identified, of which there are 186 E1, 63 E2, and 51 E3 events. The mean amplitudes and vertical wavelengths of these waves are calculated to be about 7.9 K and 34 km for the E1, 5.7 K and 29 km for the E2, and 5.1 K and 27 km for the E3, having the standard deviations of 1.5 K and 6.5 km, 0.6 K and 5.6 km, and 0.5 K and 6.7 km. Occurrences of the E1 cases are not observed to depend on season, but the large-amplitude (>8 K) cases occur more often during solstices than at equinoxes. Similarly, the E2 and E3 cases are observed to occur most often in January-February and May-August. Among these waves, 199 cases (66%) are found to have the corresponding variations in the equatorial ionosphere with amplitudes ≥4.2% relative to the mean TEC values (corresponding to 90th percentile). Most of these waves have long vertical wavelengths and large amplitudes (˜3 times more than short vertical wavelength and small-amplitude waves). Because no seasonal or solar cycle dependence on the frequency at which these waves have corresponding variations in the ionosphere at this TEC perturbation threshold is observed, we conclude that there is no seasonal and solar

  7. 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

  8. 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

  9. 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.

  10. 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. PMID:27154596

  11. 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.

  12. 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. PMID:25590683

  13. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle: effect of a 3-day, high-fat diet.

    PubMed

    Jordy, Andreas B; Serup, Annette K; Karstoft, Kristian; Pilegaard, Henriette; Kiens, Bente; Jeppesen, Jacob

    2014-11-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hypercaloric and high-fat diet regime. Muscle biopsies were taken before and after the diet intervention, and giant sarcolemmal vesicles were prepared. The high-fat diet induced decreased insulin sensitivity, but this was not associated with a relocation of FAT/CD36 or FABPpm protein to the sarcolemma. However, FAT/CD36 and FABPpm mRNA, but not the proteins, were upregulated by increased fatty acid availability. This suggests a time dependency in the upregulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after a high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein was located intracellularly but not at the sarcolemma in humans. PMID:25163924

  14. 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

  15. 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.

  16. 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

  17. New evidence for gait abnormalities among Parkinson's disease patients who suffer from freezing of gait: insights using a body-fixed sensor worn for 3 days.

    PubMed

    Weiss, Aner; Herman, Talia; Giladi, Nir; Hausdorff, Jeffrey M

    2015-03-01

    Previous studies conducted in laboratory settings suggest that the gait pattern in between freezing of gait (FOG) episodes is abnormal among patients with Parkinson's disease (PD) who suffer from FOG (i.e., "freezers"), compared to those who do not (i.e., "non-freezers"). We evaluated whether long-term recordings also reveal gait alterations in freezers and if these features were related to freezing severity and its impact on daily function. 72 patients with PD wore a 3-D accelerometer for 3 days. Acceleration-derived gait features included quantity (e.g., the amount of walking) and quality measures (e.g., gait variability). The New FOG-Questionnaire evaluated the subject's perceptions of FOG severity and its impact. Age, gender, and disease duration were similar (p > 0.19) in the 28 freezers and 44 non-freezers. Walking quantity was similar in the two groups, while freezers walked with higher gait variability (i.e., larger anterior-posterior power spectral density width; p = 0.003) and lower gait consistency (i.e., lower vertical stride regularity; p = 0.007). Group differences were observed when comparing the typical (i.e., median), best, and worst performance among the multiple walking bouts measured. Vertical and medio-lateral gait consistency were associated with the impact of FOG on daily living (r < -0.39, p < 0.044). The present findings demonstrate that freezers have altered gait variability and consistency during spontaneous community ambulation, even during optimal performance, and that these measures are associated with the impact of FOG on daily function. Long-term recordings may provide new insights into PD and augment the monitoring of FOG and its response to therapy. PMID:25069586

  18. 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.

  19. 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

  20. Lateral flow strip assay

    SciTech Connect

    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.

  1. 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.

  2. 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.

  3. Macroautophagic cargo sequestration assays.

    PubMed

    Seglen, Per O; Luhr, Morten; Mills, Ian G; Sætre, Frank; Szalai, Paula; Engedal, Nikolai

    2015-03-01

    Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy. PMID:25576638

  4. Hyperhydration prior to a simulated second day of the 3-day moderate intensity equestrian competition does not cause arterial hypoxemia in Thoroughbred horses.

    PubMed

    Tennent-Brown, B S; Goetz, T E; Manohar, M; Hassan, A S; Freeman, D E; Bundy, J S; Evans, M R

    2006-07-01

    Dehydration and the associated impairment of cardiovascular and thermoregulatory function comprise major veterinary problems in horses performing prolonged exercise, particularly under hot and humid conditions. For these reasons, there is considerable interest in using pre-exercise hyperhydration to help maintain blood volume in the face of the excessive fluid loss associated with sweat production during prolonged exertion. However, recently it was reported that pre-exercise hyperhydration causes arterial hypoxemia in horses performing moderate intensity exercise simulating the second day of an equestrian 3-day event competition (E3DEC) which may adversely affect performance (Sosa Leon et al. in Equine Vet J Suppl 34:425-429, 2002). These findings are contrary to data from horses performing short-term maximal exertion, wherein hyperhydration did not affect arterial O2 tension/saturation. Thus, our objective in the present study was to examine the impact of pre-exercise hyperhydration on arterial oxygenation of Thoroughbred horses performing an exercise test simulating the second day of an E3DEC. Control and hyperhydration studies were carried out on seven healthy Thoroughbred horses in random order, 7 days apart. In the control study, horses received no medications. In the hyperhydration experiments, nasogastric administration of NaCl (0.425 g/kg) 5 h pre-exercise induced a plasma volume expansion of 10.9% at the initiation of exercise. This methodology for inducing hypervolemia was different from that of Sosa Leon et al. (2002). Blood-gas tensions/pH as well as plasma protein, hemoglobin and blood lactate concentrations were measured pre-exercise and during the exercise test. Our data revealed that pre-exercise hyperhydration neither adversely affected arterial O2 tension nor hemoglobin-O2 saturation at any time during the exercise test simulating the second day of an E3DEC. Further, it was observed that arterial blood CO2 tension, pH, and blood lactate

  5. Routine resite of peripheral intravenous devices every 3 days did not reduce complications compared with clinically indicated resite: a randomised controlled trial

    PubMed Central

    2010-01-01

    Background Peripheral intravenous device (IVD) complications were traditionally thought to be reduced by limiting dwell time. Current recommendations are to resite IVDs by 96 hours with the exception of children and patients with poor veins. Recent evidence suggests routine resite is unnecessary, at least if devices are inserted by a specialised IV team. The aim of this study was to compare the impact of peripheral IVD 'routine resite' with 'removal on clinical indication' on IVD complications in a general hospital without an IV team. Methods A randomised, controlled trial was conducted in a regional teaching hospital. After ethics approval, 362 patients (603 IVDs) were randomised to have IVDs replaced on clinical indication (185 patients) or routine change every 3 days (177 patients). IVDs were inserted and managed by the general hospital medical and nursing staff; there was no IV team. The primary endpoint was a composite of IVD complications: phlebitis, infiltration, occlusion, accidental removal, local infection, and device-related bloodstream infection. Results IVD complication rates were 68 per 1,000 IVD days (clinically indicated) and 66 per 1,000 IVD days (routine replacement) (P = 0.86; HR 1.03; 95% CI, 0.74-1.43). Time to first complication per patient did not differ between groups (KM with log rank, P = 0.53). There were no local infections or IVD-related bloodstream infections in either group. IV therapy duration did not differ between groups (P = 0.22), but more (P = 0.004) IVDs were placed per patient in the routine replacement (mean, 1.8) than the clinical indication group (mean, 1.5), with significantly higher hospital costs per patient (P < 0.001). Conclusions Resite on clinical indication would allow one in two patients to have a single cannula per course of IV treatment, as opposed to one in five patients managed with routine resite; overall complication rates appear similar. Clinically indicated resite would achieve savings in equipment, staff

  6. 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

  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. Robust quantitative scratch assay

    PubMed Central

    Vargas, Andrea; Angeli, Marc; Pastrello, Chiara; McQuaid, Rosanne; Li, Han; Jurisicova, Andrea; Jurisica, Igor

    2016-01-01

    The wound healing assay (or scratch assay) is a technique frequently used to quantify the dependence of cell motility—a central process in tissue repair and evolution of disease—subject to various treatments conditions. However processing the resulting data is a laborious task due its high throughput and variability across images. This Robust Quantitative Scratch Assay algorithm introduced statistical outputs where migration rates are estimated, cellular behaviour is distinguished and outliers are identified among groups of unique experimental conditions. Furthermore, the RQSA decreased measurement errors and increased accuracy in the wound boundary at comparable processing times compared to previously developed method (TScratch). Availability and implementation: The RQSA is freely available at: http://ophid.utoronto.ca/RQSA/RQSA_Scripts.zip. The image sets used for training and validation and results are available at: (http://ophid.utoronto.ca/RQSA/trainingSet.zip, http://ophid.utoronto.ca/RQSA/validationSet.zip, http://ophid.utoronto.ca/RQSA/ValidationSetResults.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975Results.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip). Supplementary Material is provided for detailed description of the development of the RQSA. Contact: juris@ai.utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26722119

  10. Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro.

    PubMed

    Jakštys, Baltramiejus; Ruzgys, Paulius; Tamošiūnas, Mindaugas; Šatkauskas, Saulius

    2015-10-01

    The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6-72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2-3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results. PMID:26077843

  11. Macrophage Inflammatory Assay

    PubMed Central

    Ylostalo, Joni H.

    2016-01-01

    Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’. In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16–18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).

  12. 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

  13. 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+.

  14. 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+.

  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. HIV-1 Fusion Assay

    PubMed Central

    Cavrois, Marielle; Neidleman, Jason; Greene, Warner C.

    2016-01-01

    The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).

  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. PMID:26498795

  19. Molecular inversion probe assay.

    PubMed

    Absalan, Farnaz; Ronaghi, Mostafa

    2007-01-01

    We have described molecular inversion probe technologies for large-scale genetic analyses. This technique provides a comprehensive and powerful tool for the analysis of genetic variation and enables affordable, large-scale studies that will help uncover the genetic basis of complex disease and explain the individual variation in response to therapeutics. Major applications of the molecular inversion probes (MIP) technologies include targeted genotyping from focused regions to whole-genome studies, and allele quantification of genomic rearrangements. The MIP technology (used in the HapMap project) provides an efficient, scalable, and affordable way to score polymorphisms in case/control populations for genetic studies. The MIP technology provides the highest commercially available multiplexing levels and assay conversion rates for targeted genotyping. This enables more informative, genome-wide studies with either the functional (direct detection) approach or the indirect detection approach. PMID:18025701

  20. 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

  1. Membrane Flotation Assay

    PubMed Central

    Vogt, Dorothee A; Ott, Melanie

    2016-01-01

    Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10–20–30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.

  2. 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...

  3. 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

  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. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays. PMID:25027375

  7. 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

  8. Transwell(®) invasion assays.

    PubMed

    Marshall, John

    2011-01-01

    The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell(®) Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of the gel and a chemoattractant on the other side of the gel, invasion is determined by counting those cells that have traversed the cell-permeable membrane having invaded towards the higher concentration of chemoattractant. In this chapter, in addition to protocols for performing Transwell invasion assays, there is consideration of the limitations of current assay designs with regard to available matrices and the absence of tumour microenvironment cells. PMID:21748672

  9. 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

  10. Development of a PCR Assay for Rapid Detection of Enterococci

    PubMed Central

    Ke, Danbing; Picard, François J.; Martineau, Francis; Ménard, Christian; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    1999-01-01

    Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR

  11. 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).

  12. 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. PMID:26108286

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

    PubMed Central

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

    2015-01-01

    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. PMID:26108286

  14. Prenatal diagnosis of the Hunter syndrome and the introduction of a new fluorimetric enzyme assay.

    PubMed

    Keulemans, J L M; Sinigerska, I; Garritsen, V H; Huijmans, J G M; Voznyi, Y V; van Diggelen, O P; Kleijer, W J

    2002-11-01

    Prenatal diagnosis of the Hunter syndrome (mucopolysaccharidosis type II; MPS II) is preferably achieved by the assay of iduronate-2-sulphate sulphatase (IDS) in uncultured chorionic villi (CV) as this allows early (12th week), rapid (2-3 days) and reliable results. We summarize the results of 174 prenatal analyses in the past 30 years, using various methods such as radiolabelled sulphate incorporation in amniotic fluid (AF) cells, glycosaminoglycan (GAG)-electrophoresis in AF and IDS assay in CV, CV-cells, AF and AF-cells. Twenty-seven fetuses with MPS II were diagnosed after finding clearly abnormal results in pregnancies with a male fetus; very low IDS activity has also been measured in some pregnancies with a (heterozygous) female fetus, emphasizing the need to combine enzyme assay with fetal sex determination. IDS activity has until recently been assessed by a cumbersome radioactive enzyme assay. Here we describe the use of a novel fluorigenic 4-methylumbelliferyl substrate, which allows a sensitive, rapid and convenient assay of IDS activity and reliable early prenatal diagnosis. This novel IDS assay was validated in retrospective analyses of 14 CV, CV-cell, AF and AF-cell samples from affected pregnancies in addition to prospective prenatal diagnosis in eight pregnancies at risk with one MPS II-affected fetus. PMID:12424767

  15. 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. PMID:26714703

  16. 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 Central

    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. PMID:21941432

  17. 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.

  18. 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.

  19. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  20. 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.

  1. 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

  2. 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

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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

  9. 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.

  10. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  11. 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.

  12. 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. PMID:26608293

  13. Protein binding assay for hyaluronate

    SciTech Connect

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

    1986-11-01

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

  14. 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.

  15. 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.

  16. A 3-day regimen with azithromycin 1.5% eyedrops for the treatment of purulent bacterial conjunctivitis in children: efficacy on clinical signs and impact on the burden of illness

    PubMed Central

    Bremond-Gignac, Dominique; Messaoud, Riadh; Lazreg, Sihem; Speeg-Schatz, Claude; Renault, Didier; Chiambaretta, Frédéric

    2015-01-01

    Purpose To compare the efficacy of azithromycin 1.5% versus tobramycin 0.3% eyedrops on clinical ocular signs and symptoms of bacterial conjunctivitis in children and to assess the parents’ satisfaction regarding the dosing regimen. Patients and methods An international, multicenter, randomized, investigator-masked, controlled clinical trial conducted in children (1 day to 18 years old) with bulbar conjunctival hyperemia and purulent discharge. Azithromycin 1.5% was administered as 1 drop twice daily for 3 days, and tobramycin 0.3% as 1 drop every 2 hours for 2 days, then 4 times daily for 5 days. Results A total of 286 patients (mean age: 3.2 years) were enrolled. In children with bacteriologically positive cultures (N=203), azithromycin produced a significantly greater improvement in conjunctival discharge (P<0.01) and a trend (P=0.054) toward improvement in conjunctival hyperemia at day 7 than did tobramycin. Complete resolution of conjunctival discharge was significantly more frequent at day 3 on azithromycin than tobramycin (P=0.005). More parents found azithromycin easier to use (in terms of treatment duration, total number of instillations, instilling drops during the day, and difficulty in performing daily activities) than tobramycin. Conclusion The azithromycin 1.5% regimen produced a rapid resolution of cardinal signs of purulent bacterial conjunctivitis with a more convenient dosage regimen. Such improved convenience is likely to improve compliance and lessen the burden of illness for patients and carers. PMID:25945033

  17. The effects of serum, lithium, ethacrynic acid, and a low external concentration of potassium on specific [3H]-ouabain binding to human lymphocytes after incubation for 3 days.

