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Sample records for 51cr release assays

  1. Differences in immunoglobulin subclasses between dye exclusion and 51Cr release complement-dependent lymphocytotoxicity assays

    SciTech Connect

    McConnachie, P.R.; Denegri, J.F.; Lower, F.E.; Torry, D.S.; McIntyre, J.A.

    1986-08-01

    Paradoxical differences previously noted between lymphocytotoxicity detected by dye exclusion at room temperature (CDCE) or by /sup 51/Cr release (CDC/sup 51/Cr) at 37/sup 0/C in maternal antipaternal complement-dependent lymphocytotoxicity have suggested that CDCE and CDC/sup 51/Cr at 37/sup 0/C, but not at 20 degrees C, may detect different immunological antibody-antigen interactions. Reactions in the two test systems against the same target cells were compared in sera from known immune dialysis patients, secondary aborting women, and refractory platelet recipients before and after heat treatment of sera, absorption with solid-phase heparin, anti-light-chain augmentation, and the addition of murine monoclonal anti-IgG subclass antibodies. The results demonstrate significant differences between the two tests using the same target and sera. Further, the results imply the presence of an inhibitor and an inhibitor of inhibitor in sera. The involvement of different immunoglobulin subclasses was shown in the two tests. These data demonstrate the necessity for further study of the nature of the differences in the mechanisms of these clinically important antibody-detecting systems.

  2. Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.

    PubMed

    Mata, Mariana M; Mahmood, Fareeha; Sowell, Ryan T; Baum, Linda L

    2014-04-01

    Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

  3. An in vitro assay for uni-directional migration inhibition employing 51Cr-labelled macrophages.

    PubMed Central

    Noronha-Blob, L; Huang, S W

    1980-01-01

    An improved in vitro technique to assay for migration inhibitory factor is presented. The method employs chromium-51-radiolabelled guinea-pig macrophages and offers significant advantages including (1) elimination of observer to observer variation and tedious measurements resulting in an objective and technically simple assay, (2) the requirement for small numbers of immune lymphocytes, (3) good sensitivity and reproducibility between successive assays performed on different days, and (4) a means of obtaining relative estimates of the 'strength' (concentration) of the factor so that comparisons with healthy individuals can be made. PMID:6998620

  4. Phagocytosis-induced 51Cr release from activated macrophages and blood mononuclears. Effect of colchicine and antioxidants

    SciTech Connect

    McGee, M.P.; Hale, A.H.

    1981-09-01

    The chromium-release test was adapted to the measurement of the cellular injury induced when activated macrophages phagocytose particulates. Macrophages obtained from rabbit lungs undergoing BCG-induced chronic inflammation released more chromium when incubated in the presence of phagocytosable particles than when incubated under resting conditions. Blood mononuclear cells, 40-60% monocytes, procured from the same BCG-injected animals, were less susceptible to phagocytosis-induced injury than the macrophages obtained from the lungs. The amount of chromium released by the activated macrophages was proportional to the number of particles present during incubation. In the presence of catalase, the amounts of chromium released by phagocytosing and resting macrophages were similar; in the presence of superoxide dismutase and cytochrome c, the amount of chromium released by phagocytosing macrophages was 13-35% less than the amount of chromium released by macrophages incubated without the antioxidants. In addition, colchicine, an inhibitor of degranulation also exerted partial inhibition of the chromium release. These results suggest that oxygen radicals and lysosomal contents contribute to the cellular injury that results from phagocytosis.

  5. Calorimetric system for high-precision determination of activity of the 51Cr neutrino source in the BEST experiment

    NASA Astrophysics Data System (ADS)

    Veretenkin, E. P.; Gavrin, V. N.; Danshin, S. N.; Ibragimova, T. V.; Kalashnikova, A. A.; Kozlova, J. P.; Martynov, A. A.

    2017-01-01

    The calorimetric system based on mass-flow calorimeter for high-precision determination of neutrino flux from 51Cr source with activity 3MCi and higher is created for experiment BEST. The achieved heat release uncertainties are less than 0.25% in the whole range of the heat power and less than 0.1% in the range of 250-500 W. Total value the uncertainty considering the uncertainty of the energy release in the 51Cr decay (0.23%) shows that the activity of 3MCi 51Cr neutrino source can be determined with accuracy better than 0.5%.

  6. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    PubMed

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G

    2012-01-01

    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay.

  7. A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay.

    PubMed

    Fassy, Julien; Tsalkitzi, Kyriaki; Salavagione, Emie; Hamouda-Tekaya, Nedra; Braud, Véronique M

    2016-12-22

    Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard (51) Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to (51) Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T-cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.

  8. Determination of activity of 51Cr on gamma radiation measurements

    NASA Astrophysics Data System (ADS)

    Gorbachev, V. V.; Gavrin, V. N.; Ibragimova, T. V.; Kalikhov, A. V.; Malyshkin, Yu. M.; Shikhin, A. A.

    2017-01-01

    A method of determining the activity of intensive distributed -sources on the measurement of the continuous spectrum of radiation, for example the internal bremsstrahlung, is developed. The recurrent formula for reconstructing of a continuous spectrum, registered in a Ge detector, at distorting it in the detector. The method of precise measurements of the spectrum of 51Cr internal bremsstrahlung using two point sources of low activity is described.

  9. Calorimetric method for determination of 51Cr neutrino source activity

    NASA Astrophysics Data System (ADS)

    Veretenkin, E. P.; Gavrin, V. N.; Danshin, S. N.; Ibragimova, T. V.; Kozlova, Yu. P.; Mirmov, I. N.

    2015-12-01

    Experimental study of nonstandard neutrino properties using high-intensity artificial neutrino sources requires the activity of the sources to be determined with high accuracy. In the BEST project, a calorimetric system for measurement of the activity of high-intensity (a few MCi) neutrino sources based on 51Cr with an accuracy of 0.5-1% is created. In the paper, the main factors affecting the accuracy of determining the neutrino source activity are discussed. The calorimetric system design and the calibration results using a thermal simulator of the source are presented.

  10. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  11. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  12. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  13. The neutral red release assay: a review.

    PubMed

    Zuang, V

    2001-01-01

    The neutral red release (NRR) assay is a cytotoxicity test that can be used to measure the immediate toxic effects of test substances on the cell membrane, resulting in the leaking of intracellular contents. The assay has already been used for several years to evaluate the cytotoxicities of various kinds of products, such as cosmetics, pharmaceuticals, industrial chemicals and household products. It has undergone in-house validation by many companies, and has been found to be particularly useful for identifying substances that are potentially capable of causing adverse reactions on coming into brief contact with the eye or the skin at relatively high concentrations, such as might occur in an adventitious splash into the eye or onto the skin, followed by a quick rinse. Because of the relatively long existence of the NRR assay, its practicality and its proven usefulness for particular purposes, ECVAM decided to review the status of the method, in order to decide whether prevalidation and formal validation studies on the test might be profitable. The review of the status of the test was carried out by performing a comprehensive review of the literature, and by conducting a survey involving companies and institutes with experience in using the test. Both the review and the survey revealed that the assay could provide extremely valuable information when it was used for particular purposes, such as for the evaluation and comparison of immediate toxic effects on the eye or the skin caused by certain products or chemicals such as surfactants. Most of those who responded in the survey favoured a prevalidation/validation study.

  14. Interferon gamma release assays: principles and practice.

    PubMed

    Lalvani, Ajit; Pareek, Manish

    2010-04-01

    The last decade has witnessed significant advances in mycobacterial genomics and cellular research which have resulted in the development of two new blood tests, the enzyme-linked immunospot assay (ELISpot) (TSPOT.TB, Oxford Immunotec, Oxford, UK) and the enzyme-linked immunosorbent assay (ELISA) (QuantiFERON-TB Gold In-Tube, Cellestis, Carnegie, Australia). These tests, which are collectively known as interferon gamma release assays (IGRAs), detect latent tuberculosis infection (LTBI) by measuring interferon (IFN)-gamma release in response to antigens present in Mycobacterium tuberculosis, but not bacille Calmette-Guerin (BCG) vaccine and most nontuberculous mycobacteria. This is done through enumeration of IFN-gamma-secreting T cells (ELISpot) or by measurement of IFN-gamma concentration (ELISA). The evidence base for these tests has expanded rapidly and now demonstrates that IGRAs are more specific than the tuberculin skin test (TST) as they are not confounded by previous BCG vaccination. In addition, with active tuberculosis (TB) as a surrogate for LTBI, it appears that the ELISA has a similar sensitivity to the TST, whereas the ELISpot is more sensitive. Using degree of exposure to TB as a surrogate for LTBI, both assays correlate at least as well with TB exposure as the TST. Recent longitudinal data have now demonstrated the prognostic power of positive IGRA results in recent contacts for the subsequent progression to active TB. Deployment of IGRAs, driven by new guidelines internationally, will impact on clinical practice in several ways. Their high specificity means that BCG-vaccinated individuals with a false-positive TST will not receive unnecessary preventive treatment, whereas improved sensitivity in individuals with weakened cellular immunity at highest risk of progressing to active TB (for example HIV-positive individuals) enables more reliable targeted testing and treatment of these vulnerable groups. The role of IGRAs in active TB is less clear but

  15. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    SciTech Connect

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L. )

    1990-06-15

    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

  16. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the /sup 51/Cr-release assay

    SciTech Connect

    Zinkernagel, R.M.; Haenseler, E.; Leist, T.; Cerny, A.; Hengartner, H.; Althage, A.

    1986-10-01

    A model for immunologically T cell-mediated hepatitis was established in mice infected with lymphocytic choriomeningitis virus (LCMV). The severity of hepatitis was monitored histologically and by determination of changes in serum levels of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and alkaline phosphatase (AP). Kinetics of histological disease manifestations, increases of liver enzyme levels in the serum, and cytotoxic T cell activities in livers and spleens all correlated and were dependent upon several parameters: LCMV-isolate; LCMV-WE caused extensive hepatitis, LCMV-Armstrong virtually none. Virus dose. Route of infection; i.v. or i.p. infection caused hepatitis, whereas infection into the footpad did not. The general genetic background of the murine host; of the strains tested, Swiss mice and A-strain mice were more susceptible than C57BL or CBA mice; BALB/c and DBA/2 mice were least susceptible. The degree of immunocompetence of the murine host; T cell deficient nu/nu mice never developed hepatitis, whereas nu/+ or +/+ mice always did. B cell-depleted anti-IgM-treated mice developed immune-mediated hepatitis comparably or even more extensively than control mice. Local cytotoxic T cell activity; mononuclear cells isolated from livers during the period of overt hepatitis were two to five times more active than equal numbers of spleen cells. Adoptive transfer of nylon wool-nonadherent anti-Thy-1.2 and anti-Lyt-2 plus C-sensitive, anti-L3T4 plus C-resistant lymphocytes into irradiated mice preinfected with LCMV-WE caused a rapid time- and dose-dependent linear increase of serum enzyme levels. This increase was caused by adoptive transfer of lymphocytes if immune cell donors and recipient mice shared class I, but not when they shared class II histocompatibility antigens.

  17. Intestinal permeability to (/sup 51/Cr)EDTA in children with Crohn's disease and celiac disease

    SciTech Connect

    Turck, D.; Ythier, H.; Maquet, E.; Deveaux, M.; Marchandise, X.; Farriaux, J.P.; Fontaine, G.

    1987-07-01

    (/sup 51/Cr)EDTA was used as a probe molecule to assess intestinal permeability in 7 healthy control adults, 11 control children, 17 children with Crohn's disease, and 6 children with untreated celiac disease. After subjects fasted overnight, 75 kBq/kg (= 2 microCi/kg) /sup 51/Cr-labeled EDTA was given by mouth; 24-h urinary excretion of (/sup 51/Cr)EDTA was measured and expressed as a percentage of the total oral dose. Mean and SD were as follows: control adults 1.47 +/- 0.62, control children 1.59 +/- 0.55, and patients with Crohn's disease or celiac disease 5.35 +/- 1.94. The difference between control children and patients was statistically significant (p less than 0.001). These results show that intestinal permeability to (/sup 51/Cr)EDTA is increased among children with active or inactive Crohn's disease affecting small bowel only or small bowel and colon, and with untreated celiac disease. The (/sup 51/Cr)EDTA permeability test could facilitate the decision to perform more extensive investigations in children suspected of small bowel disease who have atypical or poor clinical and biological symptomatology.

  18. (51Cr)EDTA intestinal permeability in children with cow's milk intolerance

    SciTech Connect

    Schrander, J.J.; Unsalan-Hooyen, R.W.; Forget, P.P.; Jansen, J. )

    1990-02-01

    Making use of ({sup 51}Cr)EDTA as a permeability marker, we measured intestinal permeability in a group of 20 children with proven cow's milk intolerance (CMI), a group of 17 children with similar complaints where CMI was excluded (sick controls), and a group of 12 control children. ({sup 51}Cr)EDTA test results (mean +/- SD) were 6.85 +/- 3.64%, 3.42 +/- 0.94%, and 2.61 +/- 0.67% in the group with CMI, the sick control, and the control group, respectively. When compared to both control groups, patients with cow's milk intolerance (CMI) showed a significantly increased small bowel permeability. We conclude that the ({sup 51}Cr)EDTA test can be helpful for the diagnosis of cow's milk intolerance.

  19. /sup 51/Cr-EDTA: a marker of early intestinal rejection in the rat

    SciTech Connect

    Grant, D.; Lamont, D.; Zhong, R.; Garcia, B.; Wang, P.; Stiller, C.; Duff, J.

    1989-05-01

    Intestinal permeability was studied after accessory intestinal transplantation in Lewis rats. Five groups were evaluated: Group 1--isografts (N = 6); Group 2--Lewis X Brown Norway F1 (LBN-F1) allografts (N = 6); Group 3--isografts treated with CsA 2 mg/kg/day X 10 days (N = 6); Group 4--LBN-F1 allografts treated with CsA 2 mg/kg/day X 10 days (N = 6); Group 5--LBN-F1 allografts treated with CsA 4 mg/kg/day X 28 days (N = 6). Chromium-labeled ethylenedimianetetraacetate (/sup 51/Cr-EDTA) was given through the proximal stoma of the graft. Renal clearance of /sup 51/Cr-EDTA and mucosal biopsies were followed post-transplant. The biopsies of the intestinal graft showed no rejection in Groups 1, 3, and 5; fulminant rejection in Group 2; and mild atypical rejection in Group 4. /sup 51/Cr-EDTA clearance was elevated in all groups during the first 7 days post-transplant. Thereafter, /sup 51/Cr-EDTA excretion fell to lower levels in the animals with histologically normal grafts (Groups 1, 3, and 5). /sup 51/Cr-EDTA excretion in Group 4 was increased with the first histological evidence of rejection on Day 14 and remained elevated until sacrifice (P less than 0.02 compared to Groups 3 and 5). A transient permeability defect occurs after intestinal grafting. Once the graft has recovered from this injury, /sup 51/Cr-EDTA is a sensitive marker for intestinal rejection.

  20. Measuring the activity of a 51Cr neutrino source based on the gamma-radiation spectrum

    NASA Astrophysics Data System (ADS)

    Gorbachev, V. V.; Gavrin, V. N.; Ibragimova, T. V.; Kalikhov, A. V.; Malyshkin, Yu. M.; Shikhin, A. A.

    2015-12-01

    A technique for the measurement of activities of intense β sources by measuring the continuous gamma-radiation (internal bremsstrahlung) spectra is developed. A method for reconstructing the spectrum recorded by a germanium semiconductor detector is described. A method for the absolute measurement of the internal bremsstrahlung spectrum of 51Cr is presented.

  1. Cytokine release assays: current practices and future directions.

    PubMed

    Finco, D; Grimaldi, C; Fort, M; Walker, M; Kiessling, A; Wolf, B; Salcedo, T; Faggioni, R; Schneider, A; Ibraghimov, A; Scesney, S; Serna, D; Prell, R; Stebbings, R; Narayanan, P K

    2014-04-01

    As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays.

  2. Intestinal permeability to (/sup 51/Cr)EDTA in children with cystic fibrosis

    SciTech Connect

    Leclercq-Foucart, J.; Forget, P.; Sodoyez-Goffaux, F.; Zappitelli, A.

    1986-05-01

    Intestinal permeability was investigated in 14 children with cystic fibrosis making use of (/sup 51/Cr)EDTA as probe molecule. Ten normal young adults and 11 children served as controls. After oral administration of (/sup 51/Cr)EDTA, 24 h urine was collected. Urinary radioactivity was calculated and results expressed as percentage of oral dose excreted in 24 h urine. Mean and SEM were as follows: 2.51 +/- 0.21, 2.35 +/- 0.24, and 13.19 +/- 1.72 for control children, normal adults, and cystic fibrosis patients, respectively. The permeability differences between cystic fibrosis patients and either control children or control adults are significant (p less than 0.001).

  3. Studies with nonradioisotopic sodium chromate. II. Single- and double-label sup 52 Cr/ sup 51 Cr posttransfusion recovery estimations

    SciTech Connect

    Heaton, W.A.; Keegan, T.; Hanbury, C.M.; Holme, S.; Pleban, P. )

    1989-10-01

    A recently developed nonradioisotopic 52Cr technique was used to measure either red cell volume or posttransfusion recovery of stored red cells. The experimental method uses Zeeman electrothermal atomic absorption spectrophotometry to measure red cell chromium. Results from the 52Cr method were compared with those from 51Cr single-label and 125I-albumin/51Cr double-label procedures using 49-day AS-1 red cell concentrates drawn and prepared according to standard procedures. In the first group of five donors, red cell volume was estimated concurrently with both 52Cr-labeled fresh red cells and 125I-albumin. The latter measured plasma volume from which red cell volume was estimated on the basis of the hematocrit (125I red cell volume). 51Cr-labeled stored red cells were transfused to measure posttransfusion recoveries. The correlation between 52Cr and 125I red cell volumes was significant (r = 0.68, p less than 0.01), and, in this group, the differences were not significant (p less than 0.05). Twenty-four-hour posttransfusion recoveries of 51Cr-labeled stored red cells averaged 66 +/- 5 percent when measured with the 125I/51Cr technique and 69 +/- 8 percent when measured with the 52Cr/51Cr method. In the second group of five donors, red cell volume was estimated by the 125I-albumin technique, and the posttransfusion recovery of stored red cells was quantitated by 51Cr- and 52Cr-labeled stored cells simultaneously. In this group, posttransfusion recoveries with 125I/51Cr averaged 73 +/- 7 percent; with 125I/52Cr, they averaged 75 +/- 10 percent. Using the single-label method of calculation, recoveries averaged 76 +/- 7 and 75 +/- 10 percent for the 51Cr and 52Cr methods, respectively.

  4. Current status of new SAGE project with 51Cr neutrino source

    NASA Astrophysics Data System (ADS)

    Gavrin, V.; Cleveland, B.; Danshin, S.; Elliott, S.; Gorbachev, V.; Ibragimova, T.; Kalikhov, A.; Knodel, T.; Kozlova, Yu.; Malyshkin, Yu.; Matveev, V.; Mirmov, I.; Nico, J.; Robertson, R. G. H.; Shikhin, A.; Sinclair, D.; Veretenkin, E.; Wilkerson, J.

    2015-03-01

    A very short-baseline neutrino oscillation experiment with an intense 51Cr neutrino source is currently under construction at the Baksan Neutrino Observatory of the Institute for Nuclear Research RAS (BNO). The experiment, which is based on the existing SAGE experiment, will use an upgraded Gallium-Germanium Neutrino Telescope (GGNT) and an artificial 51Cr neutrino source with activity ˜3 MCi to search for transitions of active neutrinos to sterile states with Δ m 2 ˜1 eV2. The neutrino source will be placed in the center of a liquid Ga metal target that is divided into two concentric zones, internal and external. The average path length of neutrinos in each zone will be the same and the neutrino capture rate will be measured separately in each zone. The oscillation signature, which comes from the ratio of events in the near and far gallium volumes, will be largely free of systematic errors, such as may occur from cross section and source strength uncertainties, and will provide a clean signal of electron neutrino disappearance into a sterile state at baselines of about 0.6 and 2.0 m. The sensitivity to the disappearance of electron neutrinos is expected to be a few percent. Construction of this set of new facilities, including a two-zone tank for irradiation of 50 tons of Ga metal with the intense 51Cr source, as well as additional modules of the GGNT counting and extraction systems, is close to completion. To check the new facilities they will first be used for SAGE solar neutrino measurements.

  5. Calorimetric method for determination of {sup 51}Cr neutrino source activity

    SciTech Connect

    Veretenkin, E. P. Gavrin, V. N.; Danshin, S. N.; Ibragimova, T. V.; Kozlova, Yu. P.; Mirmov, I. N.

    2015-12-15

    Experimental study of nonstandard neutrino properties using high-intensity artificial neutrino sources requires the activity of the sources to be determined with high accuracy. In the BEST project, a calorimetric system for measurement of the activity of high-intensity (a few MCi) neutrino sources based on {sup 51}Cr with an accuracy of 0.5–1% is created. In the paper, the main factors affecting the accuracy of determining the neutrino source activity are discussed. The calorimetric system design and the calibration results using a thermal simulator of the source are presented.

  6. Reversibility of increased intestinal permeability to 51Cr-EDTA in patients with gastrointestinal inflammatory diseases

    SciTech Connect

    Jenkins, R.T.; Jones, D.B.; Goodacre, R.L.; Collins, S.M.; Coates, G.; Hunt, R.H.; Bienenstock, J.

    1987-11-01

    Intestinal permeability in adults with inflammatory gastrointestinal diseases was investigated by measuring the 24-h urinary excretion of orally administered /sup 51/Cr-EDTA. Eighty controls along with 100 patients with Crohn's disease, 46 patients with ulcerative colitis, 20 patients with gluten-sensitive enteropathy, and 18 patients with other diseases were studied. In controls, the median 24-h excretion was 1.34%/24 h of the oral dose. Patients with Crohn's disease (median 2.96%/24 h), ulcerative colitis (median 2.12%/24 h), and untreated gluten-sensitive enteropathy (median 3.56%/24 h) had significantly elevated urinary excretion of the probe compared to controls (p less than 0.0001). Increased 24-h urinary excretion of /sup 51/Cr-EDTA had a high association with intestinal inflammation (p less than 0.0001). Test specificity and sensitivity were 96% and 57%, respectively. A positive test has a 96% probability of correctly diagnosing the presence of intestinal inflammation, whereas a negative test has a 50% probability of predicting the absence of disease.

  7. Dietary absorption of sediment-bound fluoranthene by a deposit-feeding gastropod using the {sup 14}C:{sup 51}Cr dual-labeling method

    SciTech Connect

    Forbes, V.E.; Forbes, T.L.

    1997-05-01

    Polycyclic aromatic hydrocarbons (PAH) constitute a broad class of toxic, environmentally persistent, particle-reactive organic compounds that are ubiquitous in aquatic and terrestrial ecosystems. This study was designed to measure ingestion and dietary absorption of the PAH, fluoranthene, by two genotypes of a deposit-feeding gastropod using the {sup 14}C:{sup 51}Cr dual-labeling method. Sediment processing rate, fluoranthene ingestion rate, selective ingestion of fluoranthene-containing particles, and fluoranthene absorption rate varied as a function of snail body size and genotype. Absorption efficiency of sediment-bound fluoranthene did not vary as a function of body size but differed between genotypes, averaging 42 to 46% for Clone A and 22 to 36% for Clone B. The authors could detect no significant metabolism of ingested fluoranthene to dissolved organic carbon or CO{sub 2} during 24 h following its ingestion. The {sup 14}C:{sup 51}Cr dual-labeling method provides a powerful approach for investigating the dietary absorption of sediment-bound contaminants by (1) allowing the calculation of ingestion selectivity, sediment processing rate, contaminant ingestion rate, and absorption efficiency in individual, small invertebrates; (2) permitting estimation of the fraction of ingested/absorbed contaminant that is metabolized and released via different routes following its ingestion; and (3) facilitating evaluation of the relative importance of porewater versus ingested sediment as routes of contaminant uptake by animals.

  8. Preliminary results from the {sup 51}Cr neutrino source experiment in GALLEX

    SciTech Connect

    Hampel, W.; Heusser, G.; Kiko, J.

    1996-09-01

    The GALLEX collaboration performed a second {sup 51}Cr neutrino source experiment during fall 1995. The full results from this second source experiment will not be available before the end of 1996. Meanwhile, we present a short description and preliminary results in this informal note. The (preliminary) value of the activity obtained form direct measurements has been found equal to (68.7 {+-}0.7) PBq (with 1-sigma error). This value, which is about 10% higher than the activity of the first source, was achieved by optimizing the irradiation conditions in the Silo{acute e} reactor and doing a longer irradiation of the enriched chromium. Preliminary results show that the ratio, R, of the radiochemically determined activity from {sup 71}Ge counting (57.1 {+-} PBq) to the directly measured activity is (0.83 {+-} 0.10). The combined value of R for the two source experiments is (0.92 {+-} 0.08).

  9. Bioaccumulation of 51Cr, 63Ni and 14C in Baltic Sea benthos.

    PubMed

    Kumblad, L; Bradshaw, C; Gilek, M

    2005-03-01

    The Baltic Sea is a species-poor, semi-enclosed, brackish sea, whose sediments contain a wide range of contaminants, including sediment-associated metals and radionuclides. In this study, we have examined and compared bioaccumulation kinetics and assimilation efficiencies of sediment-associated (51)Cr, (63)Ni and (14)C in three key benthic invertebrates (the deposit-feeding Monoporeia affinis, the facultative deposit-feeding Macoma baltica, and the omnivorous Halicryptus spinulosus). Our results demonstrate that (i) all radionuclides were accumulated, (ii) the different radionuclides were accumulated to various extents, (iii) small changes in organic carbon concentration can influence the accumulation, and (iv) the degree of accumulation differed only slightly between species. These processes, together with sediment resuspension and bioturbation, may remobilise trace metals from the sediment to the water and to higher trophic levels, and therefore should be taken into account in exposure models and ERAs.

  10. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  11. Measuring the activity of a {sup 51}Cr neutrino source based on the gamma-radiation spectrum

    SciTech Connect

    Gorbachev, V. V. Gavrin, V. N.; Ibragimova, T. V.; Kalikhov, A. V.; Malyshkin, Yu. M.; Shikhin, A. A.

    2015-12-15

    A technique for the measurement of activities of intense β sources by measuring the continuous gamma-radiation (internal bremsstrahlung) spectra is developed. A method for reconstructing the spectrum recorded by a germanium semiconductor detector is described. A method for the absolute measurement of the internal bremsstrahlung spectrum of {sup 51}Cr is presented.

  12. New flow cytometric assays for monitoring cell-mediated cytotoxicity.

    PubMed

    Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

    2010-06-01

    The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the (51)Cr-release assay and IFN-gamma ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the (51)Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

  13. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  14. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  15. Fluorescence-based retention assays reveals sustained release of vascular endothelial growth factor from bone grafts.

    PubMed

    Kang, Wonmo; Yun, Ye-Rang; Lee, Dong-Sung; Kim, Tae-Hyun; Kim, Joong-Hyun; Kim, Hae-Won; Jang, Jun-Hyeog

    2016-01-01

    The sustained release of growth factors following their implantation in vivo is essential for successful outcomes in bone tissue engineering. In this study, we evaluated the release kinetics and delivery efficacies of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, incorporated into calcium phosphate bone grafts (BGs). We evaluated the release profile of VEGF from BGs using a novel fluorescence-based retention assay, which revealed that VEGF loaded on BGs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. In contrast, an ELISA-based release assay showed VEGF to have an early burst-release profile for the first week. However, the biological activity of VEGF released from the BGs was preserved over the 7-week release period, which is consistent with the sustained-release profile observed in the fluorescence-based retention assay. Furthermore, the in vivo bone-forming action of the VEGF-loaded BGs was well demonstrated in a rat subcutaneous model. Taken together, the sustained release of VEGF loaded onto BGs was effective in stimulating proliferation, angiogenesis and osteogenesis, suggesting the ultimate value of VEGF-engineered BGs for bone tissue engineering.

  16. Permeability of the small intestine to (/sup 51/Cr)EDTA in children with acute gastroenteritis or eczema

    SciTech Connect

    Forget, P.; Sodoyez-Goffaux, F.; Zappitelli, A.

    1985-06-01

    Increased gut permeability to macromolecules is thought to be an important factor in the development of food hypersensitivity. The latter can develop in the course of acute gastroenteritis and could play a role in infantile eczema. The authors studied gut permeability in 10 normal adults, 11 control children, 7 children with acute gastroenteritis, and 8 patients with infantile eczema, making use of (/sup 51/Cr)EDTA as probe molecule. (/sup 51/Cr)EDTA was given orally (50-100 microCi); 24-h urinary excretion of (/sup 51/Cr)EDTA was measured and expressed as a percentage of the oral dose. Mean and standard error were 2.35 +/- 0.24, 2.51 +/- 0.21, 9.96 +/- 3.44, and 10.90 +/- 2.05 in normal adults, control children, and gastroenteritis and eczema patients, respectively. Differences between controls and either gastroenteritis (p less than 0.001) or eczema (p less than 0.001) patients are significant. The results support the hypothesis that increased gut permeability could play a role in food hypersensitivity.

  17. Radiolabeled red cell viability. I. Comparison of /sup 51/Cr, /sup 99m/Tc, and /sup 111/In for measuring the viability of autologous stored red cells

    SciTech Connect

    Marcus, C.S.; Myhre, B.A.; Angulo, M.C.; Salk, R.D.; Essex, C.E.; Demianew, S.H.

    1987-09-01

    The simultaneous determination of autologous /sup 99m/Tc red cell (RBC) and /sup 51/Cr RBC viability at 24 hours was measured in 19 normal volunteers whose RBCs had been stored in additive media (Nutracel) for 42 or 49 days. The ratio of the /sup 51/Cr:/sup 99m/Tc value was 1.23. In this experiment we also calculated /sup 51/Cr RBC viability by both the single-isotope method (extrapolation) and the double-isotope method (using /sup 125/I human serum albumin for an independent plasma volume) in the same volunteers. The corresponding viability values were not significantly different. The simultaneous determination of autologous /sup 111/In-oxine RBC and /sup 51/Cr RBC viability at 24 hours was measured in 19 other normal volunteers whose RBCs had been stored in citrate-phosphate-dextrose-adenine (CPDA-1) for 1 or 15 days. The ratio of the /sup 51/Cr:/sup 111/In value was 1.1. Use of these 24-hour viability ratios as conversion factors permits direct comparison of /sup 99m/Tc or /sup 111/In RBC viability with a /sup 51/Cr standard, and therefore expands the application of these newer RBC radiolabels.

  18. Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr-labeled platelets differ

    SciTech Connect

    Heyns A du, P.; Badenhorst, P.N.; Loetter, M.G.P.; Pieters, H.; Wessels, P.; Kotze, H.F.

    1986-01-01

    Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr-labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.

  19. Analysis of histamine release assays using the Bootstrap.

    PubMed

    Coberly, William A; Price, Joseph A

    2005-01-01

    The data from several types of bioassays is usually presented as a quotient as an intuitive parameter and a means of comparing results between experiments. For the example we considered here, we look at experiments with an experiment-wide negative control used to generate percent activity quotients from each experimental group. We asked if there was a valid means to statistically evaluate the transformed rather than the raw data. The experimental system chosen was a dose response of the agonist compound 48/80, which causes release of histamine from mast cells, thus providing test data from replicates of n=24. Descriptive statistics, the Ryan-Joiner test for normality of distribution of data, and normal probability plots confirm the normality of the distribution of data at each dose level. In parametric analysis, when the control group was treated as an errorless constant, there was a distinct consistent bias in the standard error of the data of 10% or less, which was not present if the control group's mean was treated as a variable with experimental error. This would be of minor interest in qualitative studies and might be safely ignored, but might be of considerable importance in quantitative assessments of activity using confidence intervals. When using Bootstrap estimates of standard error and probability plots of the bootstrap samples, the transformed data does not deviate significantly from normality. The standard, bias-corrected percentile limits (BCa), and empirical percentile methods gave very similar results when using resampling statistics to generate the transformed data from groups of n=6. Sample size can be as low as n=4 and still provide useful results. Thus, we have shown that resampling (i.e., bootstrap, Monte Carlo method, computer-intensive methods) can produce the data transform as well as provide confidence intervals using this type of raw data in small groups (n=4 to 6), giving improved statistical analysis of the transformed data (ratio

  20. Mannitol clearance for the determination of glomerular filtration rate-a validation against clearance of (51) Cr-EDTA.