    PubMed Central

    Rapeport, W G; Aronson, J K; Grahame-Smith, D G; Harper, C

    1986-01-01

    We have quantified specific [3H]-ouabain binding sites in normal human lymphocytes, and have measured the changes in the numbers of those sites which occur in response to various stimuli. We have confirmed previous findings that incubation for 72 h in the presence of fetal calf serum causes an increase in [3H]-ouabain binding, and that this does not occur if the cells are incubated in fetal calf serum which has first been dialysed. During incubation of the lymphocytes for 3 days in the presence of dialysed fetal calf serum each of the following stimuli caused an increase in specific [3H]-ouabain binding: addition of ethacrynic acid (1 mumol l-1), addition of lithium (1 mmol l-1), and reduction of the external potassium concentration (to 0.75 mmol l-1). By analogy with the similar results in HeLa cells reported by others, we suggest that the increase in [3H]-ouabain binding may, in the case of ethacrynic acid and the reduction of the external potassium concentration, be initiated by an increase in the intracellular sodium concentration. The mechanisms whereby fetal calf serum and lithium cause an increase in [3H]-ouabain binding are not clear. PMID:3768239

  18. Predictive assays in radiation therapy

    SciTech Connect

    West, C.M.L.

    1994-12-31

    There are reports of promising correlations between patient response to radiotherapy and laboratory measurements of tumor radiosensitivity, fibroblast radiosensitivity, tumor proliferation, and tumor oxygenation status. These all need to be substantiated in large clinical studies. The development of rapid, reliable assays, in particular for determining intrinsic radiosensitivity, would greatly facilitate this work. If the results illustrated in the figures in the chapter can be combined and shown to be feasible on a routine clinical basis, then radiobiologists would be able to provide radiotherapists with a useful aid for the individualization of patient treatment. 162 refs., 6 figs., 6 tabs.

  19. Automated cytopathic effect (CPE) assays.

    PubMed

    McAleer, W J; Miller, W J; Hurni, W M; Machlowitz, R A; Hilleman, M R

    1983-07-01

    An automated CPE procedure has been developed that increases the precision and ease of performing titrations of measles, mumps and rubella viruses in vaccine materials. By this procedure, additions of cell suspensions and reagents and the dilution of samples are performed automatically by a modified Dynatiter instrument, using 96-well microtitre plates. Cell monolayers are stained with carbolfuchsin dye to eliminate the need for microscopic examination. Finally, the trays are read in an optical scanner and the end points calculated automatically by a programmable calculator. The increased accuracy and precision attained by performing greater numbers of replicate assays at reasonable cost will be of particular value to vaccine manufacturers. PMID:6885830

  20. Indirect conductimetric assay of antibacterial activities.

    PubMed

    Sawai, J; Doi, R; Maekawa, Y; Yoshikawa, T; Kojima, H

    2002-11-01

    The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. PMID:12407467

  1. The chemistry behind antioxidant capacity assays.

    PubMed

    Huang, Dejian; Ou, Boxin; Prior, Ronald L

    2005-03-23

    This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays. PMID:15769103

  2. 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

  3. 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.

  4. 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.

  5. The validity of androgen assays

    PubMed Central

    Carruthers, Malcolm; Trinick, Tom R.; Wheeler, Michael J.

    2007-01-01

    Problems in the measurement of androgens and in interpreting results have been reviewed and classified as follows: Preanalytical factors The exact sampling conditions in relation to circadian and seasonal variations, diet, alcohol, physical activity and posture. Physiological and medical factors Androgen levels vary according to the patient's general health, stress, sexual activity and smoking habits. Analytical variables Sample preservation and storage variables are often unknown. The different androgen assays used have widely differing accuracy and precision and are subject to large inter-laboratory variation, which especially in women and children can render the results of routinely available direct immunoassays meaningless. Interpretation of results Laboratory reference ranges vary widely, largely independent of methodology, and fail to take into account the log-normal distribution of androgen values, causing errors in clinical diagnosis and treatment. Other unknowns are antagonists such as SHBG, estrogens, catecholamines, cortisol, and anti-androgens. As well as age, androgen receptor polymorphisms play a major role in regulating androgen levels and resistance to their action. Conclusions Though laboratory assays can support a diagnosis of androgen deficiency in men, they should not be used to exclude it. It is suggested that there needs to be greater reliance on the history and clinical features, together with careful evaluation of the symptomatology, and where necessary a therapeutic trial of androgen treatment given. PMID:17701661

  6. In vitro Tumorsphere Formation Assays

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells and within in vivo tumors (Lo et al., 2012; Liu et al., 2009). While several cell lines can be used for tumorsphere formation assay (e.g. primary mammary tumor cells from Her2/neu-transgenic mice, MCF7, BT474 and HCC1954), some cell lines may not form typical tumorsphere structures and may be difficult to count or classify definitively as tumorspheres.

  7. 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.

  8. Proteasome Assay in Cell Lysates

    PubMed Central

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  9. 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.

  10. 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.

  11. Oxygen Control For Bioreactors And In-vitro Cell Assays

    NASA Astrophysics Data System (ADS)

    Nock, V.; Blaikie, R. J.; David, T.

    2009-07-01

    Dissolved oxygen (DO) is an important parameter in biomedical and cell-culture applications. Several studies have found cell survival and function to be intimately linked to oxygen concentration. Laminar flow, as observed in microfluidic devices, provides an ideal environment to manipulate and control concentration gradients. In this paper we demonstrate the first characterization of integrated fluorescence-based oxygen sensors for DO measurement within a cell-culture bioreactor device. Solid-state PtOEPK/PS sensor patterns were integrated into the PDMS-based bioreactor and calibrated for detection of DO concentration with a superimposed layer of collagen and Ishikawa human endometrial cancer cells. The sensor signal of the layer subjacent to the cells was found to follow a Stern-Volmer model and the intensity ratio was measured to I0/I100 = 3.9 after 3 days in culture. The device provides a novel tool for the control and spatially-resolved measurement of oxygen levels in cellular assays and cell-culture applications.

  12. 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.

  13. 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

  14. Northwest Australia's Saladin crude assayed

    SciTech Connect

    Rhodes, A.K.

    1993-10-18

    High-quality Saladin crude oil from offshore Western Australia has been assayed. The 48.2[degree] API, 0.02 wt % sulfur crude's characteristics--determined in 1990--are presented here for the first time. The estimated 30--40 million bbl field, south of Barrow Island, is produced from two platforms in 58 ft of water in block TP 3. Production began in late 1989 from three platforms with three wells each and from two wells drilled directionally from Thevenard Island. The paper lists data on the following properties: API gravity, density, sulfur content, pour point, flash point, viscosity, salinity, heat of combustion, ash content, asphaltene content, wax content, and metal content for the whole crude and various fractions.

  15. Steroid Assays in Paediatric Endocrinology

    PubMed Central

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation. Conflict of interest:None declared. PMID:21274330

  16. Evaluating 6 ricin field detection assays.

    PubMed

    Slotved, Hans-Christian; Sparding, Nadja; Tanassi, Julia Tanas; Steenhard, Nina R; Heegaard, Niels H H

    2014-01-01

    This study presents data showing the performance of 6 commercial detection assays against ricin around concentrations specified as detection limits by the producers. A 2-fold dilution series of 20 ng/ml ricin was prepared and used for testing the lateral-flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. Three of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. We conclude that when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality. PMID:24978020

  17. 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.

  18. 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.

  19. Radiometric assays for glycerol, glucose, and glycogen.

    PubMed

    Bradley, D C; Kaslow, H R

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays. PMID:2817333

  20. Radiometric assays for glycerol, glucose, and glycogen

    SciTech Connect

    Bradley, D.C.; Kaslow, H.R. )

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with (32P)ATP and glycerokinase, residual (32P)ATP is hydrolyzed by heating in acid, and free (32P)phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.

  1. 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.

  2. 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.

  3. 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.

  4. LT-HSC Methylcellulose Assay

    PubMed Central

    Kerenyi, Marc A.

    2016-01-01

    Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an LT-HSC itself (self-renews) to maintain the LT-HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells (Orkin and Zon, 2008). Quantification of phenotypic LT-HSCs can be performed by multi-color flow cytometry and the gold standard for assessment of LT-HSC self-renewal and function is competitive bone marrow transplantation (Miller et al., 2008). Although these methods are irreplaceable to determine LT-HSC abundance and functionality, they have their disadvantages and limitations. For example, competitive bone marrow transplantation is typically monitored as a function of peripheral blood donor contribution over 12–16 weeks. While reduced peripheral blood donor contribution by itself signifies impairment in the stem/progenitor cells compartment, it cannot unambiguously discriminate between reduced LT-HSC self-renewal, impaired LT-HSC differentiation or compromised progenitor cell differentiation. Here we describe an LT-HSCs methylcellulose colony-forming assay, as a fast complementary in vitro method to directly assess LT-HSC differentiation capacity. As described in Kerenyi et al. (2013), this technique acts as a powerful tool to differentiate between LT-HSC or progenitor cell differentiation defects.

  5. Exploring the dark side of MTT viability assay of cells cultured onto electrospun PLGA-based composite nanofibrous scaffolding materials.

    PubMed

    Qi, Ruiling; Shen, Mingwu; Cao, Xueyan; Guo, Rui; Tian, Xuejiao; Yu, Jianyong; Shi, Xiangyang

    2011-07-21

    One major method used to evaluate the biocompatibility of porous tissue engineering scaffolding materials is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT cell viability assay is based on the absorbance of the dissolved MTT formazan crystals formed in living cells, which is proportional to the number of viable cells. Due to the strong dye sorption capability of porous scaffolding materials, we propose that the cell viability determined from the MTT assay is likely to give a false negative result. In this study, we aim to explore the effect of the adsorption of MTT formazan on the accuracy of the viability assay of cells cultured onto porous electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, HNTs (halloysite nanotubes)/PLGA, and CNTs (multiwalled carbon nanotubes)/PLGA composite nanofibrous mats. The morphology of electrospun nanofibers and L929 mouse fibroblasts cultured onto the nanofibrous scaffolds were observed using scanning electron microscopy. The viability of cells proliferated for 3 days was evaluated through the MTT assay. In the meantime, the adsorption of MTT formazan onto the same electrospun nanofibers was evaluated and the standard concentration-absorbance curve was obtained in order to quantify the contribution of the adsorbed MTT formazan during the MTT cell viability assay. We show that the PLGA, and the HNTs- or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to 35.6-50.2% deviation from the real cell viability assay data. The better dye sorption capability of the nanofibers leads to further deviation from the real cell viability. Our study gives a general insight into accurate MTT cytotoxicity assessment of various porous tissue engineering scaffolding materials, and may be applicable to other colorimetric assays for analyzing the biological properties of porous scaffolding materials. PMID:21647502

  6. Improvement of Microbial Assays of Vitamins

    PubMed Central

    Heed, Edward J.

    1972-01-01

    A method for the improvement of microbial assays of vitamins, which involves the addition of a surfactant to the incubated test, was developed. This surfactant tends to eliminate bacterial clumping, giving a uniform suspension of single cells, thereby making the turbidity readings less erratic and the actual assay standard curves more closely related to the desired theoretical curve. PMID:4627232

  7. Microtissue Culture Plaque Assay for Herpesvirus saimiri

    PubMed Central

    Chan, Emerson W.; Dunkel, Virginia C.

    1973-01-01

    A microtissue culture method for the plaque assay of Herpesvirus saimiri has been developed. Virus titrations carried out in Microtest II tissue culture plates (Falcon) yielded reproducible results that agreed well with those obtained by employing macrocultures. The described method is quantitative, reproducible, economical, and suitable for routine assay of large numbers of virus samples. Images PMID:4201642

  8. A bioluminescent assay for measuring glucose uptake.

    PubMed

    Valley, Michael P; Karassina, Natasha; Aoyama, Natsuyo; Carlson, Coby; Cali, James J; Vidugiriene, Jolanta

    2016-07-15

    Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts. PMID:27130501

  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. Fluorescence polarization assays in signal transduction discovery.

    PubMed

    Sportsman, J Richard; Daijo, Janet; Gaudet, Elizabeth A

    2003-05-01

    Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP. PMID:12678698

  11. 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....

  12. 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....

  13. 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....

  14. 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....

  15. 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....

  16. 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....

  17. 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....

  18. 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....

  19. 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....

  20. 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....

  1. Micronucleus assay in aquatic animals.

    PubMed

    Bolognesi, Claudia; Hayashi, Makoto

    2011-01-01

    Aquatic pollutants produce multiple consequences at organism, population, community and ecosystem level, affecting organ function, reproductive status, population size, species survival and thus biodiversity. Among these, carcinogenic and mutagenic compounds are the most dangerous as their effects may exert a damage beyond that of individual and may be active through several generations. The application of genotoxicity biomarkers in sentinel organisms allows for the assessment of mutagenic hazards and/or for the identification of the sources and fate of the contaminants. Micronucleus (MN) test as an index of accumulated genetic damage during the lifespan of the cells is one of the most suitable techniques to identify integrated response to the complex mixture of contaminants. MN assay is today widely applied in a large number of wild and transplanted aquatic species. The large majority of studies or programmes on the genotoxic effect of the polluted water environment have been carried out with the use of bivalves and fish. Haemocytes and gill cells are the target tissues most frequently considered for the MN determination in bivalves. The MN test was widely validated and was successfully applied in a large number of field studies using bivalves from the genera Mytilus. MN in fish can be visualised in different cell types: erythrocytes and gill, kidney, hepatic and fin cells. The use of peripheral erythrocytes is more widely used because it avoids the complex cell preparation and the killing of the animals. The MN test in fish erythrocytes was validated in laboratory with different species after exposure to a large number of genotoxic agents. The erythrocyte MN test in fish was also widely and frequently applied for genotoxicity assessment of freshwater and marine environment in situ using native or caged animals following different periods of exposure. Large interspecies differences in sensitivity for MN induction were observed. Further validation studies are

  2. Assays for Determination of Protein Concentration.

    PubMed

    Olson, Bradley J S C

    2016-01-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. PMID:27248579

  3. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  4. 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

  5. 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

  6. 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

  7. 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

  8. A lateral electrophoretic flow diagnostic assay

    PubMed Central

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

    2015-01-01

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a “lateral e-flow assay” and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings. PMID:25608872

  9. Methods for threshold determination in multiplexed assays

    DOEpatents

    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.

  10. 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.

  11. Practical assay for nitrite and nitrosothiol as an alternative to the Griess assay or the 2,3-diaminonaphthalene assay.

    PubMed

    Shen, Yanming; Zhang, Quanjuan; Qian, Xuhong; Yang, Youjun

    2015-01-20

    Nitrite is a heavily assayed substrate in the fields of food safety, water quality control, disease diagnosis, and forensic investigation and more recently in basic biological studies on nitric oxide physiology and pathology. The colorimetric Griess assay and the fluorimetric 2,3-diaminonaphthalene (DAN) assay are the current gold standards for nitrite quantification. They are not without limitations, yet have amazingly survived 156 and 44 years, respectively, due to the lack of a practical alternative. Both assays exhibit slow detection kinetics due to inactivation of nucleophiles under strongly acidic media, require an extensive incubation time for reaction to go completion, and hence offer a limited detection throughput. By converting an intermolecular reaction of the Griess assay intramolecularly, we designed a novel probe (NT555) for nitrite detection, which displays superior detection kinetics and sensitivity. NT555 was constructed following our "covalent-assembly" probe design principle. Upon detection, it affords a gigantic bathochromic shift of the absorption spectrum and a sensitive turn-on fluorescence signal from a zero background, both of which are typical of an "assembly" type probe. Overall, NT555 has addressed various difficulties associated with the Griess and the DAN assays and represents an attractive alternative for practical applications. PMID:25519711

  12. 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.

  13. Precision estimates for tomographic nondestructive assay

    SciTech Connect

    Prettyman, T.H.

    1995-12-31

    One technique being applied to improve the accuracy of assays of waste in large containers is computerized tomography (CT). Research on the application of CT to improve both neutron and gamma-ray assays of waste is being carried out at LANL. For example, tomographic gamma scanning (TGS) is a single-photon emission CT technique that corrects for the attenuation of gamma rays emitted from the sample using attenuation images from transmission CT. By accounting for the distribution of emitting material and correcting for the attenuation of the emitted gamma rays, TGS is able to achieve highly accurate assays of radionuclides in medium-density wastes. It is important to develope methods to estimate the precision of such assays, and this paper explores this problem by examining the precision estimators for TGS.