    PubMed

    Kiss, Katalin; Molnár, Miklós; Söndergaard, Sören; Molnár, Gyula; Ricksten, Sven-Erik

    2016-06-03

    We studied the agreement between plasma clearance of mannitol and the reference method, plasma clearance of (51) Cr-EDTA in outpatients with normal to moderately impaired renal function. Forty-one patients with a serum creatinine <200 μmol l(-1) entered the study. (51) Cr-EDTA clearance was measured with the standard bolus injection technique and glomerular filtration rate (GFR) was calculated by the single-sample method described by Jacobsson. Mannitol, 0·25 g kg(-1) body weight (150 mg ml(-1) ), was infused for 4-14 min and blood samples taken at 1-, 2-, 3- and 4-h (n = 24) or 2-, 3-, 3·5- and 4-h after infusion (n = 17). Mannitol in serum was measured by an enzymatic method. Plasma clearance for mannitol and its apparent volume of distribution (Vd) were calculated according to Brøchner-Mortensen. Mean plasma clearance (±SD) for (51) Cr-EDTA was 59·7 ± 18·8 ml min(-1) . The mean plasma clearance for mannitol ranged between 57·0 ± 20·1 and 61·1 ± 16·7 ml min(-1) and Vd was 21·3 ± 6·2% per kg b.w. The between-method bias ranged between -0·23 and 2·73 ml min(-1) , the percentage error between 26·7 and 39·5% and the limits of agreement between -14·3/17·2 and -25·3/19·9 ml min(-1) . The best agreement was seen when three- or four-sample measurements of plasma mannitol were obtained and when sampling started 60 min after injection. Furthermore, accuracy of plasma clearance determinations was 88-96% (P30) and 41-63% (P10) and was highest when three- or four-sample measurements of plasma mannitol were obtained, including the first hour after the bolus dose. We conclude that there is a good agreement between plasma clearances of mannitol and (51) Cr-EDTA for the assessment of GFR.

  1. Use of /sup 51/Cr-labeled mononuclear cells for measuring the cellular immune response in mouse lungs

    SciTech Connect

    Zarkower, A.; Scheuchenzuber, W.J.; Ferguson, F.G.

    1981-02-01

    Spleen cells labeled with /sup 51/Cr were used in sensitized syngeneic mice to measure the degree of mononuclear cell infiltration into antigen-challenged tissues. With this method, increased cellular infiltration was found after footpad challenge of mice sensitized with sheep erythrocyte, Escherichia coli, and BCG antigens. Cellular response also was determined by using this technique in the lungs of mice sensitized with sheep erythrocytes and BCG. This procedure offers the opportunity to measure cellular infiltration, whether due to cellular or humoral influences, in tissues not easily accessible to conventional immunological manipulation.

  2. Use of 51Cr-labeled mononuclear cells for measuring the cellular immune response in mouse lungs

    SciTech Connect

    Zarkower, A.; Scheuchenzuber, W.J.; Ferguson, F.G.

    1981-02-01

    Spleen cells labeled with 51Cr were used in sensitized syngeneic mice to measure the degree of mononuclear cell infiltration into antigen-challenged tissues. With this method, increased cellular infiltration was found after footpad challenge of mice sensitized with sheep erythrocyte, Escherichia coli, and BCG antigens. Cellular response also was determined by using this technique in the lungs of mice sensitized with sheep erythrocytes and BCG. This procedure offers the opportunity to measure cellular infiltration, whether due to cellular or humoral influences, in tissues not easily accessible to conventional immunological manipulation.

  3. A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity.

    PubMed

    Wagner, Bettina; Childs, Bronwen A; Erb, Hollis N

    2008-12-15

    Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE receptors to the surfaces of mast cells and basophils induces the release of inflammatory mediators. One of these mediators is histamine. The histamine was then detected by a colorimetric enzyme-linked immunosorbent assay. The histamine assay was used to test 33 horses with skin hypersensitivity and 20 clinically healthy control animals for histamine release from their peripheral blood basophils after stimulation with Culicoides allergen extract or monoclonal anti-IgE antibody. An increased histamine release was observed in the horses with skin hypersensitivity compared to the control group after allergen-specific stimulation with Culicoides extract (p=0.023). In contrast, stimulation with anti-IgE induced similar amounts of released histamine in both groups (p=0.46). For further evaluation of the HRA, we prepared a receiver operating-characteristic (ROC) curve and performed a likelihood-ratio analysis for assay interpretation. Our results suggested that the assay is a valuable diagnostic tool to identify sensitization to Culicoides allergens in horses. Because some of the clinically healthy horses also showed sensitization to Culicoides extract, the assay cannot be used to distinguish allergic from non-allergic animals. The observation that sensitization is sometimes detectable in non-affected animals suggested that clinically healthy horses use immune mechanisms to control the

  4. Continuous automated assay of alpha-amylase release from superfused rat salivary gland.

    PubMed

    Templeton, D

    1980-11-01

    A method of continuous automated amylase assay is described. This relies on the absorption of iodine by starch to produce a blue color that can be quantified colorimetrically. Digestion of the starch by amylase released from parotid tissue slices reduces the intensity of the color formed, allowing quantification of the amylase released. The assay in sensitive to 0.05 U/amylase/ml and linear up to 13 U/ml. Typical tissue responses to acetyl-beta-methylcholine and isoprenaline are presented.

  5. Simple visual assay for determination of Pasteurella haemolytica cytotoxin neutralizing antibody titers in cattle sera.

    PubMed Central

    Gentry, M J; Confer, A W; Kreps, J A

    1985-01-01

    A simple visual assay is described for determining the capacity of bovine serum to neutralize the cytotoxin produced by Pasteurella haemolytica serotype 1. The test was reproducible from day to day with different target cell populations and cytotoxin preparations. Cytotoxin neutralization titers obtained by the visual assay were comparable to those determined by the trypan blue exclusion and 51Cr-release methods. The visual assay was used to measure neutralization titers of bovine sera obtained from vaccination experiments and fractions of purified serum obtained by gel filtration. The major advantages of the visual assay over other assays are that it is rapid, inexpensive, and does not use radioisotopes. It also does not require specialized equipment, making it adaptable to most laboratories. Images PMID:3905853

  6. A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.

    PubMed

    Jeffery, Unity; Gray, Robert D; LeVine, Dana N

    2016-11-21

    Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

  7. Comparative gastrointestinal blood loss associated with placebo, aspirin, and nabumetone as assessed by radiochromium (/sup 51/Cr)

    SciTech Connect

    Lussier, A.; Davis, A.; Lussier, Y.; Lebel, E.

    1989-03-01

    Nabumetone differs from most other nonsteroidal anti-inflammatory drugs. It is presented to the gut as a nonacidic prodrug, and is metabolized to its active form after absorption. Studies in animals and humans suggest it is less irritating to the gastrointestinal mucosa. This study compared the gastrointestinal microbleeding induced by nabumetone to aspirin (acetylsalicylic acid, ASA), and placebo in a double blind parallel study using chromium /sup 51/Cr labelled red cells to quantitate fecal blood loss (FBL) in healthy volunteers. Thirty subjects were randomized to treatment with nabumetone (2000 mg), ASA (3.6 g) or placebo for 21 days following a 7 day placebo period. Six subjects served as untreated controls. FBL in nabumetone treated subjects was not significantly different to placebo or untreated subjects. In contrast, ASA-treated subjects exhibited significantly increased FBL than the other 3 groups (P less than .0001).

  8. Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with 125I bovine serum albumin and 51Cr-erythrocyte tracers

    USGS Publications Warehouse

    Gingerich, W.H.; Pityer, R.A.

    1989-01-01

    Total, packed cell and, plasma volume estimates were made for the whole body and selected tissues of rainbow trout by the simultaneous injection of radiolabelled trout erythrocyte (51Cr-RBC) and radioiodinated bovine serum albumin (125I-BSA) tracers. Blood volumes were estimated with both markers separately by the tracer-hematocrit method and as the combination of the 51Cr-RBC packed cell and 125I-BSA plasma volumes. Mean whole body blood volume was significantly less when calculated from the 51Cr-RBC tracer data (3.52±0.78 ml/100 g; ±SD) than when calculated with the 125I-BSA tracer (5.06±0.86 ml/100 g) or as the sum of the two volumes combined (4.49±0.60 ml/100 g). The whole body hematocrit (28±5%), estimated as the quotient of the 51Cr-RBC volume divided by the sum of the 125I-BSA and the 51Cr-RBC volumes, also was significantly less than the dorsal aortic microhematocrit (36±4%). Estimates of total blood volumes in most tissues were significantly smaller when calculated from the51Cr-RBC data than when calculated by the other two methods. Tissue blood volumes were greatest in highly vascularized and well perfused tissues and least in poorly vascularized tissues. The relative degree of vascularization among tissues generally remained the same regardless of whether the red cell or the plasma tracer was used to calculated blood volume. It is not clear whether the expanded plasma volume is the result of the distribution of erythrocyte-poor blood into the secondary circulation or the result of extravascular exchange of plasma proteins.

  9. Ricin Activity Assay by Direct Analysis in Real Time Mass Spectrometry Detection of Adenine Release

    DTIC Science & Technology

    2010-02-01

    direct analysis in real time mass spectrometry. The release of adenine from the inhomo- geneous substrate herring sperm DNA by ricin was determined to...chain catalyzes cleavage at adenosine 4324 (in rat RNA) of 28S rRNA to release adenine.10 This action inhibits protein synthesis, leading to cell...death. In addition to RNA, herring sperm DNA (hsDNA) is a substrate for ricin.11 We chose to employ hsDNA for this assay because it is relatively stable

  10. A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans.

    PubMed

    Wolf, Babette; Morgan, Hannah; Krieg, Jennifer; Gani, Zaahira; Milicov, Adriana; Warncke, Max; Brennan, Frank; Jones, Stewart; Sims, Jennifer; Kiessling, Andrea

    2012-12-01

    The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine

  11. Clinical evaluation of a /sup 51/Cr-labeled red blood cell survival test for in vivo blood compatibility testing

    SciTech Connect

    Pineda, A.A.; Dharkar, D.D.; Wahner, H.W.

    1984-01-01

    Modified red blood cell survival studies with use of 51Cr were performed in three groups of subjects. Group 1 consisted of normal subjects who were given labeled autologous blood, group 2 were subjects in need of blood transfusions and given labeled ABO and Rh crossmatch-compatible blood, and group 3 were patients in need of blood transfusion but in whom problems arose in finding compatible blood. The results of the studies suggest that for patients with blood compatibility problems, normal red blood cell survival values at 1 hour do not exclude the possibility of severe hemolysis 24 hours later. Thus, if a 1-hour test result is normal, the procedure should be extended routinely to 24 hours. Moreover, the test can be used to evaluate the clinical importance of antibodies. We showed that anti-Yka and anti-Lan were clinically significant, but high-titer, low-avidity antibodies, anti-Kna, anti-I, and anti-HI were clinically insignificant in the cases studied. This finding emphasizes the importance of an in vivo test for the final compatibility evaluation in complicated blood replacement problems.

  12. Dried plasma spots in the diagnosis of tuberculosis: IP-10 release assay on filter paper

    PubMed Central

    Aabye, Martine G.; Latorre, Irene; Diaz, Jessica; Maldonado, Jose; Mialdea, Irene; Eugen-Olsen, Jesper; Ravn, Pernille; Dominguez, Jose; Ruhwald, Morten

    2013-01-01

    Interferon (IFN)-γ release assays (IGRAs) are probably the most accurate tests for the detection of latent Mycobacterium tuberculosis infection, but IGRAs are labour intensive and the transport of samples over longer distances is difficult. IFN-γ-induced protein (IP)-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays have comparable performance to IGRAs. The aim of this study was to explore the diagnostic potential of a novel IP-10 release assay based on dried plasma spots (DPS). The presence of IP-10 and IFN-γ was determined in plasma and in DPS by ELISA. Diagnostic algorithms for plasma and DPS tests for IP-10 were developed on a training cohort comprising 60 tuberculosis (TB) patients and 59 healthy controls. Diagnostic accuracy was assessed in a validation cohort comprising 78 TB patients and 98 healthy controls. Plasma was measured in Spain and DPS samples were sent to Denmark using the conventional postal service for analysis. IP-10 was readily detectable in both plasma and DPS, and correlation was excellent (r2 = 0.95). QuantiFERON-TB Gold In-Tube (QFT-TB) and IP-10 in DPS and plasma rendered comparable sensitivity (78%, 82% and 84%, respectively), specificity (100%, 97% and 97%, respectively) and indeterminate rates (p>0.55). The DPS-based IP-10 test has comparable diagnostic accuracy to the QFT-TB and samples can be sent via conventional mail over long distances for analysis without affecting the results. PMID:23349445

  13. Interferon Gamma Release Assays for Latent Tuberculosis: What Are the Sources of Variability?

    PubMed Central

    Gaur, Rajiv L.; Pai, Madhukar

    2016-01-01

    Interferon gamma release assays (IGRAs) are blood-based tests intended for diagnosis of latent tuberculosis infection (LTBI). IGRAs offer logistical advantages and are supposed to offer improved specificity over the tuberculin skin test (TST). However, recent serial testing studies of low-risk individuals have revealed higher false conversion rates with IGRAs than with TST. Reproducibility studies have identified various sources of variability that contribute to nonreproducible results. Sources of variability can be broadly classified as preanalytical, analytical, postanalytical, manufacturing, and immunological. In this minireview, we summarize known sources of variability and their impact on IGRA results. We also provide recommendations on how to minimize sources of IGRA variability. PMID:26763969

  14. [³H]serotonin release assay using antigen-stimulated rat peritoneal mast cells.

    PubMed

    Skaper, Stephen D; Facci, Laura

    2012-01-01

    The concentration of nerve growth factor (NGF) is elevated in a number of inflammatory and autoimmune states in conjunction with increased accumulation of mast cells. Mast cells, which are of hematopoietic lineage, and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells themselves are capable of producing and responding to NGF. Here we describe a protocol for the isolation and culture of peritoneal-derived rat mast cells, together with a [(3)H]serotonin release assay which is useful in assessing the effects of antigens and neurotrophic factors on mast-cell activation.

  15. Time interval to conversion of interferon-gamma release assay after exposure to tuberculosis.

    PubMed

    Lee, S W; Oh, D K; Lee, S H; Kang, H Y; Lee, C-T; Yim, J-J

    2011-06-01

    The proper interval for repeating an interferon-γ release assay (IGRA) among tuberculosis contacts with initially negative results is unknown. The interval for IGRA conversion after exposure to patients with active pulmonary tuberculosis in an outbreak setting was evaluated. In a platoon of 32 soldiers, four active pulmonary tuberculosis patients, in addition to one index patient, were diagnosed during a contact investigation. For the other 27 contacts, a tuberculin skin test (TST) and QuantiFERON® TB-Gold In-Tube (QFT-GIT) assay were performed. For soldiers with a negative result on the initial QFT-GIT assay, the test was repeated at 2, 4, 8, 14, 18 and 30 weeks until positive conversion occurred. When conversion was identified, the subject was treated for latent tuberculosis infection. Initially, 17 (63.0%) soldiers gave positive QFT-GIT results, whereas 21 (77.8%) showed positive TST results. Among 10 participants with initially negative QFT-GIT results, three showed conversion at 2 weeks, three at 4 weeks and three at 14 weeks. Conversion did not occur during the 30-week observation period in one contact. Based on the tuberculosis exposure time-points among the contacts, IGRA conversion generally occurred 4-7 weeks after exposure, although it could occur as late as 14-22 weeks after exposure.

  16. Experimental and theoretical study for the production of 51Cr using p, d, 3He and 4He projectiles on V, Ti and Cr targets

    NASA Astrophysics Data System (ADS)

    Solieman, A. H. M.; Al-Abyad, M.; Ditroi, F.; Saleh, Z. A.

    2016-01-01

    Production of 51Cr (T1/2 = 27.7 d) have been studied experimentally through the reaction of proton and 3He on natV and natTi targets respectively by using a variable energy cyclotrons. Reaction cross sections were obtained at different energies using the stacked-foil technique. High resolution gamma ray spectrometers were used for measuring the γ-ray spectra. Comparison between the present experimental results and the previously reported data has been carried out and discussed. The possibility of producing 51Cr with reasonable yield using different projectiles and different natural targets was studied and reported. Excitation functions for the reactions of proton, deuteron, 3He and 4He particles on natural vanadium, titanium and chromium targets have been evaluated using two theoretical codes TALYS-1.6 and EMPIRE-3.1. The recommended cross-sections and the integral yields as well were obtained.