  14. 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

  15. LIMITATIONS OF THE FLUORESCENT PROBE VIABILITY ASSAY

    EPA Science Inventory

    Cell viability commonly is determined flow cytometrically by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. FDA is taken up by the viable cell and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF). F fluorescence intensity is...

  16. Variables Affecting Two Electron Transport System Assays

    PubMed Central

    Burton, G. Allen; Lanza, Guy R.

    1986-01-01

    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methanol was unsatisfactory. Phosphate buffer (0.06 M) produced higher activity than did either U.S. Environmental Protection Agency reconstituted hard water or Tris buffer sediment diluents. Intracellular formazan crystals were dissolved within minutes when in contact with immersion oil. Greater crystal production (respiration) detected by a tetrazolium salt assay occurred at increased substrate concentrations. Test diluents containing macrophyte exudates produced greater activity than did phosphate buffer, U.S. Environmental Protection Agency water, or ultrapure water diluents. Both assays showed decreases in sediment or bacterial activity through time. PMID:16347067

  17. Scrape Loading/Dye Transfer Assay.

    PubMed

    Babica, Pavel; Sovadinová, Iva; Upham, Brad L

    2016-01-01

    The scrape loading/dye transfer (SL/DT) technique is a simple functional assay for the simultaneous assessment of gap junctional intercellular communication (GJIC) in a large population of cells. The equipment needs are minimal and are typically met in standard cell biology labs, and SL/DT is the simplest and quickest of all the assays that measure GJIC. This assay has also been adapted for in vivo studies. The SL/DT assay is also conducive to a high-throughput setup with automated fluorescence microscopy imaging and analysis to elucidate more samples in shorter time, and hence can serve a broad range of in vitro pharmacological and toxicological needs. PMID:27207291

  18. 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. ...

  19. 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

  20. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  1. CONTROL ASSAY DEVELOPMENT: METHODOLOGY AND LABORATORY VERIFICATION

    EPA Science Inventory

    The report describes Control Assay Development (CAD), a data acquisition program designed to evaluate the potential applicability of various treatment processes for the control of solid, liquid, and gaseous emissions from coal conversion plants. The CAD program described could be...

  2. 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.

  3. Rapid assays for environmental and biological monitoring.

    PubMed

    Szurdoki, F; Jaeger, L; Harris, A; Kido, H; Wengatz, I; Goodrow, M H; Székács, A; Wortberg, M; Zheng, J; Stoutamire, D W; Sanborn, J R; Gilman, S D; Jones, A D; Gee, S J; Choudary, P V; Hammock, B D

    1996-05-01

    Rapid, inexpensive, sensitive, and selective enzyme-linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s-triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury. PMID:8642182

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Immunoperoxidase inhibition assay for rabies antibody detection.

    PubMed

    Batista, H B C R; Lima, F E S; Maletich, D; Silva, A C R; Vicentini, F K; Roehe, L R; Spilki, F R; Franco, A C; Roehe, P M

    2011-06-01

    An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG-peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the "simplified fluorescence inhibition microtest" (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available. PMID:21458492

  9. An ultrafiltration assay for lysyl oxidase.

    PubMed

    Shackleton, D R; Hulmes, D J

    1990-03-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware. PMID:1971160

  10. Comparative responses of three rat strains (DA/Han, Sprague-Dawley and Wistar) to treatment with environmental estrogens.

    PubMed

    Diel, P; Schmidt, S; Vollmer, G; Janning, P; Upmeier, A; Michna, H; Bolt, H M; Degen, G H

    2004-04-01

    The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Although much attention has been paid to identifying protocol variables and reproducibility between laboratories the question whether toxicodynamic and toxicokinetic variations of different strains may affect their sensitivity to estrogenic stimuli has been rarely addressed. We have compared the estrogenic activity of the environmental chemicals genistein (GEN), bisphenol A (BPA) and p- tert-octylphenol (OCT) in DA/Han (DA), Sprague-Dawley (SD) and Wistar (WIS) rats after repeated oral application. Rats were treated per os for 3 days with different doses of these weakly estrogenic compounds and the potent reference estrogen ethinylestradiol (EE). Then uterine wet weight, thickness of the uterine epithelium, uterine gene expression of clusterin (CLU), and thickness of the vaginal epithelium were examined as parameters for estrogenic potency of the test compounds in the three strains of rats. The uterotrophic response to treatment with BPA, OCT and GEN was similar in the three strains, and allowed us to rank them as GEN being more potent than OCT, and BPA being the weakest estrogen. This was confirmed by analysis of other biological endpoints, despite some differences in the magnitude of their response among strains and to distinct compounds. For instance, the uterus wet weight response to EE treatment indicated lower sensitivity of SD rats than that of DA and WIS rats, but this was not observed for responses of the uterine or vaginal epithelium. Moreover, blood concentrations were assessed at the time of killing and related to biological responses: plasma levels of total and unconjugated BPA and GEN depended upon the dose administered and varied to some extent within treatment groups and among the three rat strains. However, there was no good correlation in the three strains between individual compound concentrations analysed 24 h after the

  11. 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

  12. 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

  13. Novel Phakopsora pachyrhizi Extracellular Proteins Are Ideal Targets for Immunological Diagnostic Assays

    PubMed Central

    McMahon, Michael B.; Edwards, H. Herb; Boerma, Britney L.; Lewis Ivey, Melanie L.; Miller, Sally A.; Dorrance, Anne E.

    2012-01-01

    Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots. PMID:22447596

  14. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

    PubMed Central

    Shin, Yoojin; Han, Sewoon; Jeon, Jessie S.; Yamamoto, Kyoko; Zervantonakis, Ioannis K.; Sudo, Ryo; Kamm, Roger D.; Chung, Seok

    2014-01-01

    This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel incorporating chambers between surface-accessible microchannels. Using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flows and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 days to fabricate the system and experiments can run for up to several weeks. PMID:22678430

  15. 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

  16. 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

  17. Evaluation of three gentamicin serum assay techniques

    SciTech Connect

    Matzke, G.R.; Gwizdala, C.; Wery, J.; Ferry, D.; Starnes, R.

    1982-01-01

    This investigation was designed to compare the enzyme-modified immunoassay (Syva--EMIT) with a radioimmunoassay (New England Nuclear--RIA) and the radiometric assay (Johnston--BACTEC) to determine the optimal assay for use in our aminoglycoside dosing service. The serum concentration determinations obtained via the three assay methods were analyzed by linear regression analysis. Significant positive correlations were noted between the three assay techniques (p less than 0.005) during both sample collection phases. The coefficients of determination for EMIT vs BACTEC and RIA vs BACTEC were 0.73 and 0.83 during phase 1, respectively, and 0.65 and 0.68 during phase 2, respectively. The slope of the regression lines also varied markedly during the two phases; 0.49 and 0.42 for EMIT and for RIA vs BACTEC, respectively, during phase 1 compound with 1.12 and 0.77, respectively, during phase 2. The differences noted in these relationships during phase 1 and 2 may be related to the alteration of the pH of the control sera utilized in the BACTEC assay. In contrast, RIA vs EMIT regression analysis indicated that existence of a highly significant relationship (p less than 0.0005 and r2 . 0.90). The EMIT technique was the easiest and most accurate for determination of serum gentamicin concentrations, whereas the BACTEC method was judged unacceptable for clinical use.

  18. Comet assay to sense neutron 'fingerprint'.

    PubMed

    Gajendiran, N; Tanaka, K; Kamada, N

    2000-09-18

    The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125-1 Gy) and compared with the effects resulting from irradiation with (60)Co gamma. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by (60)Co gamma. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation. PMID:11024477

  19. An improved molecular assay for Tritrichomonas foetus.

    PubMed

    Grahn, R A; BonDurant, R H; van Hoosear, K A; Walker, R L; Lyons, L A

    2005-01-01

    Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment. PMID:15619373

  20. Microplate Assay for Colletotrichum Spore Production

    PubMed Central

    Slade, S. J.; Harris, R. F.; Smith, C. S.; Andrews, J. H.; Nordheim, E. V.

    1987-01-01

    A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs. PMID:16347310

  1. Microbiological assay of ketoconazole in shampoo.

    PubMed

    Staub, Inara; Schapoval, Elfrides E S; Bergold, Ana M

    2005-03-23

    Ketoconazole, an anti-fungal agent, is often incorporated in several pharmaceutical forms and in shampoo formulation it is known to be effective against fungal infection on the scalp. This paper describes a method to quantify ketoconazole in shampoo by comparing the cylinder plate assay and the HPLC method. The test organism used for the agar diffusion assay was Candida albicans ATCC 10231. Three different concentrations of ketoconazole were used for the diffusion assay. A mean zone diameter was obtained for each concentration. A standard curve was obtained by plotting the three values derived from the zone diameters. A prospective validation of the method showed that the method was linear (r = 0.9982), precise (R.S.D. = 2.57%) and accurate. The results obtained by the two methods were statistically evaluated by analysis of variance (ANOVA) and the results obtained indicate that there is no significant difference between these two methods. PMID:15725566

  2. Assays of Transendothelial Migration in vitro

    PubMed Central

    Luscinskas, F. William

    2009-01-01

    The inflammatory response is critical for our ability to heal wounds and fight off foreign microorganisms. Uncontrolled inflammation is also at the root of most pathology. Recruitment of leukocytes to the site of inflammation plays a defining role in the inflammatory response, and migration of leukocytes across endothelium is arguably the point of no return of the inflammatory response. Assays to study the transmigration of leukocytes have and will continue to shed light on the regulation of this vital response. Assays of transendothelial migration in vitro allow the controlled observation of this phenomenon as well as experiments to study its regulation. In this chapter we describe in vitro assays of transendothelial migration that have been used successfully in the authors’ laboratories for decades and have proven to be reproducible, reliable, and predictive of the behavior of leukocytes and endothelial cells in models of inflammation in vivo. PMID:18772016

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. A High-Throughput Radiometric Kinase Assay.

    PubMed

    Duong-Ly, Krisna C; Peterson, Jeffrey R

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  8. 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.

  9. Nanoparticles for Use in Enzyme Assays.

    PubMed

    Kim, Young-Pil; Kim, Hak-Sung

    2016-02-01

    Nanoparticles (NPs) have created new ways to enhance the performance of classical biosensors in analytical sciences. NPs with unprecedented physiochemical properties can serve both as excellent carriers of bioreceptors and as signal enhancers, leading to improved assay platforms with high sensitivity and selectivity. Because enzymes play central roles in many cellular functions, specific and precise assays of their functions are of great significance in medical science and biotechnology. Here we review recent advances in NP-based biosensors and their use in enzyme assays. With fast and specific responses to enzyme-mediated reactions, NPs transduce and amplify the initial responses into various types of signals, such as electrochemical, optical and magnetic ones. Translation of their potential should lead to functionalized NPs finding wide applications in diagnostics, drug development and biotechnology. PMID:26662229

  10. Robot speeds assays and enhances safeguards

    SciTech Connect

    Phelan, P.F.; Powell, W.D.; Blankenship, R.W.

    1990-01-01

    At the Los Alamos National Laboratory Plutonium Facility, a robotics system utilizing a gantry robot and an automated inventory system operates five calorimeters and two gamma isotopic assay instruments. This system has significantly improved safeguards, because the opportunity for diversion has been greatly reduced. Not only is the accountability much more timely because throughput has doubled but the special nuclear material has been made physically more secure in several ways. First, items awaiting assay are kept in the inventory system, whose doors remain locked whenever the robot is unattended. An alarm sounds if the doors are unlocked without authorization. Second, light curtains surround the robot's work envelope and pressure-sensitive pads cover the floor to detect entry into the assay area. Third, the robot weighs each item whenever it is moved, and the result is compared with the weight that was measured when the item was first put into inventory. 2 refs., 3 figs.

  11. Harmonization of the intracellular cytokine staining assay.

    PubMed

    Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H

    2012-07-01

    Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables. PMID:22714399

  12. 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/

  13. Method validation and clinical utility of chromogenic factor VIII assay compared to one-stage assay.

    PubMed

    Gouws, Wilmare; Botha, Elsabie; Visser, Adele

    2014-01-01

    The chromogenic FVIII assay is currently considered the gold standard for quantitation of factor VIII levels in both haemophilia A patients and as part of screening for thrombophilia. A method validation and evaluation of clinical utility within a routine diagnostic laboratory was undertaken by comparing the currently used one-stage assay to a commercially available chromogenic assay (Siemens, Johannesburg, South Africa). In total, 60 samples were included in this study to encompass the whole diagnostic range of the assay. Both low and high values showed very good correlation on linear regression analysis with correlation coeffients of 0.949 and 0.888 respectively. However, the lower detection limit of the Siemens Chromogenic assay was 1.5 IU/dL rendering it impossible to utilize in the setting of classifying a haemophilia A patient in terms of disease severity. Although the Siemens FVIII chromogenic assay shows excellent correlation to the currently used one-stage assay, the relatively high detection limit restrict implementation as a stand-alone assay in a routine diagnostic laboratory. PMID:23504571

  14. Operant assays for assessing pain in preclinical rodent models: highlights from an orofacial assay.

    PubMed

    Murphy, Niall P; Mills, Richard H; Caudle, Robert M; Neubert, John K

    2014-01-01

    Despite an immense investment of resources, pain remains at epidemic proportions. Given this, there has been an increased effort toward appraising the process by which new painkillers are developed, focusing specifically on why so few analgesics make it from the benchside to the bedside. The use of behavioral assays and animal modeling for the preclinical stages of analgesic development is being reexamined to determine whether they are truly relevant, meaningful, and predictive. Consequently, there is a strengthening consensus that the traditional reflex-based assays upon which several decades of preclinical pain research has been based are inadequate. Thus, investigators have recently turned to the development of new preclinical assays with improved face, content, and predictive validity. In this regard, operant pain assays show considerable promise, as they are more sensitive, present better validity, and, importantly, better encompass the psychological and affective dimensions of pain that trouble human pain sufferers. Here, we briefly compare and contrast reflex assays with operant assays, and we introduce a particular operant orofacial pain assay used in a variety of experiments to emphasize how operant pain assays can be applied to preclinical studies of pain. PMID:25103871

  15. Bicinchoninic acid (BCA) assay in low volume.

    PubMed

    Bainor, Anthony; Chang, Lyra; McQuade, Thomas J; Webb, Brian; Gestwicki, Jason E

    2011-03-15

    The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format. PMID:21078286

  16. 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

  17. 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.

  18. 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. PMID:26551162

  19. A Rapid and Quantitative Recombinase Activity Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

  20. 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.

  1. 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…

  2. 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.

  3. INTERLABORATORY COMPARISON OF CHOLINESTERASE ASSAY MEASUREMENTS

    EPA Science Inventory

    Twelve wildlife toxicology laboratories participated in an interlaboratory survey of cholinesterase (ChE) assays to determine comparability of absolute ChE values and estimates of ChE inhibition from organophosphorus insecticide-dosed birds and to examine the type and consistency...

  4. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  5. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  6. 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...

  7. 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...

  8. 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...

  9. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  10. 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...

  11. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  12. 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...

  13. 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...

  14. 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...

  15. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  16. 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...

  17. 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...

  18. 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...

  19. A radiometric assay for HIV-1 protease

    SciTech Connect

    Hyland, L.J.; Dayton, B.D.; Moore, M.L.; Shu, A.Y.; Heys, J.R.; Meek, T.D. )

    1990-08-01

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

  20. 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…

  1. DEVELOPMENT OF AN 'IN VITRO' NEUROTOXICITY ASSAY

    EPA Science Inventory

    The aim of this research project was to investigate the possibility of developing the neurotoxic esterase (NTE) assay into a totally in vitro system to enable neurotoxicity assessment to be carried out rapidly on large numbers of compounds. The purified enzyme was to be immobiliz...

  2. Wafer-scale Mitochondrial Membrane Potential Assays

    PubMed Central

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

    2012-01-01

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

  3. 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.

  4. Homogeneous assay technology based on upconverting phosphors.

    PubMed

    Kuningas, Katri; Rantanen, Terhi; Ukonaho, Telle; Lövgren, Timo; Soukka, Tero

    2005-11-15

    Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening. PMID:16285685

  5. 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

  6. Paraprotein interference with turbidimetric gentamicin assay

    PubMed Central

    Bassett, Kendra; Brown, Nigel

    2015-01-01

    Introduction Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays. Materials and methods A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser. Results The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery. Conclusion IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol. PMID:25672475

  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. PMID:25410901

  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. 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.

  10. 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.

  11. 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... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  12. 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... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  13. 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... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  14. 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... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  15. 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... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  16. 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

  17. Liquid chromatography-tandem mass spectrometric assay for the light sensitive survivin suppressant sepantronium bromide (YM155) in mouse plasma.

    PubMed

    Dolman, M Emmy M; den Hartog, Ilona J M; Molenaar, Jan J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2014-04-01

    A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for sepantronium bromide (YM155), an inhibitor of survivin, was developed and validated. Under reduced light exposure, plasma samples were pre-treated using protein precipitation with acetonitrile containing AT7519 as internal standard. After dilution with water, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.5-100ng/ml calibration range with r(2)=0.9981±0.0007 using double logarithmic calibration (n=5). Within day precisions (n=6) were 3.6-8.8% and between day (3 days; n=18) precisions 6.5-11.1%. Accuracies were between 92 and 111% for the whole calibration range. The light sensitive drug sepantronium was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine plasma drug levels in mice after administration of sepantronium bromide by continuous infusion from subcutaneously implanted osmotic pumps. PMID:24518133

  18. 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. PMID:26332947

  19. 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

  20. 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.

  1. Nondestructive assay methods for solids containing plutonium

    SciTech Connect

    Macmurdo, K.W.; Gray, L.W.; Gibbs, A.

    1984-06-01

    Specific nondestructive assay (NDA) methods, e.g. calorimetry, coincidence neutron counting, singles neutron counting, and gamma ray spectrometry, were studied to provide the Savannah River Plant with an NDA method to measure the plutonium content of solid scrap (slag and crucible) generated in the JB-Line plutonium metal production process. Results indicate that calorimetry can be used to measure the plutonium content to within about 3% in 4 to 6 hours by using computerized equilibrium sample power predictive models. Calorimetry results confirm that a bias exists in the present indirect measurement method used to estimate the plutonium content of slag and crucible. Singles neutron counting of slag and crucible can measure plutonium to only +-30%, but coincidence neutron counting methods improve measurement precision to better than +-10% in less than ten minutes. Only four portions of a single slag and crucible sample were assayed, and further study is recommended.

  2. DNA Origami Seesaws as Comparative Binding Assay.

    PubMed

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

    2016-06-16

    The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami-based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw-like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays. PMID:27038073

  3. 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

  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. 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

  6. 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.

  7. System and method for assaying a radionuclide

    DOEpatents

    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.

  8. System and method for assaying radiation

    DOEpatents

    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.

  9. 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.

  10. High-Throughput Cell Toxicity Assays.

    PubMed

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  11. 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. PMID:19454791

  12. 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

  13. Small Sample Assay Station users guide

    SciTech Connect

    Adams, E.L.; Bourret, S.; Meier, M.M.

    1981-03-01

    A system for acquisition of delayed neutron data, based on an LSI-11 with 28 K words of memory, is described. Hardware features are a six-channel scaler and level sensor to determine the state of the experiment; and normal peripherals include dual floppy-disk drive, line printer, and CRT terminal. The software for experiment control and for the analysis of data is presented. The protocol for assays that optimally utilize the system is suggested.

  14. Yemen's light, sweet Alif crude assayed

    SciTech Connect

    Rhodes, A.K.

    1994-05-23

    Crude oil from Yemen's Alif field has been assayed. The light sweet crude, also known as Marib, is part of the Marib al-Jawf concession in northern Yemen. Alif field was discovered in 1984 by Hunt Oil Co. The field was declared commercial in November 1985. Alif production averaged 118,500 b/d in 1992. Physical and chemical properties are listed for the whole crude and its fractions.

  15. 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.

  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. Assaying the reporter gene chloramphenicol acetyltransferase

    SciTech Connect

    Crabb, D.W.; Minth, C.D.; Dixon, J.E.

    1989-01-01

    These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product.

  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. 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.

  20. The Engineered SVA Trans-mobilization Assay.

    PubMed

    Bock, Anja; Schumann, Gerald G

    2016-01-01

    Mammalian genomes harbor autonomous retrotransposons coding for the proteins required for their own mobilization, and nonautonomous retrotransposons, such as the human SVA element, which are transcribed but do not have any coding capacity. Mobilization of nonautonomous retrotransposons depends on the recruitment of the protein machinery encoded by autonomous retrotransposons. Here, we summarize the experimental details of SVA trans-mobilization assays which address multiple questions regarding the biology of both nonautonomous SVA elements and autonomous LINE-1 (L1) retrotransposons. The assay evaluates if and to what extent a noncoding SVA element is mobilized in trans by the L1-encoded protein machinery, the structural organization of the resulting marked de novo insertions, if they mimic endogenous SVA insertions and what the roles of individual domains of the nonautonomous retrotransposon for SVA mobilization are. Furthermore, the highly sensitive trans-mobilization assay can be used to verify the presence of otherwise barely detectable endogenously expressed functional L1 proteins via their marked SVA trans-mobilizing activity. PMID:26895056

  1. 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

  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, 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.

  4. High-content screening for biofilm assays.

    PubMed

    Peng, Fubing; Hoek, Eric M V; Damoiseaux, Robert

    2010-08-01

    The authors describe a novel high-throughput screening platform that provides rapid, reliable, quantitative assessment of biofilm formation and removal on engineered surfaces. Unlike traditional biofilm assays based on plate readers, this assay platform is based on high-content screening, which allows for multiplexing to simultaneously quantify the number of bacterial adhesions per unit area and the viability of adhered cells using fluorescent dye combinations. This platform is fully automated and has a throughput of more than 10,000 wells per day. The authors used this platform to examine the influence of different assay buffer systems on bacterial adhesion, viability, and removal on cross-linked polyvinyl alcohol coating films synthesized directly onto the bottoms of 384-well plates. The results indicated that water chemistry, bacteria cell type, and film chemistry combine to govern biofilm formation. In general, both reversible and irreversible bacterial adhesion increased with the extent of cross-linking in coating films, which correlates strongly with coating film cross-linking degree and hydrophobicity, which is closely related. The high-throughput platform offers a powerful tool for rapid evaluation of fouling-resistant coating films in addition to elucidation of fundamental mechanisms governing bacterial adhesion. PMID:20639506

  5. Allele-specific expression assays using Solexa

    PubMed Central

    Main, Bradley J; Bickel, Ryan D; McIntyre, Lauren M; Graze, Rita M; Calabrese, Peter P; Nuzhdin, Sergey V

    2009-01-01

    Background Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts. Results We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation. Conclusion We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets. PMID:19740431

  6. 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

  7. Tumor promotion: models and assay systems.

    PubMed

    Fitzgerald, D J; Yamasaki, H

    1990-01-01

    Tumor promotion is defined operationally from two-stage models of experimental carcinogenesis. It is, therefore, in a strict sense, possible to identify tumor promoters only from such models. The development and use of in vitro two-stage cell transformation assays was a logical extension toward in vitro short-term testing for tumor promoters. Another approach is to apply mechanistic knowledge of the tumor promotion process in developing end points for such assays. In this context, we have been examining the role of blocked gap-junctional intercellular communication (GJIC) in tumor promotion, using in vitro and in vivo systems. Many promoters have been shown to block GJIC in vitro; our studies support the idea that inhibition of GJIC does play an important role in the promotion stage of BALB/c 3T3 cell transformation. In animal studies, we have shown that the rat liver tumor promoter phenobarbital can decrease the level of expression of the 32 Kd gap junction protein gene specifically in liver upon systemic exposure in rats. Further examination of the role of GJIC in tumor promotion is indeed warranted. Also, deployment of in vitro GJIC and transformation assay systems should provide useful short-term tests for detecting tumor promoting activity of environmental chemicals. PMID:1973858

  8. [Interference of ethylene glycol on lactate assays].

    PubMed

    Graïne, H; Toumi, K; Roullier, V; Capeau, J; Lefèvre, G

    2007-01-01

    Ethylene glycol is broken down to three main organic acids: glycolic acid, glyoxylic acid and oxalic acid which cause severe metabolic acidosis. Effect of these three acids on lactate assays was evaluated in five blood gas analysers and two clinical chemistry analysers. For all systems, no influence of oxalic acid on lactate results could be demonstrated. No interference of glycolic acid could be observed on lactate assay performed with Rapid Lab 1265 (R: 104,9 +/- 12,1%), Vitros 950 (R: 105,7 +/- 5,3 %) and Architect ci8200 (R: 104,9 +/- 4,7%), but on the contrary, CCX 4, OMNI S, ABL 725 and 825 demonstrated a concentration-dependent interference. No interference of glyoxylic acid could be observed with Vitros 950, but a positive interference could be observed with ABL 725 and 825, OMNI S, CCX4 and Architect ci8200 A linear relationship between apparent lactate concentration found with ABL 725 and 825, OMNI S, CCX 4, and glyoxylic acid could be observed (0,94 < r < 0,99), a weaker interference being observed with Rapid Lab 1265 and Architect ci 8200. Our results demonstrated that in case of ethylene glycol poisoning, cautious interpretation of lactate assay should be done, since wrong results of lactacidemia could lead to misdiagnostic and delay patient treatment. PMID:17627925

  9. Quality control of antibodies for assay development.

    PubMed

    Schumacher, Sarah; Seitz, Harald

    2016-09-25

    Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test. PMID:26873787

  10. 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.

  11. In vitro Cell Migration and Invasion Assays

    PubMed Central

    Justus, Calvin R.; Leffler, Nancy; Ruiz-Echevarria, Maria; Yang, Li V.

    2014-01-01

    Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup. PMID:24962652

  12. High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay.

    PubMed

    Jones, P A; King, A V

    2003-01-01

    Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2-3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring approximately 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 microg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 microg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 microg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring <1 mg test material, giving a substantial increase in productivity. Any material selected for further development and inclusion in a formulation may require further confirmatory testing, e.g. using a human skin model assay for phototoxicity. PMID:14599466

  13. 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.

  14. Evaluation of Chikungunya Diagnostic Assays: Differences in Sensitivity of Serology Assays in Two Independent Outbreaks

    PubMed Central

    Yap, Grace; Pok, Kwoon-Yong; Lai, Yee-Ling; Hapuarachchi, Hapuarachchige-Chanditha; Chow, Angela; Leo, Yee-Sin; Tan, Li-Kiang; Ng, Lee-Ching

    2010-01-01

    Background The sensitivity and specificity of two in-house MAC-ELISA assays were tested and compared with the performance of commercially-available CTK lateral flow rapid test and EUROIMMUN IFA assays for the detection of anti-Chikungunya virus (CHIKV) IgM. Each MAC-ELISA assay used a whole virus-based antigen derived from genetically distinct CHIKV strains involved in two chikungunya disease outbreaks in Singapore (2008); a January outbreak strain with alanine at amino acid residue 226 of the E1 glycoprotein (CHIKV-A226) and a May-to-September outbreak strain that possessed valine at the same residue (CHIKV-226V). We report differences in IgM detection efficacy of different assays between the two outbreaks. The sensitivities of two PCR protocols were also tested. Methods and Findings For sera from January outbreak, the average detection threshold of CTK lateral flow test, MAC-ELISAs and EUROIMMUN IFA assays was 3.75, 4.38 and 4.88 days post fever onset respectively. In contrast, IgM detection using CTK lateral flow test was delayed to more than 7 days after fever onset in the second outbreak sera. However, MAC-ELISA using CHIKV-226V detected IgM in the second outbreak sera 3.96 days after fever onset, which was approximately one day earlier compared to the same assay using CHIKV-A226 (4.86 days). Specificity was 100% for both commercial assays, and 95.6% for the in-house MAC-ELISAs. For sensitivity determination of the PCR protocols, the probe-based real time RT-PCR method was found to be 10 times more sensitive than one based on SYBR Green. Conclusion Our findings suggested that the two strains of CHIKV using variants A226 and 226V resulted in variation in sensitivities of the assays evaluated. We postulated that the observed difference in antigen efficacy could be due to the amino acid substitution differences in viral E1 and E2 envelope proteins, especially the E1-A226V substitution. This evaluation demonstrates the importance of appraisal of different

  15. 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.

  16. Automated Protein Assay Using Flow Injection Analysis

    NASA Astrophysics Data System (ADS)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  17. 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.

  18. 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.

  19. Assays of thyroid-stimulating antibody

    SciTech Connect

    McKenzie, J.M.; Zakarija, M.

    1985-01-01

    A comparison is presented of the two major assay methods of thyroid-stimulating antibody (TSAb) of Graves' disease. The basic procedures involve: (1) some index of thyroid stimulation, usually in vitro, using TSAb to indicate its activity; and (2) indirect recognition by assessment of the inhibition of binding of radioiodinated thyrotropin (TSH) to a preparation of its receptor, i.e., TSH-binding inhibition or TBI. There is potential for misinterpretation of data acquired by testing patients' sera by one or the other basic procedure.

  20. 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.

  1. 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)

  2. 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.

  3. A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

    PubMed

    Kim, Yong Hyun; Sathiyanarayanan, Ganesan; Kim, Hyun Joong; Bhatia, Shashi Kant; Seo, Hyung-Min; Kim, Jung-Ho; Song, Hun-Seok; Kim, Yun-Gon; Park, Kyungmoon; Yang, Yung-Hun

    2015-12-28

    A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way. PMID:26282689

  4. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

    PubMed Central

    VanDussen, Kelli L; Marinshaw, Jeffrey M; Shaikh, Nurmohammad; Miyoshi, Hiroyuki; Moon, Clara; Tarr, Phillip I; Ciorba, Matthew A; Stappenbeck, Thaddeus S

    2014-01-01

    Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualized medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (i.e., barrier function and host-microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n = 65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within two weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarized monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusion This culture system will facilitate the study of inter-individual, functional studies of human intestinal epithelial cells, including host-microbial interactions. PMID:25007816

  5. 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. PMID:26019209

  6. Developing predictive assays: the phenotypic screening "rule of 3".

    PubMed

    Vincent, Fabien; Loria, Paula; Pregel, Marko; Stanton, Robert; Kitching, Linda; Nocka, Karl; Doyonnas, Regis; Steppan, Claire; Gilbert, Adam; Schroeter, Thomas; Peakman, Marie-Claire

    2015-06-24

    Phenotypic drug discovery approaches can positively affect the translation of preclinical findings to patients. However, not all phenotypic assays are created equal. A critical question then follows: What are the characteristics of the optimal assays? We analyze this question and propose three specific criteria related to the disease relevance of the assay-system, stimulus, and end point-to help design the most predictive phenotypic assays. PMID:26109101

  7. Performance Characteristics of Xpert Flu/RSV XC Assay

    PubMed Central

    Popowitch, Elena B.

    2015-01-01

    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. PMID:26019209

  8. 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

  9. Reporting biological assay screening results for maximum impact.

    PubMed

    Bolton, Evan

    2015-07-01

    A very large corpus of biological assay screening results exist in the public domain. The ability to compare and analyze this data is hampered due to missing details and lack of a commonly used terminology to describe assay protocols and assay endpoints. Minimum reporting guidelines exist that, if followed, would greatly enhance the utility of biological assay screening data so it may be independently reproduced, readily integrated, effectively compared, and rapidly analyzed. PMID:26194585

  10. 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. PMID:22964101

  11. Comparison of Luminescence ADP Production Assay and Radiometric Scintillation Proximity Assay for Cdc7 Kinase

    PubMed Central

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

    2013-01-01

    Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [33P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [33P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay. PMID:21564015

  12. Digital microfluidic assay for protein detection.

    PubMed

    Mok, Janine; Mindrinos, Michael N; Davis, Ronald W; Javanmard, Mehdi

    2014-02-11

    Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase. PMID:24449893

  13. 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

  14. 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. 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.