  17. Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

    PubMed

    El-Hawiet, Amr; Shoemaker, Glen K; Daneshfar, Rambod; Kitova, Elena N; Klassen, John S

    2012-01-03

    Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~10(3) M(-1)) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ~10 μM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

  18. Interferon-γ release assays for diagnosis of tuberculosis infection and disease in children.

    PubMed

    Starke, Jeffrey R

    2014-12-01

    Tuberculosis (TB) remains an important problem among children in the United States and throughout the world. Although diagnosis and treatment of infection with Mycobacterium tuberculosis (also referred to as latent tuberculosis infection [LTBI] or TB infection) remain the lynchpins of TB prevention, there is no diagnostic reference standard for LTBI. The tuberculin skin test (TST) has many limitations, including difficulty in administration and interpretation, the need for a return visit by the patient, and false-positive results caused by significant cross-reaction with Mycobacterium bovis-bacille Calmette-Guérin (BCG) vaccines and many nontuberculous mycobacteria. Interferon-γ release assays (IGRAs) are blood tests that measure ex vivo T-lymphocyte release of interferon-γ after stimulation by antigens specific for M tuberculosis. Because these antigens are not found on M bovis-BCG or most nontuberculous mycobacteria, IGRAs are more specific tests than the TST, yielding fewer false-positive results. However, IGRAs have little advantage over the TST in sensitivity, and both methods have reduced sensitivity in immunocompromised children, including children with severe TB disease. Both methods have a higher positive predictive value when applied to children with risk factors for LTBI. Unfortunately, neither method distinguishes between TB infection and TB disease. The objective of this technical report is to review what IGRAs are most useful for: (1) increasing test specificity in children who have received a BCG vaccine and may have a false-positive TST result; (2) using with the TST to increase sensitivity for finding LTBI in patients at high risk of developing progression from LTBI to disease; and (3) helping to diagnose TB disease.

  19. Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays

    PubMed Central

    Miwa, Satomi; Treumann, Achim; Bell, Amy; Vistoli, Giulio; Nelson, Glyn; Hay, Sam; von Zglinicki, Thomas

    2016-01-01

    Amplex Red is a fluorescent probe that is widely used to detect hydrogen peroxide (H2O2) in a reaction where it is oxidised to resorufin by horseradish peroxidase (HRP) as a catalyst. This assay is highly rated amongst other similar probes thanks to its superior sensitivity and stability. However, we report here that Amplex Red is readily converted to resorufin by a carboxylesterase without requiring H2O2, horseradish peroxidase or oxygen: this reaction is seen in various tissue samples such as liver and kidney as well as in cultured cells, causing a serious distortion of H2O2 measurements. The reaction can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at concentrations which do not disturb mitochondrial function nor the ability of the Amplex Red-HRP system to detect H2O2.In vitro experiments and in silico docking simulations indicate that carboxylesterases 1 and 2 recognise Amplex Red with the same kinetics as carboxylesterase-containing mitochondria. We propose two different approaches to correct for this problem and re-evaluate the commonly performed experimental procedure for the detection of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data. PMID:26577176

  20. Cost-effectiveness of interferon-gamma release assay for entry tuberculosis screening in prisons.

    PubMed

    Kowada, A

    2013-10-01

    The incidence of active tuberculosis (TB) and latent tuberculosis infection (LTBI) in inmates and prison staff is higher than that in the general population. Mycobacterium tuberculosis-specific interferon-gamma release assays (IGRAs) provide more accurate diagnosis of M. tuberculosis infection with higher specificity than the tuberculin skin test (TST). To assess the cost effectiveness of QuantiFERON®-TB Gold In-Tube (QFT) compared to TST, TST followed by QFT and chest X-ray, we constructed Markov models using a societal perspective on the lifetime horizon. The main outcome measure of effectiveness was quality-adjusted life-years (QALYs) gained. The incremental cost-effectiveness was compared. The QFT-alone strategy was the most cost-effective for entry TB screening in prisons in developed countries. Cost-effectiveness was not sensitive to the rates of BCG vaccination, LTBI, TB, HIV infection and multidrug-resistant TB. Entry TB screening using an IGRA in prisons should be considered on the basis of its cost-effectiveness by public health intervention.

  1. Evaluation of a domestic interferon-gamma release assay for detecting Mycobacterium tuberculosis infection in China.

    PubMed

    Liu, Yongliang; Ou, Mingzhan; He, Shuizhen; Li, Xiaofei; Lin, Yanyan; Xiong, Junhui; Zhang, Jun; Ge, Shengxiang

    2015-07-01

    Interferon-gamma release assays (IGRAs) have been demonstrated to be useful in the diagnosis of Mycobacterium tuberculosis (MTB) infection. However, IGRAs have not been recommended for clinical usage in most low-income countries due to the shortage of clinical data available resulting from their high test cost. Recently, a cheaper domestic TB-IGRA was approved in China. In this study, we compared TB-IGRA with QuantiFERON-TB Gold In-Tube (QFT-GIT) for MTB infection diagnosis in 253 active TB patients, 48 non-TB lung disease patients, 115 healthcare workers and 216 healthy individuals. The proportion of positive TB-IGRA results in active TB patients, patients with non-TB lung disease, healthcare workers and healthy individuals was 88.3%, 27.1%, 40.9% and 17.6%, respectively, which was similar to the results of QFT-GIT, with an overall agreement of 95% (κ = 0.89) and a high correlation between their responses (r = 0.85, p < 0.001) being observed. In conclusion, the TB-IGRA has comparable clinical performance with QFT-GIT.

  2. Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection - United States, 2010.

    PubMed

    Mazurek, Gerald H; Jereb, John; Vernon, Andrew; LoBue, Phillip; Goldberg, Stefan; Castro, Kenneth

    2010-06-25

    n 2005, CDC published guidelines for using the QuantiFERON-TB Gold test (QFT-G) (Cellestis Limited, Carnegie, Victoria, Australia) (CDC. Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR;54[No. RR-15]:49-55). Subsequently, two new interferon gamma (IFN- gamma) release assays (IGRAs) were approved by the Food and Drug Administration (FDA) as aids in diagnosing M. tuberculosis infection, both latent infection and infection manifesting as active tuberculosis. These tests are the QuantiFERON-TB Gold In-Tube test (QFT-GIT) (Cellestis Limited, Carnegie, Victoria, Australia) and the T-SPOT.TB test (T-Spot) (Oxford Immunotec Limited, Abingdon, United Kingdom). The antigens, methods, and interpretation criteria for these assays differ from those for IGRAs approved previously by FDA. For assistance in developing recommendations related to IGRA use, CDC convened a group of experts to review the scientific evidence and provide opinions regarding use of IGRAs. Data submitted to FDA, published reports, and expert opinion related to IGRAs were used in preparing these guidelines. Results of studies examining sensitivity, specificity, and agreement for IGRAs and TST vary with respect to which test is better. Although data on the accuracy of IGRAs and their ability to predict subsequent active tuberculosis are limited, to date, no major deficiencies have been reported in studies involving various populations. This report provides guidance to U.S. public health officials, health-care providers, and laboratory workers for use of FDA-approved IGRAs in the diagnosis of M. tuberculosis infection in adults and children. In brief, TSTs and IGRAs (QFT-G, QFT-GIT, and T-Spot) may be used as aids in diagnosing M. tuberculosis infection. They may be used for surveillance purposes and to identify persons likely to benefit from treatment. Multiple additional recommendations are provided that address quality control, test

  3. Experience with a gastrointestinal marker (51CrCl3) in a combined study of ileal function using 75SeHCAT and 58CoB12 measured by whole body counting.

    PubMed Central

    Smith, T; Bjarnason, I

    1990-01-01

    Introduction of a radioactive gastrointestinal marker (51CrCl3) into a combined study (75SeHCAT + 58CoB12) of ileal function by whole body counting has been undertaken. The technique was assessed in 23 subjects (15 patients with inflammatory bowel disease, six on non-steroidal anti-inflammatory drugs for rheumatoid arthritis, and two normal subjects). Mean (SD) 51CrCl3 retention was only 4.1 (6.0)% on day 4, and was similar on day 7 in subjects given a second dose of 51CrCl3 on day 4. Only one subject had more than 20% of 51CrCl3 retention after four days. A 51CrCl3 correction method adequately corrected for colonic hold up of 58CoB12, when compared with final equilibrium values of 58CoB12 retention. Use of the non-absorbed 58CoB12 fraction as a gastrointestinal marker gave good agreement with the 51CrCl3 method in correcting 75SeHCAT values. In all subjects studied, corrections for colonic retention of 75SeHCAT on day 4, were small (less than 7% of dose) and did not affect the assessment of any subject. In conclusion, an additional gastrointestinal marker such as 51CrCl3 is unnecessary in our combined study since that role can be effected, when indicated by the non-absorbed 58CoB12 fraction. PMID:2128070

  4. Highly Sensitive and Multiple Enzyme Activity Assay Using Reagent-release Capillary-Isoelectric Focusing with Rhodamine 110-based Substrates.

    PubMed

    Sueyoshi, Kenji; Nogawa, Yuto; Sugawara, Kasumi; Endo, Tatsuro; Hisamoto, Hideaki

    2015-01-01

    In this study, a simple and highly sensitive enzyme activity assay based on reagent-release capillary-isoelectric focusing is described. Reagent-release capillaries containing a fluorescent substrate, which produces fluorescent products possessing an isoelectric point after reaction with enzymes, provides a simple procedure. This is because it allows to spontaneously inject a sample solution into the capillary by capillary action, mixing reagents, and subsequently concentrating the fluorescent products based on isoelectric focusing. Fluorescent rhodamine 110 and its monoamide derivative, which were generated as a final product and an intermediate, respectively, were then focused and separated by reagent-release capillary-isoelectric focusing. After 30 min of enzyme reactions, two focused fluorescent bands were clearly isolated along the prepared capillaries. Employing the focused band of rhodamine 110 monoamide allowed for highly sensitive detection of enzyme activity in the 10 pg mL(-1) order, while that of the conventional assay using a microplate was in the ng mL(-1) order. Furthermore, arraying reagent-release capillaries of different substrates on a chip allowed for simultaneous multi-assay of enzyme activity with good sensitivity in the pg mL(-1) order for each protein.

  5. Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

    PubMed Central

    Shafer-Weaver, Kimberly A; Sayers, Thomas; Kuhns, Douglas B; Strobl, Susan L; Burkett, Mark W; Baseler, Michael; Malyguine, Anatoli

    2004-01-01

    Background This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. Methods We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard 51Cr-release assays were made using human LAK cells. Results Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. Conclusions

  6. Development of a phenotypic assay for characterisation of ethanologenic yeast strain sensitivity to inhibitors released from lignocellulosic feedstocks.

    PubMed

    Greetham, D; Wimalasena, T; Kerruish, D W M; Brindley, S; Ibbett, R N; Linforth, R L; Tucker, G; Phister, T G; Smart, K A

    2014-06-01

    Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.

  7. New, tritium-release assay for 25-hydroxyvitamin D-1. cap alpha. -hydroxylase

    SciTech Connect

    Brown, A.J.; Perlman, K.; DeLuca, H.F.

    1986-05-01

    A new, rapid assay for 25-hydroxyvitamin D (25-OH-D)-1..cap alpha..-hydroxylase has been developed using 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ as substrate. This compound was prepared by reduction of 1-oxo-25-hydroxycyclovitamin D/sub 3/ with (/sup 3/H)NaBH/sub 4/, separation of the 1..cap alpha..- and 1..beta..-hydroxy products by HPLC, subsequent treatments with methylsulfonylchloride and lithium aluminum hydride, cycloreversion, and saponification. The 1..cap alpha..- and 1..beta..-tritiated substrates were tested in the solubilized and reconstituted chick 1..cap alpha..-hydroxylase system. After incubation, the reaction mixture was passed through a reversed phase silica cartridge to separate (/sup 3/H)H/sub 2/O from the labeled substrate. The cartridges were then washed with methanol to elute all vitamin D metabolites, and the amount of 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ was measured by HPLC. In addition, identical reaction mixtures using 25-OH-(26,27-/sup 3/H)D/sub 3/ as substrate were extracted and analyzed by HPLC for 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/. Reactions with 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ produced (/sup 3/H)H/sub 2/O comparable to the amount of 1,25-(OH)/sub 2/(26,27-/sup 3/H)D/sub 3/ and negligible (/sup 3/H) in 1,25-(OH)/sub 2/D/sub 3/. Conversely, reactions with 25-OH-(1..beta..-/sup 3/H)D/sub 3/ produced negligible (/sup 3/H)H/sub 2/O but produced 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ comparable to that from reactions with 25-OH-(26,27-/sup 3/H)D/sub 3/. The results indicate that 1..cap alpha..-hydroxylation specifically displaces the 1..cap alpha..-hydrogen of 25-OH-D/sub 3/ and that the release of the 1..cap alpha..-/sup 3/H provides an accurate measure of vitamin D 1..cap alpha..-hydroxylation.

  8. Executive summary of the guidelines for the use of interferon-γ release assays in the diagnosis of tuberculosis infection.

    PubMed

    Santin, Miguel; García-García, José-María; Rigau, David; Altet, Neus; Anibarro, Luis; Casas, Irma; Díez, Nuria; García-Gasalla, Mercedes; Martínez-Lacasa, Xavier; Penas, Antón; Pérez-Escolano, Elvira; Sánchez, Francisca; Domínguez, José

    2016-05-01

    Interferon-gamma release assays are widely used for the diagnosis of tuberculosis infection in Spain. However, there is no consensus on their application in specific clinical scenarios. To develop a guideline for their use, a panel of experts comprising specialists in infectious diseases, respiratory diseases, microbiology, pediatrics and preventive medicine, together with a methodologist, conducted a systematic literature search, summarized the findings, rated the quality of the evidence, and formulated recommendations following the GRADE (Grading of Recommendations of Assessment Development and Evaluations) methodology. This document provides evidence-based guidance on the use of interferon-gamma release assays for the diagnosis of tuberculosis infection in patients at the risk of tuberculosis or suspected of having active disease. The guidelines will be applicable to specialist and primary care, and public health.

  9. Executive Summary of the Guidelines for the Use of interferon-gamma Release Assays in the Diagnosis of Tuberculosis Infection.

    PubMed

    Santin, Miguel; García-García, José-María; Rigau, David; Altet, Neus; Anibarro, Luis; Casas, Irma; Díez, Nuria; García-Gasalla, Mercedes; Martínez-Lacasa, Xavier; Penas, Antón; Pérez-Escolano, Elvira; Sánchez, Francisca; Domínguez, José

    2016-09-01

    Interferon-gamma release assays are widely used for the diagnosis of tuberculosis infection in Spain. However, there is no consensus on their application in specific clinical scenarios. To develop a guide-line for their use, a panel of experts comprising specialists in infectious diseases, respiratory diseases, microbiology, pediatrics and preventive medicine, together with a methodologist, conducted a systematic literature search, summarized the findings, rated the quality of the evidence, and formulated recommendations following the Grading of Recommendations of Assessment Development and Evaluations methodology. This document provides evidence-based guidance on the use of interferon-gamma release assays for the diagnosis of tuberculosis infection in patients at risk of tuberculosis or suspected of having active disease. The guidelines will be applicable to specialist and primary care, and public health.

  10. Mycobacterium kansasii Infection in a Patient Receiving Biologic Therapy-Not All Reactive Interferon Gamma Release Assays Are Tuberculosis.

    PubMed

    Saleem, Nasir; Saba, Raya; Maddika, Srikanth; Weinstein, Mitchell

    2017-04-01

    Mycobacterium kansasii, a nontuberculous mycobacterium, can lead to lung disease similar to tuberculosis. Immunotherapeutic biologic agents predispose to infections with mycobacteria, including M kansasii. T-cell-mediated interferon gamma release assays like QuantiFERON-TB Gold Test (QFT) are widely used by clinicians for the diagnosis of infections with Mycobacterium tuberculosis; however, QFT may also show positive result with certain nontuberculous mycobacterial infections. We report a case of M kansasii pulmonary infection, with a positive QFT, in an immunocompromised patient receiving prednisone, leflunomide and tocilizumab, a humanized anti-interleukin-6 receptor monoclonal antibody. This case highlights the risk of mycobacterial infections with the use of various biologic agents and the need for caution when interpreting the results of interferon gamma release assays.

  11. Drug release assays from new chitosan/pHEMA membranes obtained by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Casimiro, M. H.; Gil, M. H.; Leal, J. P.

    2007-12-01

    With the purpose of obtaining a biocompatible and microbiologically safe matrix that simultaneously could be used as wound dressing material and as a controlled drug release system, membranes with different thickness and different contents in chitosan and hydroxyethyl methacrylate (HEMA) have been prepared by γ irradiation from a 60Co source. Antibiotic release experiments were performed before or after irradiation over amoxicillin loaded chitosan/pHEMA membranes in physiological saline solution, and monitored by UV-Vis spectrometry. Results point out a fast amoxicillin release with similar release profile in all studied membranes. The amount of released drug was shown to be dependent on membranes network crosslinking due composition, radiation and membrane thickness.

  12. Bioelimination of /sup 51/Cr and /sup 85/Sr by cockroaches, Gromphadorhina portentosa (Orthoptera: Blaberidae), as affected by mites, Gromphadorholaelaps schaeferi (parasitiformes: laelapidae)

    SciTech Connect

    Schowalter, T.D.; Crossley, D.A. Jr.

    1982-03-01

    This paper describes rates of Chromium-51 and Strontium-85 assimilation and bioelimination by the hissing cockroach, Gromphadorhina portentosa (Schaum), when the symbiotic mite, Gromphadorholaelaps schaeferi Till, was present or removed. Mite-infested cockroaches had significantly higher rates of /sup 51/Cr elimination relative to mite-free cockroaches, implying more rapid gut clearance times. We did not find a significant mite effect on /sup 85/Sr elimination by the host, but mite effects could have been masked by the apparently unique process of nutrient assimilation and elimination by G. portentosa. Conventional models of radioactive tracer bioelimination predict a rapid initial loss of tracer due to gut clearance, followed by a slower loss due to excretion of assimilated tracer. Our results indicated that assimilated /sup 85/Sr was eliminated earlier than unassimilated /sup 85/Sr was lost by defecation.

  13. Bioelimination of /sup 51/Cr and /sup 85/Sr by cockroaches, Gromphadorhina portentosa (orthoptera: blaberidae), as affected by mites, Gromphadorholaelaps schaeferi (parasitiformes: laelapidae)

    SciTech Connect

    Schowalter, T.D.; Crossley, D.A. Jr.

    1982-03-01

    The rates of Chromium-51 and Strontium-85 assimilation and bioelimination by the hissing cockroach, Gromphadorhina portentosa (Schaum) are described when the symbiotic mite, Gromphadorholaelaps schaeferi Till, was present or removed. Mite-infested cockroaches had significantly higher rates of /sup 51/Cr elimination relative to mite-free cockroaches, implying more rapid gut clearance times. The authors did not find a significant mite effect on /sup 85/Sr elimination by the host, but mite effects could have been masked by the apparently unique process of nutrient assimilation and elimination by G. portentosa. Conventional models of radioactive tracer bioelimination predict a rapid initial loss of tracer due to gut clearance, followed by a slower loss due to excretion of assimilated tracer. The results indicated that assimilated /sup 85/Sr was eliminated earlier than unassimilated /sup 85/Sr, which was lost by defecation.

  14. MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

    PubMed

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory

    2009-08-31

    criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.

  15. Assay method for polymer-controlled antibiotic release from allograft bone to target orthopaedic infections - biomed 2010.

    PubMed

    Sevy, Justin O; Slawson, Matthew H; Grainger, David W; Brooks, Amanda E

    2010-01-01

    To mitigate and circumvent orthopaedic-associated infection, systematic oral and parenteral antibiotic therapy is often used; however, efficacy is limited due to dosing, systemic side-effects, patient compliance, effective delivery, treatment length, and resistant bacteria. A more effective method may be sustained local drug delivery of antibiotics at the wound site, using delivery vehicles that control release rates. In the case of bone for example, this could be clinically familiar bone graft. Unfortunately, without a rate-control strategy, local antibiotic delivery from allograft displays a prominent burst release: a large amount of drug payload is released as a bolus within 72 hours and depleted. Although his offers effective immediate killing, persitor bacteria remain an infection risk. Notably, drug resistance is a problem at reduced antibiotic levels. To allow better local dosing modulation, a degradable polycaprolactone (PCL) polymer allograft coating is used to modulate local delivery of the antibiotic, tobramycin. This polymer/antibiotic hybrid coats the porous structure of the cancellous bone graft, providing a substantial drug reservoir and allowing controlled release of antibiotic over extended time. PCL/tobramycin-coated bone fragments of different PCL molecular weights and variable drug loads are assayed in vitro for drug release. Tobramycin concentration is determined based on derivatization of its 5 primary amine groups with a fluorescent reagent, phthaldialdehyde (OPA). Tobramycin concentrations in release media can be calculated based on a standard curve with a reasonable accuracy and dynamic range.

  16. A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin.

    PubMed

    Colepicolo, P; Camarero, V C; Nicolas, M T; Bassot, J M; Karnovsky, M L; Hastings, J W

    1990-02-01

    Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.

  17. Experimental bacterial pneumonia in rabbits: polymorphonuclear leukocyte margination and sequestration in rabbit lungs and quantitation and kinetics of /sup 51/Cr-labeled polymorphonuclear leukocytes in E. coli-induced lung lesions

    SciTech Connect

    Cybulsky, M.I.; Movat, H.Z.

    1982-12-01

    A relationship between the circulating and marginal polymorphonuclear leukocyte (PMN) pools was documented using /sup 51/Cr-labeled leukocytes as a marker. /sup 51/Cr-leukocytes marginating in the lungs were found to decrease following a first-order exponential decline, while /sup 51/Cr radioactivity accumulated in the liver and the spleen. Intravenously administered endotoxin caused a rapid selective disappearance of PMNs from the circulation. The percentage of infused /sup 51/Cr cells disappearing was equal to the percentage of disappearance of host cells. The PMNs were found to sequester in the lungs, with peak sequestration of labeled cells occurring 5 min after an endotoxin challenge. Over the next 25 min the /sup 51/Cr radioactivity in the lungs declined. Large numbers of PMNs, probably newly derived from the bone marrow, were observed histologically to be sequestered in the lung vasculature 90 min after an endotoxin dose, while the early sequestration of circulating leukocytes could not be assessed histologically. Pulmonary inflammatory lesions were induced selectively with Escherichia coli in the left lower lobes of rabbits, leaving the right lower lobes as intrinsic controls. PMN-accumulation into the lesions was quantitated using /sup 51/Cr-labeled blood leukocytes. With the aid of /sup 125/I-labeled E. coli, a logarithmic dose-response relationship was found between the number of E. coli and of PMNs. Over a 6-hr period circulating PMNs were found to accumulate in a lesion in the left lower lobe, whereas in the control right lower lobe, leukocyte radioactivity declined. These findings were confirmed with the aid of lavages of the right and left lungs. Two peaks of PMN-accumulation were found by studying leukocyte kinetics: a larger peak between 0 and 6 hr and a smaller peak 18-24 hr after instillation of the microorganisms. Histologic studies confirmed the accumulation of leukocytes, and by 3 weeks showed a complete resolution of the lesions.

  18. Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications.

    PubMed

    Georgiou, Christos D; Zisimopoulos, Dimitrios; Panagiotidis, Konstantinos; Grintzalis, Konstantinos; Papapostolou, Ioannis; Quinn, Richard C; McKay, Christopher P; Sun, Henry J

    2016-02-01

    This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2 and their quantification by an O2 electrode based on the stoichiometry of the involved reactions. The intermediate product O₂˙⁻ from the hydrolysis of metal superoxides is converted by cytochrome c to O2 and by superoxide dismutase (SOD) to ½ mol O2 and ½ mol H2O2, which is then converted by catalase (CAT) to ½ mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to ½ mol O2 by CAT. The assay method was validated in a sealed sample chamber by using a liquid-phase Clark-type O2 electrode with known concentrations of O₂˙⁻ and H2O2, and commercial metal superoxide and peroxide mixed with Mars analog Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, when using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(-3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by γ radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, which demonstrates the suitability of these enzymes for planetary missions, for example, on Mars or Europa.

  19. Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications

    NASA Technical Reports Server (NTRS)

    Georgiou, Christos D.; Zisimopoulos, Dimitrios; Panagiotidis, Konstantinos; Grintzalis, Kontantinos; Papapostolou, Ioannis; Quinn, Richard C.; McKay, Christopher P.; Sun, Henry J.

    2015-01-01

    This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2, and their quantification by an O2 electrode based on the stoichiometry of the involved reactions: The intermediate product O2 from the hydrolysis of metal superoxides is converted by cytochrome c to O2, and also by superoxide dismutase (SOD) to 1/2 mol O2 and 1/2 mol H2O2, which is then converted by catalase (CAT) to 1/2 mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to 1/2 mol O2 by CAT. The assay-method was validated in a sealed sample chamber using a liquid-phase Clark-type O2 electrode with known concentrations of O2 and H2O2, and with commercial metal superoxide and peroxide mixed with Mars analogue Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(exp. -3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by gamma-radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, demonstrating the suitability of these enzymes for planetary missions, e.g., in Mars or Europa.

  20. Serological assays as alternatives to the Ph Eur challenge test for batch release of tetanus vaccines for human use.

    PubMed

    Winsnes, R; Hendriksen, C; Sesardic, D; Akkermans, A; Daas, A

    1999-01-01

    According to the European Pharmacopoeia (Ph Eur) monograph on Tetanus Vaccine (adsorbed) (1997: 0452), assessment of potency is based on a challenge test in guinea pigs or mice. The end-point is taken as paralysis or death. The test requires a large number of animals and causes severe distress. The aim of the present study was to refine the test, and reduce the number of animals needed, for batch release purposes. Serological assays having the potential of being internationally accepted, have been compared with Ph Eur assays. The study included five tetanus vaccines of various combinations and produced by different manufacturers. The results indicated an excellent correlation between enzyme-linked immunosorbent assay (ELISA) and the challenge test (about 93% predictive value), as well as between the toxin binding inhibition (ToBI) test and the challenge test (about 95% predictive value) and between ELISA and the ToBI test (r = 0.92). Antitoxin concentrations determined by ELISA and ToBI were generally in the same range. An overall good correlation was also seen for serum pools of the guinea pigs injected with equal vaccine doses, between toxin neutralisation test in mice (TNT) and ELISA (r = 0.93) as well as between TNT and ToBI (r = 0.97). The ultimate goal of this project was to determine whether serological assays can be used for testing combined vaccines, particularly for tetanus and diphtheria components, using sera from the same animals.

  1. The effects of sigma ligands on protein release from lacrimal acinar cells: a potential agonist/antagonist assay.

    PubMed

    Schoenwald, R D; Barfknecht, C F; Shirolkar, S; Xia, E

    1995-03-03

    Sigma receptor antagonists have been proposed as leading clinical candidates for use in various psychotic disorders. Prior to clinical testing, it is imperative that a new agent be correctly identified as an antagonist and not an agonist since the latter may worsen the psychosis. For sigma-ligands many behavioral and pharmacological assays have been developed in an attempt to classify agonist/antagonist activity. These assays evaluate a response or a behavior in an animal model that can be related to clinical efficacy. However, is the action by the presumed antagonist a consequence of sigma-receptor activity? Previously we have identified sigma-receptors in acinar cells of the main lacrimal gland of the New Zealand white rabbit and have measured protein release after the addition of various N,N-disubstituted phenylalkylamine derivatives known to be sigma-ligands by receptor binding studies. Although protein release from acinar cells has been attributed to either muscarinic or alpha-adrenergic stimulation, protein release from sigma-receptor stimulation was also confirmed. In the reported studies here, we isolated and incubated acinar cells with varying concentrations of known sigma-ligands and measured protein concentration. A knowledge of the receptor profile for the disubstituted phenylalkylamines permitted experiments to be designed in which various alpha, muscarinic, serotonergic, and dopaminergic antagonists could be added in equimolar concentrations. Under the conditions of these experiments, statistically significant increases in protein release for sigma-ligands could be attributed to stimulation of sigma-receptors. Haloperidol, an apparent sigma-antagonist, caused a statistically significant decrease in protein release and also inhibited protein release when tested with a known sigma-ligand, AF2975 [N,N-dimethyl-2-phenylethylamine]. In this system, stimulation and inhibition of protein release were defined as agonist and antagonist behavior, respectively

  2. Identifying Carbohydrate Ligands of a Norovirus P Particle using a Catch and Release Electrospray Ionization Mass Spectrometry Assay

    NASA Astrophysics Data System (ADS)

    Han, Ling; Kitova, Elena N.; Tan, Ming; Jiang, Xi; Klassen, John S.

    2014-01-01

    Noroviruses (NoVs), the major cause of epidemic acute gastroenteritis, recognize human histo-blood group antigens (HBGAs), which are present as free oligosaccharides in bodily fluid or glycolipids and glycoproteins on the surfaces of cells. The subviral P particle formed by the protruding (P) domain of the NoV capsid protein serves as a useful model for the study NoV-HBGA interactions. Here, we demonstrate the application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against the P particle to rapidly identify NoV ligands and potential inhibitors. Carbohydrate libraries of 50 and 146 compounds, which included 18 and 24 analogs of HBGA receptors, respectively, were screened against the P particle of VA387, a member of the predominant GII.4 NoVs. Deprotonated ions corresponding to the P particle bound to carbohydrates were isolated and subjected to collision-induced dissociation to release the ligands in their deprotonated forms. The released ligands were identified by ion mobility separation followed by mass analysis. All 13 and 16 HBGA ligands with intrinsic affinities >500 M-1 were identified in the 50 and the 146 compound libraries, respectively. Furthermore, screening revealed interactions with a series of oligosaccharides with structures found in the cell wall of mycobacteria and human milk. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors, as estimated from the relative abundance of released ligand ions.

  3. Analysis of Ebola Virus and VLP Release Using an Immunocapture Assay

    DTIC Science & Technology

    2007-11-02

    restrictions inherent to biosafety level 4 (BSL4) containment. Central to these studies is a surrogate model for viral replication , assembly, and budding, as...supernatants. Vero-E6 cells were infected with EBOV Zaire (MOI: 0.5) and viral replication was monitored concurrently by ELISA and plaque assay at several...contrast, the cell-associated VP40 (tested in 10 l of lysate) reached a plateau after 48 h. Fig. 3B shows the replication curve of EBOV in the same

  4. Cross sections of the 56Fe(n ,α ) 53Cr and 54Fe(n ,α ) 51Cr reactions in the MeV region

    NASA Astrophysics Data System (ADS)

    Wang, Zhimin; Fan, Xiao; Zhang, Luyu; Bai, Huaiyong; Chen, Jinxiang; Zhang, Guohui; Gledenov, Yu. M.; Sedysheva, M. V.; Krupa, L.; Khuukhenkhuu, G.

    2015-10-01

    Cross sections of the 56Fe(n ,α ) 53Cr and 54Fe(n ,α )51Cr reactions were measured at En=5.5 and 6.5 MeV and En=4.0 ,4.5 ,5.5 ,and 6.5 MeV , respectively, using a double-section gridded ionization chamber as the α -particle detector. Natural iron and enriched 56Fe and 54Fe foil samples were prepared. A deuterium gas target was used to produce monoenergetic neutrons through the 2H(d ,n )3He reaction. Two rounds of experiments were performed at the 4.5-MV Van de Graaff Accelerator of Peking University. The foreground and background were measured in separate runs. The neutron flux was monitored by a B F3 long counter, and the cross sections of the 238U(n ,f ) reaction were used as the standard. Present results are compared with those of the talys-1.6 code calculations, existing measurements, and evaluations.

  5. Cytokine release assays for the prediction of therapeutic mAb safety in first-in man trials — Whole blood cytokine release assays are poorly predictive for TGN1412 cytokine storm

    PubMed Central

    Vessillier, S.; Eastwood, D.; Fox, B.; Sathish, J.; Sethu, S.; Dougall, T.; Thorpe, S.J.; Thorpe, R.; Stebbings, R.

    2015-01-01

    The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs. PMID:25960173

  6. [Up-to-date applicability of interferon-γ release assays for the diagnosis of tuberculosis].