  15. Cellulase assays: Methods from empirical mathematical models

    SciTech Connect

    Adney, W.S.; Baker, J.O.; Himmel, M.E.

    1993-12-31

    Recent advances in the assay of cellulose preparations by Sattler et al. [Biotech. Bioeng. 33, 1221-1234 (1989)] describe a relationship between extent of hydrolysis, reaction time, and enzyme concentration. This procedure permits the ranking of the effectiveness of different enyzmes and of different pretreatment methods. For this method, cellulose hydrolysis data collected from hyperbolic functions of substrate concentration versus cellulose enzyme concentration at various timed incubations are collected. A double reciprocal plot based on the relationship (Y/C{sub 6}){sup {minus}1}=(KC/{sub 0}/Y{sub max})[E]{sup {minus}1} + (Y{sub max}/C{sub 0}){sup {minus}1} where Y/C{sub 0} is the fraction of substrate hydrolyzed; [E] is given in FPU/g substrate initially added; and Y{sub max}/C{sub 0} is the where Y/C{sub 0} is the fraction of substrate hydrolyzed an an infinite enzyme concentration. The y-axis intercept in the double reciprocal plot, (Y{sub max}/C{sub 0}){sup {minus}1}, may be used to quantify the {open_quotes}quality{close_quotes} of the enzyme preparation. ideally, an enzyme should have a high Y{sub max} and a low value for KC{sub 0}/Y{sub max}. The application of these assay methods to the hydrolysis of both clean, microcrystallite cellulose (Sigmacell 50) and pretreated aspen wood metal will be presented.

  16. 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

  17. A quantitative assay for intercellular aggregation

    NASA Technical Reports Server (NTRS)

    Neelamegham, S.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    In an earlier communication (Munn et al., J Immunol. Methods 166: 11-25, 1993), we presented the initial development of a quantitative assay for monitoring the rates of cellular aggregation based on digital image processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is introduced to characterize the three-dimensional morphology of the aggregates. This index is based on temporal changes in the projected area of the cells and cell aggregates during the course of the experiment. By drawing an analogy with the kinetic theory of gases, we have also introduced a procedure to normalize for variations in cell seeding density among different experiments. In addition, the image analysis technique has been improved by introducing a background subtraction algorithm to remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysis procedure, thus minimizing user intervention and improving the reproducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte aggregation.

  18. 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

  19. 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.

  20. 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.

  1. Microfluidic integration for automated targeted proteomic assays.

    PubMed

    Hughes, Alex J; Lin, Robert K C; Peehl, Donna M; Herr, Amy E

    2012-04-17

    A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic "single-channel, multistage" immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories. PMID:22474344

  2. 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

  3. 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)

  4. 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.

  5. High Throughput Danio Rerio Energy Expenditure Assay.

    PubMed

    Williams, Savannah Y; Renquist, Benjamin J

    2016-01-01

    Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers). PMID:26863590

  6. 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

  7. 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.

  8. 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. PMID:20023265

  9. 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.

  10. 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...

  11. 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...

  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. Some target assay uncertainties for passive neutron coincidence counting

    SciTech Connect

    Ensslin, N.; Langner, D.G.; Menlove, H.O.; Miller, M.C.; Russo, P.A.

    1990-01-01

    This paper provides some target assay uncertainties for passive neutron coincidence counting of plutonium metal, oxide, mixed oxide, and scrap and waste. The target values are based in part on past user experience and in part on the estimated results from new coincidence counting techniques that are under development. The paper summarizes assay error sources and the new coincidence techniques, and recommends the technique that is likely to yield the lowest assay uncertainty for a given material type. These target assay uncertainties are intended to be useful for NDA instrument selection and assay variance propagation studies for both new and existing facilities. 14 refs., 3 tabs.

  14. 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

  15. 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.

  16. Benchmark West Texas Intermediate crude assayed

    SciTech Connect

    Rhodes, A.K.

    1994-08-15

    The paper gives an assay of West Texas Intermediate, one of the world's market crudes. The price of this crude, known as WTI, is followed by market analysts, investors, traders, and industry managers around the world. WTI price is used as a benchmark for pricing all other US crude oils. The 41[degree] API < 0.34 wt % sulfur crude is gathered in West Texas and moved to Cushing, Okla., for distribution. The WTI posted prices is the price paid for the crude at the wellhead in West Texas and is the true benchmark on which other US crudes are priced. The spot price is the negotiated price for short-term trades of the crude. And the New York Mercantile Exchange, or Nymex, price is a futures price for barrels delivered at Cushing.

  17. Assay of Endocannabinoid Oxidation by Cyclooxygenase-2.

    PubMed

    Kudalkar, Shalley N; Kingsley, Philip J; Marnett, Lawrence J

    2016-01-01

    The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA), are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive, and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter, we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in vitro and cell assays. PMID:27245906

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Indicator displacement assays inside live cells.

    PubMed

    Norouzy, Amir; Azizi, Zahra; Nau, Werner M

    2015-01-12

    The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells. PMID:25430503

  4. 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.

  5. Assaying Cell Cycle Status Using Flow Cytometry.

    PubMed

    Kim, Kang Ho; Sederstrom, Joel M

    2015-01-01

    In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation-specific marker (Ki-67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined. PMID:26131851

  6. 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.

  7. 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

  8. 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.

  9. Microfluidic Magnetic Bead Assay for Cell Detection.

    PubMed

    Liu, Fan; KC, Pawan; Zhang, Ge; Zhe, Jiang

    2016-01-01

    We present a novel cell detection device based on a magnetic bead cell assay and microfluidic Coulter counting technology. The device cannot only accurately measure cells size distribution and concentration but also detect specific target cells. The device consists of two identical micro Coulter counters separated by a fluid chamber where an external magnetic field is applied. Antibody-functionalized magnetic beads were bound to specific antigens expressed on the target cells. A high-gradient magnetic field was applied to the chamber closer to the second counter via an external cylindrical magnet. Because of the magnetic interaction between the magnetic beads and the magnetic field, target cells were retarded by the magnetic field; transit time of a target cell (bound with magnetic beads) passing through the second counter was longer than that through the first counter. In comparison, transit times of a nontarget cell remained nearly the same when it passed through both counters. Thus, from the transit time delay we can identify target cells and quantify their concentration in a cell suspension. The transit time and the size of each cell were accurately measured in terms of the width and amplitude of the resistive pulses generated from the two Coulter counters. Experiments demonstrated that for mixed cells with various target cell ratios, the transit time delay increased approximately linearly with the increasing target cell ratio. The limit of detection (LOD) of the assay was estimated to be 5.6% in terms of target cell ratio. Cell viability tests further demonstrated that most cells were viable after the detection. With the simple device configuration and easy sample preparation, this rapid and reliable method is expected to accurately detect target cells and could be applied to facilitate stem cell isolation and characterization. PMID:26636715

  10. Validation of a von Willebrand factor antigen enzyme-linked immunosorbent assay and newly developed collagen-binding assay

    PubMed Central

    Burgess, Hilary; Wood, Darren

    2008-01-01

    No single test is comprehensive enough to detect all of the variants of von Willebrand Disease (VWD), making determination of both concentration and function of von Willebrand Factor (VWF) important for an accurate diagnosis. The objective of the study was to validate a newly developed VWF collagen binding assay (VWF:CB) and VWF antigen enzyme-linked immunosorbent assay (ELISA) developed at the Ontario Veterinary College (OVC VWF:Ag). Linearity, sensitivity, and coefficients of variation were determined. The Asserachrom VWF:Ag ELISA was used as the reference assay for this study. Concordance correlation and Bland-Altman plots were used to evaluate agreement between both VWF:Ag assays. The VWF:CB accuracy was assessed by degree of association with the VWF:Ag assays, and the VWF:Ag to VWF:CB ratio. All assays were assessed for their ability to distinguish between VWD negative and VWD positive patients. Linearity, intra-assay coefficients of variation, and inter-assay coefficients of variation were acceptable for both the newly developed VWF:CB (R2 = 0.97, average CV = 4.4, and 15, respectively) and OVC VWF:Ag assays (R2 = 0.96, average CV = 7.9, and 5.9, respectively). Agreement between the OVC VWF:Ag assay and reference assay was excellent (ρc = 0.89), and although differences between assay results precluded interchangeable use of the assays, both successfully distinguished VWD positive and VWD negative dogs (P < 0.0001). The VWF:CB showed a strong association with both VWF:Ag assays (R2 = 0.86, 0.82) and VWF:Ag to VWF:CB ratios (≤ 1) were as expected. The excellent performance of both assays in this validation study confirm their reliability and potential for clinical application. PMID:19086374

  11. Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay

    PubMed Central

    Francois, Patrice; Pittet, Didier; Bento, Manuela; Pepey, Béatrice; Vaudaux, Pierre; Lew, Daniel; Schrenzel, Jacques

    2003-01-01

    A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5′-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions. PMID:12517857

  12. Containment of Bioaerosol Infection Risk by the Xpert MTB/RIF Assay and Its Applicability to Point-of-Care Settings ▿

    PubMed Central

    Banada, Padmapriya P.; Sivasubramani, Satheesh K.; Blakemore, Robert; Boehme, Catharina; Perkins, Mark D.; Fennelly, Kevin; Alland, David

    2010-01-01

    The recently introduced Xpert MTB/RIF assay (Xpert) has point-of-care potential, but its capacity for biohazard containment remained to be studied. We compared the bioaerosols generated by the Xpert assay to acid-fast bacillus (AFB) microscope slide smear preparation. The Xpert assay sample treatment reagent (SR) was also studied for its sterilizing capacity, stability, and effect on assay sensitivity after prolonged treatment. During the preparation of AFB smears, sputum samples spiked with Mycobacterium bovis BCG at 5 × 108 CFU/ml produced 16 and 325 CFU/m3 air measured with an Andersen impactor or BioSampler, respectively. In contrast, neither the sample preparation steps for the Xpert assay nor its automated processing produced any culturable bioaerosols. In testing of SR sterilizing capacity, clinical sputum samples from strongly smear-positive tuberculosis patients treated with SR at a 2:1 ratio eliminated Mycobacterium tuberculosis growth in all but 1/39 or 3/45 samples cultured on solid or liquid medium, respectively. These few unsterilized samples had a mean 13.1-day delay in the time to positive culture. SR treatment at a 3:1 ratio eliminated growth in all samples. SR retained a greater than 6-log-unit killing capacity despite storage at temperatures spanning 4 to 45°C for at least 3 months. The effect of prolonged SR sample treatment was also studied. Spiked sputum samples could be incubated in SR for up to 3 days without affecting Xpert sensitivity for M. tuberculosis detection and up to 8 h without affecting specificity for rifampin resistance detection. These results suggest that benchtop use of the Xpert MTB/RIF assay limits infection risk to the user. PMID:20720033

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

    PubMed

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

    2011-06-01

    Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology. PMID:21552635

  14. 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. PMID:24803666

  15. Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test.

    PubMed

    Hagio, Soichiro; Furukawa, Satoshi; Abe, Masayoshi; Kuroda, Yusuke; Hayashi, Seigo; Ogawa, Izumi

    2014-06-01

    Recently, the liver micronucleus (MN) assay using young adult rats with repeated administrations has been investigated by employing a new method without partial hepatectomy or in situcollagenase perfusion as the repeated dose liver MN (RDLMN) assay by Narumi et al. (2012). In our study, in order to investigate the possibility of the RDLMN assay using young adult mice instead of rats and the feasibility of employing some genotoxicity assays along with the RDLMN assay as a combination test, two genotoxic carcinogens (N,N-diethylnitrosoamine (DEN) and cisplatin (CIS)) and a nongenotoxic carcinogen (phenobarbital sodium (PHE)) were administered to mice for 15 or 29 days. Then, the liver MN assay, peripheral blood (PB) MN assay and comet assay using the liver and kidney were concurrently performed as a combination test. DEN showed positive responses to all endpoints except MN induction in PB after 15 days of repeat administration. A cross-linking agent, CIS, showed MN induction in liver after 29 days of repeat administration, and in PB after 15 and 29 days of repeat administration, although the comet assay yielded negative responses for both organs at both sampling times. PHE yielded negative responses for all endpoints. In conclusion, it is suggested that the RDLMN assay using mice is a feasible method to be integrated into the general repeated toxicity test along with the combination assays, i.e., comet assay or PB MN assay, which would help in risk assessment for carcinogenicity by comparing the results of combination assays with each other. PMID:24849678

  16. 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

  17. Comparison of Five Assays for Detection of Clostridium difficile Toxin

    PubMed Central

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

    2011-01-01

    Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

  18. A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

    PubMed

    Kravtsov, V D

    1994-01-01

    The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances. PMID:7833120

  19. Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices.

    PubMed

    Watanabe, Hiroo; Satake, Atsuko; Kido, Yasumasa; Tsuji, Akio

    2002-01-01

    Enrofloxacin has been increasingly used in veterinary medicine to treat microbial infections. A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography (HPLC) is sensitive but labor-intensive. This paper reports an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) and the development of a rapid test kit based on immunochromatography. The detection limits using the ELISA were 10 ppb for chicken liver and muscle, and 1 ppb for cattle milk, respectively. The mean recovery values were 77.3-96.0% for chicken liver, 72.4-92.0% for chicken muscle and 84.0-99.0% for cattle milk. The detection limits using the kit were ca. 100 ppb for chicken muscle and ca. 10 ppb for cattle milk, respectively. All ELISA results for assay of chicken liver, chicken muscle and cattle milk were confirmed using HPLC which is used as the routine assay. The HPLC (x) and ELISA (y) results showed close correlation for chicken liver (y = 8.7 + 0.85x, r2 = 0.99, n = 25), chicken muscle (y = -3.9 + 0.94x, r2 = 0.98, n = 25) and cattle milk (y = 18.4 + 0.92x, r2 = 0.99, n = 25). PMID:11827405

  20. 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.

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

    PubMed

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

    2015-09-01

    To avoid contamination of adventitious gammaretroviruses in biological products such as vaccines, it is necessary to check the master seed cells for manufacturing. There are several assays to detect infectious gammaretroviruses. Among these, sarcoma-positive, leukemia-negative (S+L-) assay is a classical infectivity assay, which is often recommended in governmental guidelines. The S+L- cells used in S+L- assay generate unique focus upon the infection of replication-competent gammaretroviruses. Although S+L- assay is well recognized for the detection, their applicability is questionable in some cases. On the other hand, LacZ marker rescue (LMR) assay detects infectious gammaretroviruses by transducing LacZ marker gene to the target cells, which shows lacZ-positive foci if the infectious virus is present. In this study, we compared LMR and S+L- assays for detection of a variety of endogenous and exogenous gammaretroviruses. As results, LMR assay could detect all gammaretroviruses examined. On the other hand, S+L- assay using feline S+L- cells, termed QN10S, could not detect porcine endogenous retrovirus (PERV) subgroups A/B. Further, S+L- mink cells could not detect feline leukemia virus subgroups B in addition to PERV-A/B. These data indicate that LMR assay is better suited to detect wider range of gammaretroviruses. PMID:26164289

  2. 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

  3. Fluorometric enzymatic assay of l-arginine.

    PubMed

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

    2017-01-01

    The enzymes of l-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415nm under excitation at 360nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100nM to 6μМ with a limit of detection of 34nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples. PMID:27450117

  4. 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.

  5. The synchronous active neutron detection assay system

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-08-01

    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design.