    PubMed

    Domínguez, José; Latorre, Irene

    2015-07-01

    Utility of the in-vitro immunodiagnostic methods, based on the detection of interferon-γ released by T-cells after specific Mycobacterium tuberculosis antigen stimulation (IGRA), has been an improvement in the accuracy of the latent tuberculosis infection diagnosis. IGRA have a well-known higher specificity than the tuberculin skin testing (TST). Moreover, they can obtain a larger number of positive results than the TST in immunocompromised patients. IGRA have shown a high correlation with M. tuberculosis exposure, but their positive and negative predictive value are similar than those obtained by TST. Nevertheless, given their high specificity, they allow reducing number of unnecessary preventive treatments. In addition, these in-vitro techniques are less affected than TST by the different immunosuppressing status. In this review is discussed up-to-date applicability of IGRA in different patient groups: contact studies, pediatric population, immunosuppressed patients, health care workers and active tuberculosis patients. Furthermore, it has been included possible future directions for latent tuberculosis infection and active tuberculosis diagnosis.

  7. Direct measurement of human plasma corticotropin-releasing hormone by two-site immunoradiometric assay

    SciTech Connect

    Linton, E.A.; McLean, C.; Nieuwenhuyzen Kruseman, A.C.; Tilders, F.J.; Van der Veen, E.A.; Lowry, P.J.

    1987-05-01

    A ''two-site'' immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL (mean, 15 +/- 7 (+/- SD) pg/mL; n = 58). Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term (1462 +/- 752 (+/- SD) pg/mL; n = 55), and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA.

  8. Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment

    PubMed Central

    Park, I-Nae; Shim, Tae Sun

    2017-01-01

    Background/Aims The usefulness of interferon-γ release assays (IGRAs) in monitoring to responses to anti-tuberculosis (TB) treatment is controversial. We compared the results of two IGRAs before and after anti-TB treatment in same patients with active TB. Methods From a retrospective review, we selected patients with active TB who underwent repeated QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) and T-SPOT.TB (Oxford Immunotec) assays before and after anti-TB treatment with first-line drugs. Both tests were performed prior to the start of anti-TB treatment or within 1 week after the start of anti-TB treatment and after completion of treatment. Results A total of 33 active TB patients were included in the study. On the QFN-Gold test, at baseline, 23 cases (70%) were early secreted antigenic target 6-kDa protein 6 (ESAT-6) or culture filtrate protein 10 (CFP-10) positive. On the T-SPOT. TB test, at baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Most of patients remained both test-positive after anti-TB treatment. Although changes in interferon-γ release responses over time were highly variable in both tests, there was a mean decline of 27 and 24 spot-forming counts for ESAT-6 and CFP-10, respectively on the T-SPOT.TB test (p < 0.05 for all). Conclusions Although limited by the small number of patients and a short-term follow-up, there was significant decline in the quantitative result of the T-SPOT. TB test with treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate range. PMID:27951621

  9. Renal distribution volumes of indocyanine green, ( 51 Cr)EDTA, and 24 Na in man during acute renal failure after shock. Implications for the pathogenesis of anuria.

    PubMed

    Reubi, F C; Vorburger, C; Tuckman, J

    1973-02-01

    The mechanism responsible for the anuria in acute renal failure after shock is still controversial. Suppressed glomerular filtration and/or tubular back-diffusion of the filtrate are major possible causes. In the present investigation, seven patients with acute anuria, three of these seven again in the polyuric phase, six patients with moderate renal impairment, four patients with chronic renal failure, and eight subjects with normal renal function were studied by a multiple indicator-dilution method in which the total renal blood flow and renal distribution volumes of indocyanine green, [(51)Cr]EDTA, and (24)Na were determined. In normal subjects the average values for one kidney were 582 ml/min, 42 ml, 92 ml, and 139 ml, respectively. The measurements in the patients with moderate renal impairment were similar to those in the normal subjects, but were decreased in chronic renal failure. In acute anuria, the average values were 269 ml/min, 40 ml, 101 ml, and 114 ml and the kidney volume, estimated radiographically, was increased by 40%. When expressed as milliliters per milliliters kidney, the average distribution volume of (24)Na was decreased from 0.64 to 0.38. This decrease is consistent with the hypothesis that suppressed filtration is largely responsible for the anuria and that back-diffusion is, at most, a contributory factor. The apparent contradiction between the relatively well-preserved total blood flow and the suppressed filtration may be due to a combination of afferent vasoconstriction and efferent vasodilatation. This view is supported by the observation that low filtration fractions were found in clearance measurements performed during the polyuric phase.

  10. Renal Distribution Volumes of Indocyanine Green, [51Cr]EDTA, and 24Na in Man during Acute Renal Failure after Shock. IMPLICATIONS FOR THE PATHOGENESIS OF ANURIA

    PubMed Central

    Reubi, F. C.; Vorburger, C.; Tuckman, J.

    1973-01-01

    The mechanism responsible for the anuria in acute renal failure after shock is still controversial. Suppressed glomerular filtration and/or tubular back-diffusion of the filtrate are major possible causes. In the present investigation, seven patients with acute anuria, three of these seven again in the polyuric phase, six patients with moderate renal impairment, four patients with chronic renal failure, and eight subjects with normal renal function were studied by a multiple indicator-dilution method in which the total renal blood flow and renal distribution volumes of indocyanine green, [51Cr]EDTA, and 24Na were determined. In normal subjects the average values for one kidney were 582 ml/min, 42 ml, 92 ml, and 139 ml, respectively. The measurements in the patients with moderate renal impairment were similar to those in the normal subjects, but were decreased in chronic renal failure. In acute anuria, the average values were 269 ml/min, 40 ml, 101 ml, and 114 ml and the kidney volume, estimated radiographically, was increased by 40%. When expressed as milliliters per milliliters kidney, the average distribution volume of 24Na was decreased from 0.64 to 0.38. This decrease is consistent with the hypothesis that suppressed filtration is largely responsible for the anuria and that back-diffusion is, at most, a contributory factor. The apparent contradiction between the relatively well-preserved total blood flow and the suppressed filtration may be due to a combination of afferent vasoconstriction and efferent vasodilatation. This view is supported by the observation that low filtration fractions were found in clearance measurements performed during the polyuric phase. PMID:4630601

  11. Measurement of calcium fluxes in permeabilized cells using a ⁴⁵Ca²+ uptake and release assay.

    PubMed

    Wilcox, Robert A

    2005-01-01

    Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P(3)-induced Ca(2+) release was initially demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved the introduction of Ins(1,4,5)P(3) by rendering a cell population permeable, using microinjection techniques or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile "caged Ins(1,4,5)P(3)" (5). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores, however, these preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to cells. The author will describe a (45)Ca(2+)-release assay used to monitor Ins(1,4,5)P(3)-induced Ca(2+) mobilization from nonmitochondrial intracellular Ca(2+) stores using "cytosol-like" buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.

  12. Direct determination of lignin peroxidase released from Phanerochaete chrysosporium by in-capillary enzyme assay using micellar electrokinetic chromatography.

    PubMed

    Harada, Airi; Sasaki, Keiko; Kaneta, Takashi

    2016-04-01

    Here we describe the application of an in-capillary enzyme assay using micellar electrokinetic chromatography (MEKC) in the determination of enzyme activity in a crude culture medium containing lignin peroxidase released from Phanerochaete chrysosporium (P. chrysosporium). The method consists of a plug-plug reaction between lignin peroxidase and its substrate, veratryl alcohol, the separation of the product, veratraldehyde, from the other components including the enzyme and the culture medium, and the determination of the enzyme activity from the peak area of veratraldehyde produced by the plug-plug reaction. This method is more sensitive than conventional spectrophotometry since the background originates from the enzyme and the culture medium can be removed via MEKC separation. Veratraldehyde was separated at -10kV in a background electrolyte containing 50mM tartrate buffer (pH 2.5) and 50mM sodium dodecyl sulfate (SDS) after a plug-plug reaction in the capillary for 5min. The calibration curve of veratraldehyde was linear up to 4pmol (500μM) with a limit to quantification of 0.026pmol (3.2μM) (SN=10). The activity of lignin peroxidase was directly measured from the peak area of veratraldehyde. The activity of lignin peroxidase released from P. chrysosporium into the medium for 7 days was successfully determined to be 3.40UL(-1).

  13. Reassessment of the pharmacology of Sphingosine-1-phosphate S1P3 receptor ligands using the DiscoveRx PathHunter™ and Ca2+ release functional assays

    PubMed Central

    Riddy, DM; Stamp, C; Sykes, DA; Charlton, SJ; Dowling, MR

    2012-01-01

    BACKGROUND AND PURPOSE DiscoverRx's PathHunter™ assay measures GPCR agonist potency, via the recruitment of β-arrestin, independent of the subtype of Gα protein activated. This assay is frequently used in drug discovery although little is known about the agonist pharmacology generated. Here we have compared agonist potency, efficacy and affinity values obtained in PathHunter™ assays with those from more established radioligand binding and functional techniques. EXPERIMENTAL APPROACH Using cells expressing the human sphingosine-1-phosphate S1P3 receptor at four different densities, we compared pharmacological affinity and efficacy values of four structurally distinct ligands – FTY720-P, VPC24191, CYM5442 and the endogenous agonist S1P – obtained from competition binding, functional Ca2+ release and PathHunter™ assays. KEY RESULTS The pKi values for S1P were significantly different (9.34 ± 0.10 and 8.92 ± 0.15) in clones expressing different receptor levels using the binding assay. In the PathHunter™ and Ca2+ assays, S1P and CYM5442 were full agonists, FTY720-P was a partial agonist, while the efficacy of VPC24191 could not be detected in PathHunter™ assays. VPC23019, previously described as a S1P1/3 receptor antagonist, behaved as an S1P3 receptor partial agonist in the Ca2+ release assay. CONCLUSIONS AND IMPLICATIONS Comparison of data from the PathHunter™ assay with binding and functional Ca2+ assays suggest that PathHunter™ assays measured a different agonist-bound receptor conformation. While this assay has great utility in drug discovery, care must be taken as high-efficacy, low-affinity agonist compounds would not be detected. Therefore highly amplified, more traditional assays are necessary to identify agonists with low efficacy. PMID:22577868

  14. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  15. Entrapping efficiency and drug release profile of an oil-in-water (o/w) emulsion formulation using a polydimethylsiloxane-coated glass bead assay.

    PubMed

    Minagawa, T; Kohno, Y; Suwa, T; Tsuji, A

    1994-04-01

    Evaluation of entrapping efficiency is difficult for an o/w emulsion formulation containing a lipophilic oily drug, isocarbacyclin methyl ester (TEI-9090), by commonly employed techniques (dialysis, ultrafiltration, or gel filtration), because of its adsorption to the system materials. Employing this characteristic of TEI-9090, we developed an adsorption technique with polydimethylsiloxane-coated glass beads (PDMS-GB). The assay is based on the quantitative adsorption of unentrapped TEI-9090 to the PDMS-GB. The entrapping efficiency of a 10% soybean oil emulsion containing [3H]TEI-9090 (1 microgram/mL) assayed by this method approached 100%. The PDMS-GB assay was performed for the emulsion diluted 100 times with physiological saline at different time intervals after dilution over a period of 24 hr. A plot of [3H]TEI-9090 in the emulsion particles versus time showed rapid release within 1 hr, followed by very slow release, reaching equilibrium. Applying first-order kinetics, the data were found to fit to a biexponential equation over the first hour of release. The terminal release resembled the first-order release of the drug from the phospholipid-rich infranatant, which was separated from the creamy layer by ultracentrifugation of the emulsion and contained 35% [3H]TEI-9090. These results suggest that the drug is released from two components in the emulsion.

  16. Screening for latent TB in patients with rheumatic disorders prior to biologic agents in a 'high-risk' TB population: comparison of two interferon gamma release assays.

    PubMed

    Melath, Sunil; Ismajli, Mediola; Smith, Robin; Patel, Ishita; Steuer, Alan

    2014-01-01

    Patients with rheumatic disorders treated with TNF inhibitors are at increased risk of developing TB. There is no 'gold-standard' for the diagnosis of latent TB prior to initiation of biologic agents. We report our own experience of comparing two interferon gamma release assays (IGRAs) in screening for latent TB in a 'high-risk' TB area in patients with rheumatic disorders. The study demonstrated good concordance between the two tests. We believe the additional cost of these assays is justified in high-risk populations prior to biologic agents, with 16% of the current study population with at least one positive IGRA assay.

  17. Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

    PubMed

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

    2011-09-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

  18. Cytokine Release Assays as Tests for Exposure to Leishmania, and for Confirming Cure from Leishmaniasis, in Solid Organ Transplant Recipients

    PubMed Central

    López-Medrano, Francisco; Salto, Efrén; Fernández, Laura; San Martín, Juan Víctor; Alvar, Jorge; Aguado, Jose María; Moreno, Javier

    2015-01-01

    Spain has one of the world’s largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain) has involved 600 clinical cases since late 2009 (prevalence 0.2%). It may therefore be wise to monitor the town’s transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition. PMID:26496365

  19. Risk of tuberculosis among air passengers estimated by interferon gamma release assay: survey of contact investigations, Japan, 2012 to 2015.

    PubMed

    Ota, Masaki; Kato, Seiya

    2017-03-23

    Although the World Health Organization recommends contact investigations around air travel-associated sputum smear-positive tuberculosis (TB) patients, evidence suggests that the information thus obtained may have overestimated the risk of TB infection because it involved some contacts born in countries with high TB burden who were likely to have been infected with TB in the past, or because tuberculin skin tests were used, which are less specific than the interferon gamma release assay (IGRA) particularly in areas where Bacillus Calmette-Guérin (BCG) vaccination coverage is high. We conducted a questionnaire survey on air travel-associated TB contact investigations in local health offices of Japan from 2012 to 2015, focusing on IGRA positivity. Among 651 air travel-associated TB contacts, average positivity was 3.8% (95% confidence interval (CI): 2.5-5.6) with a statistically significant increasing trend with older age (p < 0.0094). Positivity among 0-34 year-old contacts was 1.0% (95% CI: 0.12-3.5%), suggesting their risk of TB infection is as small as among Japanese young adults with low risk of TB infection (positivity: 0.85-0.90%). Limiting the contact investigation to fewer passengers (within two seats surrounding the index case, rather than two rows) seems reasonable in the case of aircraft with many seats per row.

  20. Tuberculosis contact investigation using interferon-gamma release assay with chest x-ray and computed tomography.

    PubMed

    Fujikawa, Akira; Fujii, Tatsuya; Mimura, Satoshi; Takahashi, Ryota; Sakai, Masao; Suzuki, Shinya; Kyoto, Yukishige; Uwabe, Yasuhide; Maeda, Shinji; Mori, Toru

    2014-01-01

    Between September 2009 and January 2010, 6 members of the Japanese Eastern Army, who had completed the same training program, were diagnosed with active tuberculosis (TB) on different occasions. The Ministry of Defense conducted a contact investigation of all members who had come into contact with the infected members. The purpose of this study was to verify the efficacy of the TB screening protocol used in this investigation. A total of 884 subjects underwent interferon-gamma release assay (IGRA) and chest X-ray. The 132 subjects who were IGRA positive or with X-ray findings suggestive of TB subsequently underwent chest computer tomography (CT). Chest CT was performed for 132 subjects. Based on CT findings, 24 (2.7%) subjects were classified into the active TB group, 107 (12.1%) into the latent tuberculosis infection (LTBI) group, and 753 (85.2%) into the non-TB group. The first 2 groups underwent anti-TB therapy, and all 3 groups were followed for 2 years after treatment. Although one subject in the active TB group experienced relapse during the follow-up period, no patient in the LTBI or non-TB groups developed TB. IGRA and chest X-ray, followed by chest CT for those IGRA positive or with suspicious X-ray findings, appears to be an effective means of TB contact screening and infection prevention.

  1. Cytokine Release Assays as Tests for Exposure to Leishmania, and for Confirming Cure from Leishmaniasis, in Solid Organ Transplant Recipients.

    PubMed

    Carrillo, Eugenia; Carrasco-Antón, Nerea; López-Medrano, Francisco; Salto, Efrén; Fernández, Laura; San Martín, Juan Víctor; Alvar, Jorge; Aguado, Jose María; Moreno, Javier

    2015-01-01

    Spain has one of the world's largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain) has involved 600 clinical cases since late 2009 (prevalence 0.2%). It may therefore be wise to monitor the town's transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.

  2. The Effectiveness of Screening with Interferon-Gamma Release Assays in a University Health Care Setting with a Diverse Global Population

    ERIC Educational Resources Information Center

    Birch, Samantha J.; Golbeck, Amanda L.

    2015-01-01

    Objective: This analysis examined the effectiveness of utilizing interferon-gamma release assay (IGRA) technology in a TB (TB) screening program at a university. Participants: Participants were 2299 students at a Montana university who had presented to the university health center for TB screening during 2012 and 2013. Methods: A retrospective…

  3. Histamine release-neutralization assay for sera of patients with atopic dermatitis and/or cholinergic urticaria is useful to screen type I hypersensitivity against sweat antigens.

    PubMed

    Shindo, Hajime; Ishii, Kaori; Yanase, Yuhki; Suzuki, Hidenori; Hide, Michihiro

    2012-10-01

    We previously reported that about 80 % of patients with atopic dermatitis and 60 % with cholinergic urticaria revealed type I allergy against sweat, by means of skin test against autologous sweat and/or histamine-release test for peripheral blood basophils with semi-purified sweat antigen. In this study, we developed an assay for sera to neutralize histamine-releasing activity of semi-purified sweat antigen. The semi-purified sweat antigen was pre-incubated with serially diluted sera for 30 min at 37 °C and was subjected to histamine-release activity. Histamine release-neutralization (HRN) activities were calculated by measuring the amount of histamine release from basophils in the presence or absence of semi-purified sweat antigen. Of 62 subjects, 39 showed positive histamine release (≥5 %) from their basophils in response to semi-purified sweat antigen, and sera of 34 out of 39 subjects (87.2 %) were also positive in HRN activity (≥10 %). The specificity of the HRN assay was 0.522. Moreover, HRN activities in sera were largely correlated with degrees of histamine release from peripheral blood basophils of the same donors in response to sweat antigen. To identify the substance that neutralizes histamine-release activity, we removed IgE and IgG from the sera of HRN (+) subjects by column chromatography. The HRN activities in 30 out of 42 sera were largely reduced by the removal of IgG. On the other hand, sera of four subjects lost HRN activity by the removal of IgE, suggesting that the majority of HRN (+) subjects have serum IgG against the sweat antigen as well as IgE bound to peripheral basophils. Thus, the HRN assay maybe useful for the screening of type I allergy against sweat antigen.

  4. Omega 3 fatty acids increase spontaneous release of cytosolic components from tumor cells

    SciTech Connect

    Jenski, L.J.; Sturdevant, L.K.; Ehringer, W.D.; Stillwell, W. )

    1991-05-01

    Mice fed menhaden (fish) oil or coconut oil-rich diets were inoculated intraperitoneally with a rapidly growing leukemia, T27A. After one week, the tumor cells were harvested, and 51Cr was used to label intracellular molecules. Spontaneous release of 51Cr was used as a measure of plasma membrane permeability. Compared to cells from mice fed coconut oil (rich in saturated fatty acids), tumor cells from mice fed menhaden oil (rich in long chain polyunsaturated omega 3 fatty acids) showed an increased level of spontaneous 51Cr release, which was exacerbated by increased temperature and reduced by extracellular protein. At physiological salt concentrations, the released 51Cr was detected in particles of approximately 2700 daltons. Enhanced permeability correlated with the incorporation of dietary (fish oil) omega 3 polyunsaturated fatty acids docosahexaenoic and eicosapentaenoic acid into the tumor cells. The results demonstrate that omega 3 fatty acids are incorporated into cellular constituents of tumor cells and change properties associated with the plasma membrane. This result suggests that dietary manipulation may be used to enhance tumor cell permeability and contribute to tumor eradication.

  5. Usefulness of interferon-γ release assay for the diagnosis of latent tuberculosis infection in young children

    PubMed Central

    Kim, Young Kwang; Kim, Hae Ryun; Lee, Mi Kyung; Lim, In Seok

    2016-01-01

    Purpose Latent tuberculosis infection (LTBI) in young children may progress to severe active tuberculosis (TB) disease and serve as a reservoir for future transmission of TB disease. There are limited data on interferon-γ release assay (IGRA) performance in young children, which our research aims to address by investigating the usefulness of IGRA for the diagnosis of LTBI. Methods We performed a tuberculin skin test (TST) and IGRA on children who were younger than 18 years and were admitted to Chung-Ang University Hospital during May 2011–June 2015. Blood samples for IGRA were collected, processed, and interpreted according to manufacturer protocol. Results Among 149 children, 31 (20.8%) and 10 (6.7%) were diagnosed with LTBI and active pulmonary TB, respectively. In subjects lacking contact history with active TB patients, TST and IGRA results were positive in 41.4% (29 of 70) and 12.9% (9 of 70) subjects, respectively. The agreement (kappa) of TST and IGRA was 0.123. The control group, consisting of non-TB-infected subjects, showed no correlation between age and changes in interferon-γ concentration after nil antigen, TB-specific antigen, or mitogen stimulation in IGRAs (P=0.384, P=0.176, and P=0.077, respectively). In serial IGRAs, interferon-γ response to TB antigen increased in IGRA-positive LTBI subjects, but did not change considerably in initially IGRA-negative LTBI or control subjects. Conclusion The lack of decrease in interferon-γ response in young children indicates that IGRA could be considered for this age group. Serial IGRA tests might accurately diagnose LTBI in children lacking contact history with active TB patients. PMID:27462354

  6. Tuberculin skin test and interferon-gamma release assay use among privately insured persons in the United States.

    PubMed

    Owusu-Edusei, K; Stockbridge, E L; Winston, C A; Kolasa, M; Miramontes, R

    2017-03-28

    OBJECTIVE:

    To describe tuberculin skin test (TST) and interferon-gamma release assay (IGRA) (i.e., QuantiFERON(®)-TB [QFT] and T-SPOT(®).TB [T-SPOT]) use among privately insured persons in the United States over a 15-year period.

    METHODS:

    We used current procedural terminology (CPT) codes for the TST and IGRAs to extract out-patient claims (2000–2014) and determined usage (claims/100 000). The χ(2) test for trend in proportions was used to describe usage trends for select periods.

    RESULTS:

    The TST was the dominant (>80%) test in each year. Publication of guidelines preceded the assignment of QFT and T-SPOT CPT codes by 1 year (2006 for QFT; 2011 for T-SPOT). QFT usage was higher (P < 0.01) than T-SPOT in each year. The average annual increase in the use of QFT was higher than that of T-SPOT (35 vs. 3.8/100 000), and more so when the analytic period was 2011–2014 (65 vs. 38/100 000). However, during that 4-year period (2011–2014), TST use trended downward, with an average annual decrease of 28/100 000. The annual proportion of enrollees tested ranged from 1.1% to 1.5%.

    CONCLUSIONS:

    These results suggest a gradual shift from the use of the TST to the newer IGRAs. Future studies can assess the extent, if any, to which the shift from the use of the TST to IGRAs evolved over time.

  7. Application and interpretation of an interferon-gamma release assay: Results of an audit in a Canadian centre

    PubMed Central

    Vat, Sopharat; Ghannoum, Marc; Laflamme, Pierre; Dugas, Mario; Labrecque, Manon; Lavergne, Valéry

    2012-01-01

    BACKGROUND: Interferon-gamma release assays (IGRAs) are newly approved for diagnosing latent tuberculosis infection (LTBI). An internal audit was conducted to review the use of a newly implemented IGRA at the Hôpital du Sacré-Coeur de Montréal (Montréal, Québec) to evaluate its concordance with Canadian recommendations and its implication on diagnosis. METHODS: From April 2007 to January 2009, all Quantiferon TB Gold In-Tube (QFT, Cellestis inc, USA) tests performed in at the Hôpital du Sacré-Coeur de Montréal were retrieved. Strategies used to investigate LTBI and clinical interpretation of test results were compared with the local algorithm, which is derived from the current national guidelines. RESULTS: A total of 200 patients tested with QFT were included in the analysis. LTBI investigation and QFT testing were considered to be appropriate in 87.5% and 66.5% of patients, respectively. Overall, 67 QFT tests were performed inappropriately; 25 were performed when a LTBI investigation was not indicated and 42 were performed whe LTBI interpretation was possible with the result of the tuberculin skin test alone. Among the 175 patients investigated appropriately for LTBI, 49 QFT tests (28%) were interpreted incorrectly; 32 patients (at high risk of developing active tuberculosis) had a positive tuberculin skin test and a negative QFT result wrongly interpreted as being negative for LTBI and 13 patients should have undergone further LTBI investigations. CONCLUSION: Globally, the present study revealed that there are discrepancies on how the IGRA was employed and interpreted in a Montreal hospital and that strict compliance to the guidelines could significantly reduce errors in interpretation. PMID:24294272

  8. Costs and Consequences of Using Interferon-γ Release Assays for the Diagnosis of Active Tuberculosis in India

    PubMed Central

    Little, Kristen M.; Pai, Madhukar; Dowdy, David W.

    2015-01-01

    Background There is growing concern that interferon-γ release assays (IGRAs) are being used off-label for the diagnosis of active tuberculosis (TB) disease in many high-burden settings, including India, where the background prevalence of latent TB infection is high. We analyzed the costs and consequences of using IGRAs for the diagnosis of active TB in India from the perspective of the Indian TB control sector. Methods and Findings We constructed a decision analytic model to estimate the incremental cost and effectiveness of IGRAs for the diagnosis of active TB in India. We compared a reference scenario of clinical examination and non-microbiological tests against scenarios in which clinical diagnosis was augmented by the addition of either sputum smear microscopy, IGRA, or Xpert MTB/RIF. We examined costs (in 2013 US dollars) and consequences from the perspective of the Indian healthcare sector. Relative to sputum smear microscopy, use of IGRA for active TB resulted in 23,700 (95% uncertainty range, UR: 3,800 – 38,300) additional true-positive diagnoses, but at the expense of 315,700 (95% UR: 118,300 – 388,400) additional false-positive diagnoses and an incremental cost of US$49.3 million (95% UR: $34.9 – $58.0 million) (2.9 billion Indian Rupees). Relative to Xpert MTB/RIF (including the cost of treatment for drug resistant TB), use of IGRA led to 400 additional TB cases treated (95% UR: [-8,000] – 16,200), 370,600 (95% UR: 252,200 – 441,700) more false-positive diagnoses, 70,400 (95% UR: [-7,900] – 247,200) fewer disability-adjusted life years averted, and US$14.6 million (95%UR: [-$7.2] – $28.7 million) (854 million Indian Rupees) in additional costs. Conclusion Using IGRAs for diagnosis of active TB in a setting like India results in tremendous overtreatment of people without TB, and substantial incremental cost with little gain in health. These results support the policies by WHO and Standards for TB Care in India, which discourage the use of

  9. Clinical and diagnostic developments of a gamma interferon release assay for use in bovine tuberculosis control programs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently the Bovigam assay is used as an official supplemental test within the bovine tuberculosis eradication program. This assay measures interferon-gamma (IFN-gamma) produced by lymphocytes in response to specific antigens. The objectives of the present study were to evaluate two Mycobacterium ...

  10. Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

    PubMed

    Cai, Huili; Mullier, François; Frotscher, Birgit; Briquel, Marie-Elisabeth; Toussaint, Marie; Massin, Frédéric; Lecompte, Thomas; Latger-Cannard, Véronique

    2016-04-01

    Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

  11. International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials

    PubMed Central

    Galipeau, Jacques; Krampera, Mauro; Barrett, John; Dazzi, Francesco; Deans, Robert J.; Debruijn, Joost; Dominici, Massimo; Fibbe, Willem E.; Gee, Adrian P.; Gimble, Jeffery M.; Hematti, Peiman; Koh, Mickey B.C.; Leblanc, Katarina; Martin, Ivan; Mcniece, Ian K.; Mendicino, Michael; Oh, Steve; Ortiz, Luis; Phinney, Donald G.; Planat, Valerie; Shi, Yufang; Stroncek, David F.; Viswanathan, Sowmya; Weiss, Daniel J.; Sensebe, Luc

    2016-01-01

    Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an “open-access” manner, such as through publication or database collection. PMID:26724220

  12. Detection of a low-molecular-weight antigen on melanoma cells by a human antiserum in leukocyte-dependent antibody assays.

    PubMed

    Hersey, P; Murray, E; Werkmeister, J; McCarthy, W H

    1979-10-01

    Biochemical characterization of serologically detected human melanoma antigens was undertaken for the development of immunodiagnostic assays in melanoma. An antiserum from a human melanoma patient, which detected melanoma antigens expressed on a large proportion of different melanoma cells, was used in leucocyte-dependent cytotoxic antibody (LDA) 51Cr-release assays to monitor the purification of melanoma antigens in urea/acetate extracts of lactoperoxidase 125I-labelled melanoma cell membranes. The separation procedures included affinity chromatography on Concanavalin A, gel filtration on porous polyacrylamide beads and preparative isoelectric focusing. The fractions were also monitored by polyacrylamide electrophoresis in sodium dodecyl sulphate and by measurement of beta 2 microglobulin and carcinoembryonic antigen content. The antigens detected by this antiserum appeared to be acidic (pI 3.5) low-mol.-wt glycoproteins of approximately 15,000 daltons which were resistant to heating at 56 degrees C and digestion with neuraminidase, but susceptible to repeated freeze-thawing and trypsin digestion. They did not appear to be related to HLA antigens, beta 2 microglobulin or known foetal antigens. The nature of the antigens detected in these studies is as yet unknown, but they appear similar to those described in the sera and urine of melanoma patients in previous reports. Thes combined results and the frequent expression of these antigens on melanoma cells from different patients suggest that assays to detect this antigen may provide a valuable immunodiagnostic aid in the management of melanoma.

  13. Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes: A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications

    NASA Astrophysics Data System (ADS)

    de Lima, R.; Oliveira, J. L.; Murakami, P. S. K.; Molina, M. A. M.; Itri, R.; Haddad, P.; Seabra, A. B.

    2013-04-01

    This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

  14. Prevalence of latent tuberculosis infection in Sudan: a case–control study comparing interferon-γ release assay and tuberculin skin test

    PubMed Central

    2013-01-01

    Background Most people exposed to M. tuberculosis show no evidence of clinical disease. Five to 10% of individuals with latent infection progress to develop overt disease during their life time. Identification of people with latent TB infection will increase case detection rates and may dictate new treatment policies to control tuberculosis. This study aimed to determine LTBI point prevalence in a population from Sudan using two different diagnostic methods: the tuberculin skin test (TST) and the IFN-γ release assay (IGRA). Methods This was a prospective, community-based and case-controlled study. Following informed consent, household contacts (HHCs; n = 98) of smear-positive index cases and Community controls (CCs; 186), were enrolled. Tuberculin skin test (TST), whole blood stimulation with ESAT-6/CFP-10 ± TB7.7 antigens or purified protein derivative (PPD) and IFN-γ levels determination with ELISA were performed. The levels of IFN-γ and TST induration between the CCs and the HHCs were compared using student t-test, Chi-square and Kappa coefficient. Pearson correlation test was used to compare TST and IFN-γ. P levels of <0.05 were considered significant. Results TST induration of ≥ 10 mm gave an LTBI point prevalence of 327 cases/1000 individuals among HHCs compared to 126 cases/1000 individuals among CCs (p = 0.000). PPD-induced IFN-γ release assay gave an LTBI point prevalence of 418 cases/1000 individuals among HHCs compared to 301 cases/1000 individuals among CCs (p =0.06). On the other hand ESAT-6/CFP-10 ± TB7.7-induced IFN-γ gave an LTBI point prevalence of 429 cases/1000 individuals among HHCs compared to 268 cases/1000 individuals among CCs (p = 0.01). IFN-γ productions levels induced by ESAT-6/CPF-10 ± TB7.7 antigens in HHCS and CCs were not significantly different from those induced by PPD (p = 0.7). Conclusion IFN-γ release assay (IGRA) gave higher LTBI point prevalence compared to TST in HHCs and CCs. PPD

  15. Tiered application of the neutral red release and EpiOcular™ assays for evaluating the eye irritation potential of agrochemical formulations.