  6. The synchronous active neutron detection assay system

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-09-01

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. They are using a Schlumberger neutron generator for the direct measurement of the fissile material content in spent fuel, in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics for the detection of very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. They have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The results to data are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference.

  7. PARALLEL ASSAY OF OXYGEN EQUILIBRIA OF HEMOGLOBIN

    PubMed Central

    Lilly, Laura E.; Blinebry, Sara K.; Viscardi, Chelsea M.; Perez, Luis; Bonaventura, Joe; McMahon, Tim J.

    2013-01-01

    Methods to systematically analyze in parallel the function of multiple protein or cell samples in vivo or ex vivo (i.e. functional proteomics) in a controlled gaseous environment have thus far been limited. Here we describe an apparatus and procedure that enables, for the first time, parallel assay of oxygen equilibria in multiple samples. Using this apparatus, numerous simultaneous oxygen equilibrium curves (OECs) can be obtained under truly identical conditions from blood cell samples or purified hemoglobins (Hbs). We suggest that the ability to obtain these parallel datasets under identical conditions can be of immense value, both to biomedical researchers and clinicians who wish to monitor blood health, and to physiologists studying non-human organisms and the effects of climate change on these organisms. Parallel monitoring techniques are essential in order to better understand the functions of critical cellular proteins. The procedure can be applied to human studies, wherein an OEC can be analyzed in light of an individual’s entire genome. Here, we analyzed intraerythrocytic Hb, a protein that operates at the organism’s environmental interface and then comes into close contact with virtually all of the organism’s cells. The apparatus is theoretically scalable, and establishes a functional proteomic screen that can be correlated with genomic information on the same individuals. This new method is expected to accelerate our general understanding of protein function, an increasingly challenging objective as advances in proteomic and genomic throughput outpace the ability to study proteins’ functional properties. PMID:23827235

  8. Granuloma pouch assay for mutagenicity testing.

    PubMed

    Maier, P

    1980-11-01

    The Granuloma Pouch Assay (GPA) is an animal model in which mutagenic and carcinogenic effects of a testcompound can be detected in rapidly dividing fibroblasts of a granulation tissue in adult male rats. Growth of this tissue was initiated with a small amount of croton oil at the inside wall of a subcutaneous air pouch on the back of the animals. The test compound can be injected either into the pouch (local) or administered by systemic routes. Alkali labile DNA-lesions, chromosome aberrations, sister chromatid exchanges, point mutations and tumor development in situ were determined. The comparison of mutation frequencies after local and systemic administration of testcompounds, provide an estimation of the pharmacokinetic characteristics and the mutagenic potency of the chemical. The local application route allows the detection of locally active mutagens and of compounds which require activation by P-448 dependent mono-oxygenases. Liver mediated proximate metabolites are detectable when they are transformed into ultimate carcinogens in extrahepatic cells whereas chemicals with a strong organ specific activity are not. PMID:7235991

  9. Strategies for Assaying Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Hafner Česen, Maruša; Turk, Boris

    2016-01-01

    Late endosomal organelles have an acidic pH and contain hydrolytic enzymes to degrade cargo delivered either from the extracellular environment by endocytosis or from within the cell itself by autophagy. In the event of lysosomal membrane permeabilization (LMP), the contents of late endosomes and lysosomes can be released into the cytosol and then initiate apoptosis. Compounds that can trigger LMP are therefore candidates for the induction of apoptosis, in particular in anticancer therapy. Alternatively, drug-delivery systems, such as nanoparticles, can have side effects that can include LMP, which has toxic consequences for the cells. To determine when, to what extent, and with what consequences LMP occurs is therefore of paramount importance for the evaluation of new potentially LMP-inducing compounds. In this introduction, we provide an overview of some basic assays for assessing LMP, such as staining with lysosomotropic dyes and measurement of cysteine cathepsin activity, and discuss additional strategies for the detection of the release of endogenous lysosomal molecules or preloaded exogenous tracers into the cytosol. PMID:27250949

  10. 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

  11. 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

  12. Liquid chromatographic assay for dicloxacillin in plasma.

    PubMed

    Alderete, Oscar; González-Esquivel, Dinora F; Del Rivero, L Misael; Castro Torres, Nelly

    2004-06-15

    A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans. PMID:15135112

  13. Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Internal Wave Apparatus for Copepod Behavior Assays

    NASA Astrophysics Data System (ADS)

    Jung, S.; Haas, K. A.; Webster, D. R.

    2015-11-01

    Internal waves are ubiquitous features in coastal marine environments and have been observed to mediate vertical distributions of zooplankton in situ. Internal waves are generated through oscillations of the pycnocline in stratified waters and thereby create fine-scale hydrodynamic cues that copepods and other zooplankton are known to sense, such as fluid density gradients and velocity gradients (quantified as shear deformation rate). The role of copepod behavior in response to cues associated with internal waves is largely unknown. Thus, a coupled quantification of copepod behavior and hydrodynamic cues will provide insight to the bio-physical interaction and the role of biological versus physical forcing in mediating organism distributions. We constructed a laboratory-scale internal wave apparatus to facilitate fine-scale observations of copepod behavior in flows that replicate in situ conditions of internal waves in a two-layer stratification. Three cases are chosen with density jump ranging between 0.75 - 1.5 kg/m3. Analytical analysis of the two-layer system provides guidance of the target forcing frequency to generate a standing internal wave with a single dominate frequency of oscillation. Flow visualization and signal processing of the interface location are used to quantify the wave characteristics. A copepod behavior assay is conducted, and sample trajectories are analyzed to identify copepod response to internal wave structure.

  19. 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

  20. 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.

  1. Recent Applications for in Vitro Antioxidant Activity Assay.

    PubMed

    Bunaciu, Andrei A; Danet, Andrei Florin; Fleschin, Şerban; Aboul-Enein, Hassan Y

    2016-09-01

    This review presents some of the most recent aspects related to antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Taking into account the reactions involved, in the antioxidant activity determinations, the assays can be classified into two main types: hydrogen atom transfer reactions and electron transfer. This review focuses on analytical methods used for antioxidant activity assay, published in the period 2009-2014. PMID:26575594

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

    DOEpatents

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

    2015-12-01

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

  3. 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.

  4. A sensitive method for the assay of angiotensin

    PubMed Central

    Regoli, D.; Vane, J. R.

    1964-01-01

    A method is described for the assay of angiotensin on the rat colon. Under the conditions of the assay, the preparation is sensitive to angiotensin and relatively insensitive to 5-hydroxytryptamine, to histamine and to other substances which might be present in blood. Bradykinin and catechol amines do not interfere with the assay. Evidence is given for the specificity of the angiotensin receptors. PMID:14228135

  5. An Assay for Thiaminase I in Complex Biological Samples

    PubMed Central

    Hanes, Jeremiah W.; Kraft, Clifford E.; Begley, Tadhg P.

    2007-01-01

    An alternative method for measuring thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I resulting in a large decrease in absorbance at 411 nm. This new assay is simple and sensitive and requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high throughput analysis in 96-well format with complex biological samples. PMID:17603991

  6. Development of fluorometric reactive oxygen species assay for photosafety evaluation.

    PubMed

    Seto, Yoshiki; Ohtake, Hiroto; Kato, Masashi; Onoue, Satomi

    2016-08-01

    The present investigation involved an attempt to develop a new reactive oxygen species (ROS) assay system for the photosafety assessment of chemicals using 1,3-diphenylisobenzofuran (DPBF), a fluorescent probe for monitoring ROS generation. The assay conditions of the fluorometric ROS (fROS) assay were optimized focusing on the solvent system, concentration of DPBF, fluorescent determination, screening run time and reproducibility. The photoreactivity of 21 phototoxic and 11 non-phototoxic compounds was assessed by fROS assay, and the obtained ROS data were compared with the results from a micellar ROS (mROS) assay and in vitro/in vivo phototoxicity information to confirm the predictive capacity of the fROS assay. In the optimized fROS assay, intra-day and inter-day precision levels (coefficient of variation) were found to be below 5%, and the Z'-factor for DPBF fluorescence quenching showed a large separation between positive and negative controls. Of all tested compounds, 3 false positive and 7 false negative predictions were observed in the fROS assay, and the negative predictivity for the fROS assay was found to be lower than that for the mROS assay. Although the fROS assay has some limitations, the procedures for it were highly simplified with a marked reduction in screening run time and one analytical sample for monitoring ROS generation from compounds. The fROS assay has the potential to become a new tool for photosafety assessment at an early stage of product development. PMID:27058001

  7. 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. PMID:22796283

  8. 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

  9. An assay for measurement of protein adsorption to glass vials.

    PubMed

    Varmette, Elizabeth; Strony, Brianne; Haines, Daniel; Redkar, Rajendra

    2010-01-01

    Protein adsorption to primary packaging is one of the problems faced by biopharmaceutical drug companies. An assay was developed to quantify loss of proteins to glass vial surfaces. The assay involves the labeling of protein with a fluorescent dye, incubation of the labeled protein with the vial surface, elution of the adsorbed protein using a stripping buffer, and determination of fluorescence of the adsorbed protein using a fluorometer. The assay is simple to set up, accurate, sensitive, and flexible. The assay can be modified for indirect measurement of protein adsorption and offers an attractive alternative for researchers to quantify protein adsorption to glass vials and syringes. PMID:21502031

  10. Comparison of enzymatic and liquid chromatographic chloramphenicol assays

    SciTech Connect

    Weber, A.F.; Opheim, K.E.; Koup, J.R.; Smith, A.L.

    1981-02-01

    A radioenzymatic assay and a ''high-performance'' liquid chromatographic assay for chloramphenicol were compared by using 52 patient specimens, 24 mock unknowns, and 13 quality control samples. Both methods were found to be rapid, precise, accurate, and sensitive, and either would be suitable for monitoring chloramphenicol concentrations in small volumes of serum. Linear regression analysis of serum chloramphenicol concentrations in patients receiving chloramphenicol succinate yielded a regression equation of Y . 1.04X + 0.274 (X . high-performance liquid chromatographic assay; Y . radioenzymatic assay), with a correlation coefficient of 0.971.

  11. Rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase

    SciTech Connect

    Nordeen, S.K.; Green, P.P. III; Fowlkes, D.M.

    1987-04-01

    We present a rapid, sensitive enzymatic assay for chloramphenicol acetyltransferase (CAT) that does not require chromatography, HPLC, or autoradiography. The assay is based on the use of an inexpensive substrate, tritiated acetate, instead of (/sup 14/C)chloramphenicol. The method is adapted from one originally used by de Crombrugghe et al. and by Shaw, but with simplifications appropriate for routine use. In our hands, the method is as sensitive as the customary thin-layer chromatography assay and is far more efficient for the performance of many assays, both in terms of labor and expense.

  12. 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

  13. Cost effective assay choice for rare disease study designs.

    PubMed

    Campbell, Desmond D; Porsch, Robert M; Cherny, Stacey S; Capra, Valeria; Merello, Elisa; De Marco, Patrizia; Sham, Pak C; Garcia-Barceló, Maria-Mercè

    2015-01-01

    High throughput assays tend to be expensive per subject. Often studies are limited not so much by the number of subjects available as by assay costs, making assay choice a critical issue. We have developed a framework for assay choice that maximises the number of true disease causing mechanisms 'seen', given limited resources. Although straightforward, some of the ramifications of our methodology run counter to received wisdom on study design. We illustrate our methodology with examples, and have built a website allowing calculation of quantities of interest to those designing rare disease studies. PMID:25648394

  14. Evaluation of Pregnancy Malaria Vaccine Candidates: The Binding Inhibition Assay.

    PubMed

    Saveria, Tracy; Duffy, Patrick E; Fried, Michal

    2015-01-01

    The parasite-binding inhibition assay is designed to evaluate the acquisition of naturally acquired functional antibodies that block Plasmodium falciparum binding to endothelial or placental receptors. The assay is also used to assess functional activity by antibodies induced by immunization, for example antibodies raised against pregnancy malaria vaccine candidates like VAR2CSA. Here we describe a plate-based assay to measure the levels of adhesion-blocking antibodies. This assay format can be adapted to any lab that is minimally equipped for short-term parasite culture. PMID:26450393

  15. Chromosome and genetic testing using ChIP assay.

    PubMed

    Kohzaki, Hidetsugu; Asano, Maki

    2016-01-01

    Chromatin immunoprecipitation (ChIP) assay can be used to easily visualize information about proteins, DNA, and RNA on chromosomes and is widely used for analysis of genomes, epigenomes, mRNAs, and non-coding RNAs. The ChIP assay can detect, not only DNA-binding proteins of various organisms, but also the temporal and spatial regulating mechanisms of RNA-binding proteins. Because of these features, demand for ChIP assay is expected to grow. Here, by using yeast and Drosophila as examples, we describe the superiority of the improved ChIP assay that we have developed. PMID:27100707

  16. 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.

  17. Rapid quantitative assay for chloramphenicol acetyltransferase

    SciTech Connect

    Neumann, J.R.; Morency, C.A.; Russian, K.O.

    1987-05-01

    Measuring the expression of exogenous genetic material in mammalian cells is commonly done by fusing the DNA of interest to a gene encoding an easily-detected enzyme. Chloramphenicol acetyltransferase(CAT) is a convenient marker because it is not normally found in eukaryotes. CAT activity has usually been detected using a thin-layer chromatographic separation followed by autoradiography. An organic solvent extraction-based method for CAT detection has also been described, as well as a procedure utilizing HPLC analysis. Building on the extraction technique, they developed a rapid sensitive kinetic method for measuring CAT activity in cell homogenates. The method exploits the differential organic solubility of the substrate ((/sup 3/H) or (/sup 14/C)acetyl CoA) and the product (labeled acetylchloramphenicol). The assay is a simple one-vial, two-phase procedure and requires no tedious manipulations after the initial setup. Briefly, a 0.25 ml reaction with 100mM Tris-HCL, 1mM chloramphenicol, 0.1mM (/sup 14/C)acetyl CoA and variable amounts of cell homogenate is pipetted into a miniscintillation vial, overlaid with 5 ml of a water-immiscible fluor, and incubated at 37/sup 0/C. At suitable intervals the vial is counted and the CAT level is quantitatively determined as the rate of increase in counts/min of the labeled product as it diffuses into the fluor phase, compared to a standard curve. When used to measure CAT in transfected Balb 3T3 cells the method correlated well with the other techniques.

  18. 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.

  19. 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

  20. 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

  1. A colorimetric assay for alpha-hydroxynitrile lyase.

    PubMed

    Selmar, D; Carvalho, F J; Conn, E E

    1987-10-01

    A colorimetric assay for alpha-hydroxynitrile lyase which utilizes acetone cyanohydrin as a substrate is described. The assay is based on measurement of the HCN formed when the lyase catalyzes the dissociation of acetone cyanohydrin. The procedure was devised for use with the optically inactive acetone cyanohydrin but will be applicable to enzymes utilizing other cyanohydrins. PMID:3674409

  2. 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,…

  3. 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...

  4. A fluopol-ABPP HTS assay to identify PAD inhibitors.

    PubMed

    Knuckley, Bryan; Jones, Justin E; Bachovchin, Daniel A; Slack, Jessica; Causey, Corey P; Brown, Steven J; Rosen, Hugh; Cravatt, Benjamin F; Thompson, Paul R

    2010-10-14

    Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was identified as a potent, selective, and irreversible PAD4 inactivator. PMID:20740228

  5. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

  6. 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

  7. 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…

  8. 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. PMID:27233264

  9. Radioenzymatic assay for trimethoprim in very small serum samples

    SciTech Connect

    Yogev, R.; Melick, C.; Tan-Pong, L.

    1985-02-01

    A modification of the methotrexate radioassay kit (supplied by New England Enzyme Center) enabled determination of trimethoprim levels in 5-microliter serum samples. An excellent correlation between this assay and high-pressure liquid chromatography assay was found. These preliminary results suggest that with this method rapid determination of trimethoprim levels in very small samples (5 to 10 microliters) can be achieved.

  10. 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…

  11. Coincidence technique to reduce geometry and matrix effects in assay

    SciTech Connect

    Zucker, M.S.; Gozani, T.; Bernatowicz, H.

    1983-01-01

    Algebraic combinations of coincidence multiplicities can be formed which are relatively independent of detection efficiency, yet proportional to the amount of nuclear material being assayed. Considering these combinations, rather than the coincidence alone as signatures, has the demonstrable advantage that the assay results are comparatively independent of sample geometry or even matrix.

  12. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide.

    PubMed

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J; Sidransky, Ellen; Zheng, Wei

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  13. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

    PubMed Central

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J.; Sidransky, Ellen

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  14. 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...

  15. 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...

  16. 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...

  17. A vertical diffusion method for the microbiological assay of isoniazid

    PubMed Central

    Lloyd, Janet; Mitchison, D. A.

    1964-01-01

    A method is described for the assay of isoniazid in serum and other fluids by diffusion along slopes of Löwenstein-Jensen medium inoculated with tubercle bacilli. The method is convenient, rapid and robust, but is less accurate than diffusion systems for the assay of some other substances. PMID:14227431

  18. 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...

  19. Diagnostic assays used to control small ruminant lentiviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serological diagnostic tests such as the agar gel immunodiffusion (AGID) assay and various types of enzyme linked immunosorbent assays (ELISAs) have contributed to the reduction of small ruminant lentivirus infections worldwide. Since there are no treatments or efficacious vaccines, the serolog...

  20. 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.

  1. 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

  2. Importance of albumin binding in the assay for carnitine palmitoyltransferase.

    PubMed Central

    McCormick, K; Notar-Francesco, V J

    1983-01-01

    Alterations in the long-chain acyl-CoA binding to albumin in the carnitine palmitoyltransferase (CPT) assay appreciably affect the reaction at commonly used substrate concentrations. Since in the CPT assay the latter are typically well below saturation or Vmax. values, the measured enzyme activity depends on both the absolute quantity of albumin in the CPT assay and any biochemical modification of its binding. The present study verifies the striking dependence of the K0.5 for palmitoyl-CoA on albumin and the misleading 'activation' of the enzyme by compounds that also avidly bind to albumin. In assessing the intracellular physiological relevance of any modifier of CPT, the effects of protein binding in the assay assume particular importance. Indeed, any compound that alters CPT activity may do so, not directly, but as an assay artifact changing the free or unbound substrate concentrations. PMID:6661210

  3. Controlling equine influenza: Traditional to next generation serological assays.

    PubMed

    Kinsley, Rebecca; Scott, Simon D; Daly, Janet M

    2016-05-01

    Serological assays provide an indirect route for the recognition of infectious agents via the detection of antibodies against the infectious agent of interest within serum. Serological assays for equine influenza A virus can be applied for different purposes: diagnosing infections; subtyping isolates; surveillance of circulating strains; and to evaluate the efficacy of vaccines before they reach the market. Haemagglutination inhibition (HI) and single radial haemolysis (SRH) assays are most commonly used in the equine field. This review outlines how both these assays together with virus neutralization (VN) and ELISA are performed, interpreted and applied for the control of equine influenza, giving the limitations and advantages of each. The pseudotyped virus neutralization assay (PVNA) is also discussed as a promising prospect for the future of equine influenza virus serology. PMID:27066704

  4. 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.

  5. Bioanalytical advances in assays for C-reactive protein.

    PubMed

    Vashist, Sandeep Kumar; Venkatesh, A G; Marion Schneider, E; Beaudoin, Christopher; Luppa, Peter B; Luong, John H T

    2016-01-01

    This review presents advances in assays for human C-reactive protein (CRP), the most important biomarker of infection and inflammation for a plethora of diseases and pathophysiological conditions. Routine assays in clinical settings are based on analyzers, enzyme-linked immunosorbent assays and lateral flow assays. However, assays encompassing novel sensing schemes, improved chemistry, signal enhancement, lab-on-a-chip, microfluidics and smartphone detection, have emerged in recent years. The incorporation of immune-transducing chips or sensing interfaces with nanomaterials enables multiplexing analysis of CRP with co-existing biomarkers. However, there are still considerable challenges in the development of rapid diagnostics for both pentameric and monomeric CRP forms. PMID:26717866

  6. Solid-phase genotoxicity assay for organic compounds in soil

    SciTech Connect

    Alexander, R.R.; Chung, N.; Alexander, M.

    1999-03-01

    A genotoxicity assay was developed for samples from environments in which toxic organic compounds are largely sorbed. The assay entails measurement of the rate of mutation of a strain of Pseudomonas putida to rifampicin resistance. The ratio of induced to spontaneous mutants was a function of the concentration of a test mutagen in soil. In studies of the utility of the assay in samples amended with 2-aminofluorene as a test mutagen, the ratio of induced to spontaneous mutants declined with time. The decline paralleled the disappearance of extractable 2-aminofluorene from the soil. The ratio of induced to spontaneous mutants also feel in four other soils with dissimilar properties. The authors suggest that this solid-phase assay is more appropriate for the estimation of genotoxicants sorbed in soil than assays involving extractants or suspensions of soil or sediment samples.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    PubMed

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    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

  13. Challenge assay in vitro using lymphocyte blastogenesis for the contact hypersensitivity assay.

    PubMed

    Kashima, R; Okada, J; Ikeda, Y; Yoshizuka, N

    1993-10-01

    To confirm positivity in routine guinea pig studies, contact allergenicity was investigated by a challenge assay in vitro using a co-culture of autologous lymphocytes passed through a nylon-wool column and antigen-presenting cells (APCs) modified with or without antigen. Proliferation of the lymphocytes primed with ovalbumin and/or 2,4-dinitrochlorobenzene was antigen specific and dependent on the presence of APCs (peripheral blood monocytes, splenic macrophages and macrophages induced by liquid paraffin). For another nine haptens, primed lymphocytes proliferated significantly more than control lymphocytes; the stimulation index (SI; ratio between [3H]methylthymidine ([3H]TdR) incorporation of lymphocytes with antigen-modified APCs and [3H]TdR incorporation of lymphocytes with APCs not modified by antigen) was 1.6-4.8 in sensitized animals whereas it was about 1.0 in control animals. Sodium dodecyl sulfate did not cause lymphocyte proliferation. The SI value in vitro was correlated with both the positive rate in vivo (r = 0.736) and the mean response score in vivo (r = 0.645). Thus, it was possible to confirm that positivity in routine experiments was a true sign of allergy. A combination of this assay and short-term animal studies would provide an efficient assessment of the allergic potential of chemicals. PMID:8225135

  14. 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

  15. 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.

  16. 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. PMID:26969773

  17. 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

  18. 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 ...

  19. 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

  20. 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

  1. 384-Well Multiplexed Luminex Cytokine Assays for Lead Optimization.

    PubMed

    Tang, Huaping; Panemangalore, Reshma; Yarde, Melissa; Zhang, Litao; Cvijic, Mary Ellen

    2016-07-01

    Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturized Luminex (Austin, TX) assay platform to establish a panel of high-throughput, multiplexed assays for measuring cytokines in human whole blood. The miniaturized 384-well Luminex assay uses <25% of the assay reagents compared with the 96-well assay. The development and validation of the 384-well Luminex cytokine assays enabled high-throughput screening of compounds in primary cells using cytokines as physiologically relevant readouts. Furthermore, this miniaturized multiplexed technology platform allows for high-throughput biomarker profiling of biofluids from animal studies and patient samples for translational research. PMID:27095819

  2. Development of an inhibitive enzyme assay for copper.

    PubMed

    Shukor, M Y; Bakar, N A; Othman, A R; Yunus, I; Shamaan, N A; Syed, M A

    2009-01-01

    In this work the development of an inhibitive assay for copper using the molybdenum-reducing enzyme assay is presented. The enzyme is assayed using 12-molybdophosphoric acid at pH 5.0 as an electron acceptor substrate and NADH as the electron donor substrate. The enzyme converts the yellowish solution into a deep blue solution. The assay is based on the ability of copper to inhibit the molybdenum-reducing enzyme from the molybdate-reducing Serratia sp. Strain DRY5. Other heavy metals tested did not inhibit the enzyme at 10 mg l(-1). The best model with high regression coefficient to measure copper inhibition is one-phase binding. The calculated IC50 (concentration causing 50% inhibition) is 0.099 mg l(-1) and the regression coefficient is 0.98. The comparative LC50, EC50 and IC50 data for copper in different toxicity tests show that the IC50 value for copper in this study is lower than those for immobilized urease, bromelain, Rainbow trout, R. meliloti, Baker's Yeast dehydrogenase activity Spirillum volutans, P. fluorescens, Aeromonas hydrophilia and synthetic activated sludge assays. However the IC50 value is higher than those for Ulva pertusa and papain assays, but within the reported range for Daphnia magna and Microtox assays. PMID:20112861

  3. Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

    PubMed

    Neal, Frances; Arnold, Joanne; Rossant, Christine J; Podichetty, Sadhana; Lowne, David; Dobson, Claire; Wilkinson, Trevor; Colley, Caroline; Howes, Rob; Vaughan, Tristan J

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets. PMID:26450103

  4. Validation of erythromycin microbiological assay using an alternative experimental design.

    PubMed

    Lourenço, Felipe Rebello; Kaneko, Telma Mary; Pinto, Terezinha de Jesus Andreoli

    2007-01-01

    The agar diffusion method, widely used in antibiotic dosage, relates the diameter of the inhibition zone to the dose of the substance assayed. An experimental plan is proposed that may provide better results and an indication of the assay validity. The symmetric or balanced assays (2 x 2) as well as those with interpolation in standard curve (5 x 1) are the main designs used in the dosage of antibiotics. This study proposes an alternative experimental design for erythromycin microbiological assay with the evaluation of the validation parameters of the method referring to linearity, precision, and accuracy. The design proposed (3 x 1) uses 3 doses of standard and 1 dose of sample applied in a unique plate, aggregating the characteristics of the 2 x 2 and 5 x 1 assays. The method was validated for erythromycin microbiological assay through agar diffusion, revealing its adequacy to linearity, precision, and accuracy standards. Likewise, the statistical methods used demonstrated their accordance with the method concerning the parameters evaluated. The 3 x 1 design proved to be adequate for the dosage of erythromycin and thus a good alternative for erythromycin assay. PMID:17760348

  5. Reproducibility of an imaging based prostate cancer prognostic assay

    NASA Astrophysics Data System (ADS)

    Khan, Faisal M.; Powell, Douglas; Bayer-Zubek, Valentina; Soares, Rui; Mott, Allison; Fernandez, Gerardo; Mesa-Tejada, Ricardo; Donovan, Michael J.

    2011-03-01

    The Prostate Px prognostic assay offered by Aureon Biosciences is designed to predict progression post primary treatment for prostate cancer patients based on their diagnostic biopsy specimen. The assay is driven by the automated image analysis of biological specimens. Three different histological sections are analyzed for morphometric as well as immunofluorescence protein expression properties within areas of tumor digitally masked by expert pathologists. The assay was developed on a multi-institution cohort of up to 9 images from each of 1027 patients. The variation in histological sections, staining, pathologist tumor masking and the region of image acquisition all have the potential to significantly impact imaging features and consequently the reproducibility of the assay's results for the same patient. This study analyzed the reproducibility of the assay in 50 patients who were re-processed within 3 months in a blinded fashion as de-novo patients. The key assay results reported were in agreement in 94% of the cases. The two independent endpoints of risk classification reproduced results in 90% and 92% of the predictions. This work presents one of the first assessments of the reproducibility of a commercial assay's results given the inherent variations in images and quantitative imaging characteristics in a commercial setting.

  6. 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)

  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. Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

    PubMed Central

    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

  9. Traditional and Model Based Assay of Irregular Geometry Items

    SciTech Connect

    MOORE, FRANK S.; SALAYMEH, SALEEM

    2005-06-15

    The Analytical Development Section (ADS) of SRNL was requested to perform a waste disposal assay of two heater boxes which had been used in the HB Line dissolvers. They had been sent to SRNL for study to make recommendations on how to prevent future failure of the units when they were replaced. The study having been completed, the units needed to be characterized prior to sending to Solid Waste for disposal. An assay station consisting of a turntable, HPGe detector, CANBERRA Inspector, transmission source and a portable computer was set up to do the required assays. The assays indicate the presence of U-235, Pu-239 and Cs-137. No measurable amounts of U-235 or Pu-239 were found. Therefore the Minimum Detectable Activities for U-235 and Pu-239 were calculated. For Heater Box 1, 0.23 grams of U-235 and 0.24 grams of Pu-239. For Heater Box 2, the results were 0.21 grams of U-235 and 0.21 grams of Pu-239. This paper describes and documents the assays employed to determine the amount of U, Pu and Cs contents of the heater boxes. The paper provides results of SNM assays using traditional calibration of the system and on one based on modeling. It also provides the scientific community with data that will assist the user in determining the method of choice for assaying items with irregular geometries.

  10. 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.

  11. 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

  12. Comparative endpoint sensitivity of in vitro estrogen agonist assays.

    PubMed

    Dreier, David A; Connors, Kristin A; Brooks, Bryan W

    2015-07-01

    Environmental and human health implications of endocrine disrupting chemicals (EDCs), particularly xenoestrogens, have received extensive study. In vitro assays are increasingly employed as diagnostic tools to comparatively evaluate chemicals, whole effluent toxicity and surface water quality, and to identify causative EDCs during toxicity identification evaluations. Recently, the U.S. Environmental Protection Agency (USEPA) initiated ToxCast under the Tox21 program to generate novel bioactivity data through high throughput screening. This information is useful for prioritizing chemicals requiring additional hazard information, including endocrine active chemicals. Though multiple in vitro and in vivo techniques have been developed to assess estrogen agonist activity, the relative endpoint sensitivity of these approaches and agreement of their conclusions remain unclear during environmental diagnostic applications. Probabilistic hazard assessment (PHA) approaches, including chemical toxicity distributions (CTD), are useful for understanding the relative sensitivity of endpoints associated with in vitro and in vivo toxicity assays by predicting the likelihood of chemicals eliciting undesirable outcomes at or above environmentally relevant concentrations. In the present study, PHAs were employed to examine the comparative endpoint sensitivity of 16 in vitro assays for estrogen agonist activity using a diverse group of compounds from the USEPA ToxCast dataset. Reporter gene assays were generally observed to possess greater endpoint sensitivity than other assay types, and the Tox21 ERa LUC BG1 Agonist assay was identified as the most sensitive in vitro endpoint for detecting an estrogenic response. When the sensitivity of this most sensitive ToxCast in vitro endpoint was compared to the human MCF-7 cell proliferation assay, a common in vitro model for biomedical and environmental monitoring applications, the ERa LUC BG1 assay was several orders of magnitude less

  13. Microfluidic barcode assay for antibody-based confirmatory diagnostics.

    PubMed

    Araz, M Kursad; Apori, Akwasi A; Salisbury, Cleo M; Herr, Amy E

    2013-10-01

    Confirmatory diagnostics offer high clinical sensitivity and specificity typically by assaying multiple disease biomarkers. Employed in clinical laboratory settings, such assays confirm a positive screening diagnostic result. These important multiplexed confirmatory assays require hours to complete. To address this performance gap, we introduce a simple 'single inlet, single outlet' microchannel architecture with multiplexed analyte detection capability. A streptavidin-functionalized, channel-filling polyacrylamide gel in a straight glass microchannel operates as a 3D scaffold for a purely electrophoretic yet heterogeneous immunoassay. Biotin and biotinylated capture reagents are patterned in discrete regions along the axis of the microchannel resulting in a barcode-like pattern of reagents and spacers. To characterize barcode fabrication, an empirical study of patterning behaviour was conducted across a range of electromigration and binding reaction timescales. We apply the heterogeneous barcode immunoassay to detection of human antibodies against hepatitis C virus and human immunodeficiency virus antigens. Serum was electrophoresed through the barcode patterned gel, allowing capture of antibody targets. We assess assay performance across a range of Damkohler numbers. Compared to clinical immunoblots that require 4-10 h long sample incubation steps with concomitant 8-20 h total assay durations; directed electromigration and reaction in the microfluidic barcode assay leads to a 10 min sample incubation step and a 30 min total assay duration. Further, the barcode assay reports clinically relevant sensitivity (25 ng ml(-1) in 2% human sera) comparable to standard HCV confirmatory diagnostics. Given the low voltage, low power and automated operation, we see the streamlined microfluidic barcode assay as a step towards rapid confirmatory diagnostics for a low-resource clinical laboratory setting. PMID:23925585

  14. A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses

    PubMed Central

    Langereis, Eveline J.; Wagemans, Tom; Kulik, Wim; Lefeber, Dirk J.; van Lenthe, Henk; Oussoren, Esmee; van der Ploeg, Ans T.; Ruijter, George J.; Wevers, Ron A.; Wijburg, Frits A.; van Vlies, Naomi

    2015-01-01

    Introduction Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III). Methods Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs. Results The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them. Conclusion The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay. PMID:26406883

  15. Optimized Diagnostic Assays Based on Redox Tagged Bioreceptive Interfaces.

    PubMed

    Bedatty Fernandes, Flavio C; Patil, Amol V; Bueno, Paulo R; Davis, Jason J

    2015-12-15

    Among the numerous label free electronic biomarker assay methodologies now available, impedance based electrochemical capacitance spectroscopy (ECS), based upon mapping the perturbations in interfacial charging of redox elements incorporated into a biologically receptive interface, has recently been shown to be a convenient and highly sensitive mode of transduction and one which, additionally, requires no predoping of analytical solution. We present, herein, a data acquisition and analysis methodology based on frequency resolved immittance function analysis. Ultimately, this enables both a maximization of assay sensitivity and a reduction in assay acquisition time by an order of magnitude. PMID:26583592

  16. 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

  17. Spectrophotometric assays for measuring redox biomarkers in blood.

    PubMed

    Veskoukis, Aristidis S; Kyparos, Antonios; Paschalis, Vassilis; Nikolaidis, Michalis G

    2016-05-01

    The assessment of redox status is most frequently performed by measuring redox biomarkers. The spectrophotometer is the most commonly used analytical instrument in biochemistry. There is a huge number of spectrophotometric redox biomarkers and assays, thus distinguishing the most appropriate biomarkers and protocols is overwhelming. The aim of the present review is to propose valid and reliable spectrophotometric assays for measuring redox biomarkers in blood. It is hoped that this work will help researchers to select the most suitable redox biomarkers and assays. PMID:26809994

  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. The glycophorin A assay for somatic cell mutations in humans

    SciTech Connect

    Langlois, R.G.; Bigbee, W.L.; Jensen, R.H.

    1989-08-18

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs.

  20. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

    PubMed Central

    Zhang, Chen; Jang, Sunyoung; Amadi, Ovid C.; Shimizu, Koichi; Lee, Richard T.; Mitchell, Richard N.

    2013-01-01

    Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis). In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient. PMID:24151597

  1. Neutral red assay in minimum fungicidal concentrations of antifungal agents.

    PubMed

    Fukuda, T; Naka, W; Tajima, S; Nishikawa, T

    1996-01-01

    We assayed the fungicidal effects of antifungal agents using neutral red staining. Fungal elements of Trichophyton mentagrophytes and T. rubrum were treated with various concentrations of antifungal agents in 96-well filtration plates and then stained with neutral red. The amount of neutral red incorporated by the surviving viable cells was determined from the automated spectrophotometric readings at 550 nm. The minimum fungicidal concentrations (MFCs) of antifungal agents determined by this assay correlated well with those determined by conventional assay. This newly developed procedure should provide a rapid, reproducible, quantitative, qualitative and semi-automated susceptibility test for determination of the MFCs of the fungicidal agents. PMID:8912170

  2. 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.

  3. 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

  4. Cholinesterase based amperometric biosensors for assay of anticholinergic compounds

    PubMed Central

    Pohanka, Miroslav

    2009-01-01

    Biosensors are analytical devices being approachable for multiple analytes assay. Here, biosensors with intercepted acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) are presented as tool for assay of anticholinergic compounds such as pesticides, nerve agents and some natural toxins. Principle of assay is based on evaluation of cholinesterase activity and its pertinent decrease in presence of analyte. Nerve agents, pesticides, anticholinergic drugs useable for treatment of Alzheimer′s disease as well as myasthenia gravis and aflatoxins are enlisted as compounds simply analyzable by cholinesterase biosensors. PMID:21217847

  5. A fluorescence polarization based assay for glucose sensing

    NASA Astrophysics Data System (ADS)

    Cummins, Brian M.; Coté, Gerard L.

    2012-03-01

    A fluorescence polarization (FP) assay was developed to determine concentrations of glucose using concanavalin A (ConA) and fluorescently-labeled dextran. Predictive FP responses to glucose were elicited for different assay configurations using mathematical modeling and displayed herein. Using 4 kDa FITC-dextran, we predicted a change of 0.120 P units from 0 mg/dL glucose to 500 mg/dL. This shows the potential that a homogenous, reproducible FP assay can be engineered to measure glucose concentrations using tetrameric ConA and 4k kDa FITC-dextran.

  6. 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.

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

    PubMed Central

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

    2010-01-01

    survivorship or lifespan reduction are crucial to their success. The approximate cost of the method was US$7.50 per mosquito and 60 mosquitoes could be processed in 3 days. The assay is based on conserved genes and modified versions are likely to support similar investigations of several important mosquito and other disease vectors. PMID:20186322

  8. Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay.

    PubMed

    Xu, Yazhou; Wang, Yunjie; Xu, Yuan; Li, Jia; Liao, Hong; Zhang, Luyong; Pang, Tao

    2015-08-01

    AMP-activated protein kinase (AMPK), a conserved heterotrimeric kinase, serves as an energy sensor maintaining energy balance at both cellular and whole-body levels and plays multiple beneficial roles in carbohydrate and lipid metabolism, which makes AMPK an attractive target for diabetes and other metabolic disorders. To date, establishment of the physiologically relevant biochemical assay for AMPK has not been reported. Here we developed a phosphorylated AMPK protection assay based on a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, using the protein phosphatase 2A (PP2A) to dephosphorylate AMPK. The partially dephosphorylated AMPK by PP2A had lower activity than phosphorylated AMPK. This specific TR-FRET assay for AMPK was optimized in the 384-well format and produced similar EC(50) values for AMPK activators AMP and A769662 and a similar IC(50) value for AMPK inhibitor compound C, as previously reported. Under the optimized conditions, the assay Z' factor calculated over 160 data points has an optimal value greater than 0.5, which is suitable for high-throughput screening. In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions. PMID:25956678

  9. Evidence of ATP assay as an appropriate alternative of MTT assay for cytotoxicity of secondary effluents from WWTPs.

    PubMed

    Yang, Yang; Lu, Yun; Wu, Qian-Yuan; Hu, Hong-Ying; Chen, Ying-Hua; Liu, Wan-Li

    2015-12-01

    Biological tests are effective and comprehensive methods to assess toxicity of environmental pollutants to ensure the safety of reclaimed water. In this study, the canonical MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cytotoxicity of dissolved organic matters (DOMs) of secondary effluents from wastewater treatment plants (WWTPs). It was surprising that most concentrated DOMs treated HepG2 cells yielded much higher signal compared with vehicle control regardless of difference of treatment technologies and seasons. However, there was actually no obvious enhancement of the cell proliferation by microscopy. In order to find out potential reason for the discrepancy, another three assays were performed. The results of ATP assay and flow cytometry showed expected toxicity, which was consistent with microscopy and previous studies, while DNA assay did not exhibit apparent change in treated cells. The possible mechanisms of abnormal MTT signal could be that some materials in secondary effluents isolated by solid extraction with HLB resin directly reacted with MTT and/or enhanced the activity of mitochondrial dehydrogenase. Therefore, the MTT assay is not suitable to assess cytotoxicity of complex mixtures such as secondary effluents, while ATP assay is an optional sensitive method. This study also suggests the importance of choosing both suitable extraction methods and detection assays for toxicity evaluation of component-unknown environmental samples. PMID:26410194

  10. Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

    SciTech Connect

    Ratcliffe, W.A.; Corrie, J.E.; Dalziel, A.H.; Macpherson, J.S.

    1982-06-01

    Researchers compared two direct radioimmunoassays for progesterone in 50 microL of unextracted serum or plasma with assays involving extraction of serum. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11 alpha hemisuccinyl conjugate and the radioligand /sup 125/I-labeled progesterone 11 alpha-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r greater than 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. Researchers conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum.

  11. Comparison of an Enzyme-Linked Immunosorbent Assay with an Immunochromatographic Assay for Detection of Lincomycin in Milk and Honey.

    PubMed

    Cao, Shanshan; Song, Shanshan; Liu, Liqiang; Kong, Na; Kuang, Hua; Xu, Chuanlai

    2015-01-01

    An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic assay were constructed for the detection of lincomycin (LIN) in both milk and honey samples based on the monoclonal antibody named 5F6. The half-maximum inhibition of ELISA was 0.3 ng/mL after optimizing pH and ionic strength conditions; the limit of detection was 0.07 ng/mL. The cross-reactivity with clindamycin was 0.6%. LIN recovery in spiked milk and honey samples ranged from 84.6% to 115.6% with intra-assay coefficient variations of 1.7-25.4% and inter-assay coefficient variations of 2.7-8.9%. The detection limits were estimated as 2.1 µg/L for milk and 2.1 µg/kg for honey samples. The immunochromatographic assay revealed a LIN cut-off value of 10 ng/mL in PBS, 5 ng/mL in milk, and 120 ng/g in honey, and a visual lower detection limit of 2.5 ng/mL, 1 ng/mL and 30 ng/g in PBS, milk and honey, respectively. The immunochromatographic assay is preferred for large-scale practical application for its simpler pretreatment and satisfied sensitivity compared with ELISA assay. PMID:26107744

  12. The clinical utility of mass spectrometry based protein assays.

    PubMed

    Lassman, Michael E; McAvoy, Thomas; Chappell, Derek L; Lee, Anita Y; Zhao, Xuemei X; Laterza, Omar F

    2016-08-01

    Reports of mass spectrometry based assays for peptides and proteins have become increasingly common in the literature. The growing interest of mass spectrometry for use in clinical laboratories has been primarily driven by the inherent selectivity of the platform relative to more traditional platforms such as immunoassays. However, the adoption of mass spectrometry for peptide and protein analysis in the clinic has been relatively slow compared its adoption in non-clinical laboratories such as in biomarker discovery efforts or within laboratories that support pharmaceutical and academic research. Here, we review some of the successful reports of MS based assays for human proteins in multiple stages of assay research, and describe how and why the platform was employed in order to demonstrate where and when mass spectrometry based assays will have value in the future. PMID:27259466

  13. [Research Progress on Cytometric Bead Assay for Platelet Antibody Detection].

    PubMed

    Ling, Yun; Kong, Xin; Chen, Bao-An

    2015-08-01

    Anti-platelet specific antibody is one of the most important reasons leading to thrombocytopenia and megakaryocyte dysmaturity. The detection of platelet autoantibodies is an important step in the diagnosis of ITP because of the absence of specific clinic feature. The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) has become a "gold standard" for determination of PLT specific antibody, which has high specificity and low sensitivity. However, this assay is time-consuming and tedious work. Routine use of this assay in hospital is difficult. Recently, some researches reporded the cytometric bead assay that has higher sensitivity than MAIPA, and so probably solves the problem of time-consuming partly, that also can use different beads for simultaneous detection. This review focuses on recent progress of the cytometric bead assay. PMID:26314475

  14. Click Chemistry-Mediated Nanosensors for Biochemical Assays

    PubMed Central

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

    2016-01-01

    Click chemistry combined with functional nanoparticles have drawn increasing attention in biochemical assays because they are promising in developing biosensors with effective signal transformation/amplification and straightforward signal readout for clinical diagnostic assays. In this review, we focus on the latest advances of biochemical assays based on Cu (I)-catalyzed 1, 3-dipolar cycloaddition of azides and alkynes (CuAAC)-mediated nanosensors, as well as the functionalization of nanoprobes based on click chemistry. Nanoprobes including gold nanoparticles, quantum dots, magnetic nanoparticles and carbon nanomaterials are covered. We discuss the advantages of click chemistry-mediated nanosensors for biochemical assays, and give perspectives on the development of click chemistry-mediated approaches for clinical diagnosis and other biomedical applications. PMID:27217831

  15. Molecular amplification assays for the detection of flaviviruses.

    PubMed

    Lanciotti, Robert S

    2003-01-01

    Over the past 10 years, a number of molecular amplification assays have been developed for the detection of flaviviruses. Most of these assays utilize the reverse transcriptase-polymerase chain reaction (RT-PCR) as the amplification format with detection by either agarose gel electrophoresis and ethidium bromide staining or hybridization with molecular probes. Recently, a modification of the standard RT-PCR using fluorescent-labeled oligonucleotide probes for detection (TaqMan) has been described. As a result, several assays for detecting flaviviruses have been developed using this approach. In addition, another amplification format, nucleic acid sequence based amplification (NASBA), has been developed and utilized for the detection of several flaviviruses. The various assay formats will be described and their utility discussed. PMID:14714430

  16. Quantitation of microRNAs using a modified Invader assay.

    PubMed

    Allawi, Hatim T; Dahlberg, James E; Olson, Sarah; Lund, Elsebet; Olson, Marilyn; Ma, Wu-Po; Takova, Tsetska; Neri, Bruce P; Lyamichev, Victor I

    2004-07-01

    The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses. PMID:15208450

  17. Dodecylresorufin (C12R) Outperforms Resorufin in Microdroplet Bacterial Assays.

    PubMed

    Scheler, Ott; Kaminski, Tomasz S; Ruszczak, Artur; Garstecki, Piotr

    2016-05-11

    This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets. PMID:27100211

  18. The sperm chromatin structure assay: a review of clinical applications.

    PubMed

    Love, Charles C

    2005-10-01

    The sperm chromatin structure assay (SCSA) was introduced by as a method to determine the susceptibility of sperm DNA to denaturation and how those results related to fertility. This initial study used human sperm and was followed by studies in bulls and boars . This assay was one of the first to introduce the technique of flow cytometry, which has the ability to evaluate specific sperm compartments of large numbers of sperm in a short time, as a methodology to evaluate sperm quality and further define the relationship of sperm quality to fertility. For any assay to be of use clinically, it must not only be validated and adapted for the species of interest, but guidelines that associate specific levels of fertility with assay results must be defined. This review will describe how our laboratory uses the SCSA for clinical diagnosis of reduced fertility in the stallion. PMID:16140481

  19. Nanostar Clustering Improves the Sensitivity of Plasmonic Assays.

    PubMed

    Park, Yong Il; Im, Hyungsoon; Weissleder, Ralph; Lee, Hakho

    2015-08-19

    Star-shaped Au nanoparticles (Au nanostars, AuNS) have been developed to improve the plasmonic sensitivity, but their application has largely been limited to single-particle probes. We herein describe a AuNS clustering assay based on nanoscale self-assembly of multiple AuNS and which further increases detection sensitivity. We show that each cluster contains multiple nanogaps to concentrate electric fields, thereby amplifying the signal via plasmon coupling. Numerical simulation indicated that AuNS clusters assume up to 460-fold higher field density than Au nanosphere clusters of similar mass. The results were validated in model assays of protein biomarker detection. The AuNS clustering assay showed higher sensitivity than Au nanosphere. Minimizing the size of affinity ligand was found important to tightly confine electric fields and improve the sensitivity. The resulting assay is simple and fast and can be readily applied to point-of-care molecular detection schemes. PMID:26102604

  20. Improved assays for xenosensor activation based on reverse transfection.

    PubMed

    Küblbeck, Jenni; Anttila, Teemu; Pulkkinen, Juha T; Honkakoski, Paavo

    2015-10-01

    Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z' factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies. PMID:26187274