    PubMed

    Settivari, Raja S; Amado, Ricardo Acosta; Corvaro, Marco; Visconti, Nicolo R; Kan, Lynn; Carney, Edward W; Boverhof, Darrell R; Gehen, Sean C

    2016-11-01

    Agrochemical formulations have been underrepresented in validation efforts for implementing alternative eye irritation approaches but represent a significant opportunity to reduce animal testing. This study assesses the utility of the neutral red release assay (NRR) and EpiOcular™ assay (EO) for predicting the eye irritation potential of 64 agrochemical formulations relative to Draize data. In the NRR, formulations with an NRR50 value ≤ 50 mg/mL were categorized as UN GHS Cat 1 and those >250 mg/mL were classified as UN GHS Non Classified (NC). The accuracy, sensitivity, and specificity were 78, 85 and 76% and 73, 85 and 61% for identifying UN GHS 1 and NC formulations, respectively. Specificity was poor for formulations with NRR50 > 50 to ≤250 mg/mL. The EO (ET-40 method) was explored to differentiate formulations that were UN GHS 1/2 and UN GHS NC. The EO resulted in accuracy, sensitivity, and specificity of 65%, 58% and 75% for identifying UN GHS NC formulations. To improve the overall performance, the assays were implemented using a tiered-approach where the NRR was run as a first-tier followed by the EO. The tiered-approach resulted in improved accuracy (75%) and balanced sensitivity (73%) and specificity (77%) for distinguishing between irritating and non-irritating agrochemical formulations.

  16. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    PubMed

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan

    2009-10-01

    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  17. Evaluation of the Immune Response to Interferon Gamma Release Assay and Tuberculin Skin Test Among BCG Vaccinated Children in East of Egypt: A Cross-Sectional Study.

    PubMed

    Beshir, Mohamed Refaat; Zidan, Alaa Ebrahim; El-Saadny, Hosam Fathi; Ramadan, Raghdaa Abdelaziz; Karam, Nehad Ahmed; Amin, Ezzat Kamel; Mohamed, Marwa Zakaria; Abdelsamad, Nahla Mohamed

    2016-04-01

    Bacille Calmette-Guérin vaccine (BCG) vaccination is used routinely in most of countries, especially developing one. The efficacy of the BCG vaccination generally decreases with time. The tuberculin skin test (TST) is a most popular diagnostic test for suspicion of tuberculosis (TB) in children till now, but it has many false positives. The interferon-gamma release assay (IGRA) is more specific than TST for detection of childhood TB, as it is more specific to Mycobacterium tuberculosis.Evaluate the interferon gamma response and TST reaction in BCG vaccinated children in east of Egypt.150 children were included in the study aged 1 month to 12 years; the collected data from the children included, full history taking, clinical examination, examination for the presence or absence of BCG scar under direct light. All the children had performed TST, IGRA.TST was done for all studied group reveal 51.3% with size of reaction <5 mm, 39.3% with size of reaction = 5 to 9 mm while 9.3% with size of reaction ≥10 mm. Mean size of reaction was 4.07 mm. Interferon gamma release assay was done for all studied group reveal 5 children (3.3%) with positive test. There was significant difference between the size of TST reaction and age (P < 0.01) with old children were more frequent to show positive reaction. Also, children with age range 1 month to 1 year were frequently have negative IGRA test, while children with age range 4 years to 12 years were frequently have positive test (P < 0.01). There was moderate agreement between IGRA and TST results (Kappa [κ] = 0.475). With high agreement between IGRA and TST results in children with absent BCG scar (κ = 1000).Therefore, Interferon gamma release assays have higher specificity and lower cross-reactions with BCG vaccination and nontuberculous Mycobacteraie than TST.

  18. A Survey of Monitoring and Assay Systems for Release of Metals from Radiation Controlled Areas at LANL

    SciTech Connect

    Gruetzmacher, K. M.; MacArthur, D. W.

    2002-02-28

    At Los Alamos National Laboratory (LANL), a recent effort in waste minimization has focused on scrap metal from radiological controlled areas (RCAs). In particular, scrap metal from RCAs needs to be dispositioned in a reasonable and cost effective manner. Recycling of DOE scrap metals from RCAs is currently under a self-imposed moratorium. Since recycling is not available and reuse is difficult, often metal waste from RCAs, which could otherwise be recycled, is disposed of as low-level waste. Estimates at LANL put the cost of low-level waste disposal at $550 to $4000 per cubic meter, depending on the type of waste and the disposal site. If the waste is mixed, the cost for treatment and disposal can be as high as $50,000 per cubic meter. Disposal of scrap metal as low-level waste uses up valuable space in the low-level waste disposal areas and requires transportation to the disposal site under Department of Transportation (DOT) regulations for low-level waste. In contrast, disposal as non-radioactive waste costs as little as $2 per cubic meter. While recycling is unavailable, disposing of the metal at an industrial waste site could be the best solution for this waste stream. A Green Is Clean (GIC) type verification program needs to be in place to provide the greatest assurance that the waste does not contain DOE added radioactivity. This paper is a review of available and emerging radiation monitoring and assay systems that could be used for scrap metal as part of the LANL GIC program.

  19. A survey of monitoring and assay systems for release of metals from radiation controlled areas at LANL.

    SciTech Connect

    Gruetzmacher, K. M.; MacArthur, D. W.

    2002-01-01

    At Los Alamos National Laboratory (LANL), a recent effort in waste minimization has focused on scrap metal from radiological controlled areas (RCAs). In particular, scrap metal from RCAs needs to be dispositioned in a reasonable and cost effective manner. Recycling of DOE scrap metals from RCAs is currently under a self-imposed moratorium. Since recycling is not available and reuse is difficult, often metal waste from RCAs, which could otherwise be recycled, is disposed of as low-level waste. Estimates at LANL put the cost of low-level waste disposal at $550 to $4000 per cubic meter, depending on the type of waste and the disposal site. If the waste is mixed, the cost for treatment and disposal can be as high as $50,000 per cubic meter. Disposal of scrap metal as low-level waste uses up valuable space in the low-level waste disposal areas and requires transportation to the disposal site under Department of Transportation (DOT) regulations for low-level waste. In contrast, disposal as non-radioactive waste costs as little as $2 per cubic meter. While recycling is unavailable, disposing of the metal at an industrial waste site could be the best solution for this waste stream. A Green Is Clean (GIC) type verification program needs to be in place to provide the greatest assurance that the waste does not contain DOE added radioactivity. This paper is a review of available and emerging radiation monitoring and assay systems that could be used for scrap metal as part of the LANL GIC program.

  20. From Space to the Patient: A New Cytokine Release Assay to Monitor the Immune Status of HIV Infected Patients and Sepsis Patients

    NASA Technical Reports Server (NTRS)

    Kaufmann, I.; Draenert, R.; Gruber, M.; Feuerecker, M.; Crucian, B. E.; Mehta, S. L.; Roider, J.; Pierson, D. L.; Briegel, J. M.; Schelling, G.; Sams, C. F.; Chouker, A.

    2013-01-01

    Monitoring of humans either in the healthy men under extreme environmental stress like space flight, in human immunodeficiency virus (HIV) infected patients or in sepsis is of critical importance with regard to the timing of adequate therapeutic (counter-)measures. The in vivo skin delayed-type hypersensitivity test (DTH) served for many years as a tool to evaluate cell mediated immunity. However, this standardised in vivo test was removed from the market in 2002 due to the risk of antigen stabilization. To the best of our knowledge an alternative test as monitoring tool to determine cell mediated immunity is not available so far. For this purpose we tested a new alternative assay using elements of the skin DTH which is based on an ex vivo cytokine release from whole blood and asked if it is suitable and applicable to monitor immune changes in HIV infected patients and in patients with septic shock.

  1. Exaggerated natriuretic response to isotonic volume expansion in hypertensive renal transplant recipients: evaluation of proximal and distal tubular reabsorption by simultaneous determination of renal plasma clearance of lithium and 51Cr-EDTA.

    PubMed

    Nielsen, A H; Knudsen, F; Danielsen, H; Pedersen, E B; Fjeldborg, P; Madsen, M; Brøchner-Mortensen, J; Kornerup, H J

    1987-02-01

    In fourteen hypertensive and fourteen normotensive renal transplant recipients, and in a group of thirteen healthy controls, changes in natriuresis, glomerular filtration rate (GFR), and tubular reabsorption of sodium were determined in relation to intravenous infusion of 2 mmol isotonic sodium chloride per kg body weight. An exaggerated natriuresis was demonstrated in the hypertensive renal transplant recipients. This new finding indicates that the augmented natriuresis following plasma volume expansion, which is a characteristic finding in subjects with arterial hypertension, is not mediated by the renal nerves. Investigation of the tubular reabsorption rates of sodium by simultaneous determination of the renal clearance of 51Cr-EDTA and lithium showed that in the hypertensives the changes in tubular handling of sodium were different from those registered in the normotensive subjects. The increased sodium excretion in the hypertensive renal transplant recipients was caused by an increased output of sodium from the proximal tubules which was not fully compensated for by an increased distal reabsorption. Whether this increased delivery of sodium to the distal segments was caused by changes in GFR or in the proximal tubular reabsorption of sodium could not be clarified in the present study and warrants further investigations.

  2. Living donor and recipient screening for latent tuberculosis infection by tuberculin skin test and interferon-gamma releasing assay in a country with an intermediate burden of tuberculosis.

    PubMed

    Moon, Song Mi; Park, In-Ah; Kim, Sun-Mi; Park, Su-Jin; Jung, Joo Hee; Kim, Young Hoon; Park, Jae Berm; Hong, Bumsik; Lee, Sang-Oh; Choi, Sang-Ho; Kim, Yang Soo; Woo, Jun Hee; Park, Su-Kil; Lee, Sang Koo; Park, Jung Sik; Han, Duck Jong; Kim, Sung-Han

    2013-10-01

    There are few data on donor screening for latent tuberculosis infection (LTBI) using the tuberculin skin test (TST) and interferon-gamma releasing assay (IGRA). In South Korea, most renal allografts involve living donors (average, 80%). Hence, we have an opportunity to evaluate donor and recipient screening for LTBI by TST and IGRA. All donors and recipients admitted for kidney transplantation during a 20-month period were evaluated prospectively by using TST and Mycobacterium tuberculosis-specific enzyme-linked immunosorbent spot (ELISPOT) assay. The study population consisted of 205 living donor-recipient pairs (≥16 years) including 15 (7%) who yielded indeterminate donor or recipient ELISPOT results. Of the 205 donors, 63 (31%) gave a positive TST ≥5 mm, 33 (16%) a positive TST ≥10 mm, and 96 (47%) a positive ELISPOT. Of the 205 recipients, 9 (5%) gave a positive TST ≥5 mm, 3 (2%) a positive TST ≥10 mm, and 79 (39%) had a positive ELISPOT. Of the 205 donor-recipient pairs, only 59 (29%) gave negative donor and recipient ELISPOT results and 139 (68%) negative donor and recipient TSTs (<5 mm) (P < 0.001). One third of donor-recipient pairs tends to be positive in the TST, and two thirds of the donor-recipient pairs tends to be positive in the ELISPOT. Given the high positive rate of LTBI obtained by screening donors, further studies on the clinical value of solid organ transplant donors with positive TST or ELISPOT and health economics analysis in countries with intermediate burden of TB are needed for policy decisions on isoniazid (INH) prophylaxis.

  3. Assay of 6-gingerol in CO2 supercritical fluid extracts of ginger and evaluation of its sustained release from a transdermal delivery system across rat skin.

    PubMed

    Chen, Yan; Zhang, Cuiping; Zhang, Mei; Fu, Xiaobing

    2014-07-01

    Ginger has been widely used as healthy food condiment as well as traditional Chinese medicine since antiquity. Multiple potentials of ginger for treatment of various ailments have been revealed. However, the biological half-life of 6-gingerol (a principal pungent ingredient of ginger) is only 7.23 minutes while taken orally. Delivery of ginger compositions by routes other than oral have scarcely been reported. Therefore, we studied a noninvasive transdermal drug delivery system (TDDS) of ginger to bypass hepatic first pass metabolism, avoid gastrointestinal degradation and achieve long persistent release of effective compositions. After establishment of a HPLC analysis method of 6-gingerol, assays of 6-gingerol were performed to compare two kinds of ginger extracts. Then, the characteristics of transdermal delivery of 6-gingerol in TDDS were exhibited. The results showed that the contents of 6-gingerol in two kinds of ginger extracts were significantly different. The maximal delivery percentage of 6-gingerol across rat skin at 20 h was more than 40% in different TDDS formulations. TDDS may provide long-lasting delivery of ginger compounds.

  4. Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-D slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma

    USGS Publications Warehouse

    Muller, Jennifer K.; Sepulveda, Maria S.; Borgert, Christopher J.; Gross, Timothy S.

    2005-01-01

    This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

  5. Development of a cytotoxic T-cell assay in rabbits to evaluate early immune response to human T-lymphotropic virus type 1 infection.

    PubMed

    Haynes, Rashade A H; Phipps, Andrew J; Yamamoto, Brenda; Green, Patrick; Lairmore, Michael D

    2009-12-01

    Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell lymphoma/leukemia (ATL) following a prolonged clinical incubation period, despite a robust adaptive immune response against the virus. Early immune responses that allow establishment of the infection are difficult to study without effective animal models. We have developed a cytotoxic T-lymphocyte (CTL) assay to monitor the early events of HTLV-1 infection in rabbits. Rabbit skin fibroblast cell lines were established by transformation with a plasmid expressing simian virus 40 (SV40) large T antigen and used as autochthonous targets (derived from same individual animal) to measure CTL activity against HTLV-1 infection in rabbits. Recombinant vaccinia virus (rVV) constructs expressing either HTLV-1 envelope surface unit (SU) glycoprotein 46 or Tax proteins were used to infect fibroblast targets in a (51)Cr-release CTL assay. Rabbits inoculated with Jurkat T cells or ACH.2 cells (expressing ACH HTLV-1 molecule clone) were monitored at 0, 2, 4, 6, 8, 13, 21, and 34 wk post-infection. ACH.2-inoculated rabbits were monitored serologically and for viral infected cells following ex vivo culture. Proviral load analysis indicated that rabbits with higher proviral loads had significant CTL activity against HTLV-1 SU as early as 2 wk post-infection, while both low- and high-proviral-load groups had minimal Tax-specific CTL activity throughout the study. This first development of a stringent assay to measure HTLV-1 SU and Tax-specific CTL assay in the rabbit model will enhance immunopathogenesis studies of HTLV-1 infection. Our data suggest that during the early weeks following infection, HTLV-1-specific CTL responses are primarily targeted against Env-SU.

  6. Interferon Gamma Release Assays for the Diagnosis of Latent TB Infection in HIV-Infected Individuals in a Low TB Burden Country

    PubMed Central

    Ní Cheallaigh, Clíona; Fitzgerald, Ian; Grace, Jacinta; Jagjit Singh, Gurmit; El-Eraki, Nahla; Gibbons, Noel; Keane, Joseph; Rogers, Thomas R.; Clarke, Susan; Bergin, Colm

    2013-01-01

    Background Interferon gamma release assays (IGRAs) are used to diagnose latent tuberculosis infection. Two IGRAs are commercially available: the Quantiferon TB Gold In Tube (QFT-IT) and the T-SPOT.TB. There is debate as to which test to use in HIV+ individuals. Previous publications from high TB burden countries have raised concerns that the sensitivity of the QFT-IT assay, but not the T-SPOT.TB, may be impaired in HIV+ individuals with low CD4+ T-cell counts. We sought to compare the tests in a low TB burden setting. Methodology/Principal Findings T-SPOT.TB, QFT-IT, and tuberculin skin tests (TST) were performed in HIV infected individuals. Results were related to patient characteristics. McNemar’s test, multivariate regression and correlation analysis were carried out using SPSS (SPSS Inc). 256 HIV infected patients were enrolled in the study. The median CD4+ T-cell count was 338 cells/µL (range 1–1328). 37 (14%) patients had a CD4+ T-cell count of <100 cells/µL. 46/256 (18% ) of QFT-IT results and 28/256 (11%) of T-SPOT.TB results were positive. 6 (2%) of QFT-IT and 18 (7%) of T-SPOT.TB results were indeterminate. An additional 9 (4%) of T-SPOT.TB results were unavailable as tests were not performed due to insufficient cells or clotting of the sample. We found a statistically significant association between lower CD4+ T-cell count and negative QFT-IT results (OR 1.055, p = 0.03), and indeterminate/unavailable T-SPOT.TB results (OR 1.079, p = 0.02). Conclusions/Significance In low TB prevalence settings, the QFT-IT yields more positive and fewer indeterminate results than T-SPOT.TB. Negative results on the QFT-IT and indeterminate/unavailable results on the T-SPOT.TB were more common in individuals with low CD4+ T-cell counts. PMID:23382842

  7. The Clinical Usefulness of Tuberculin Skin Test versus Interferon-Gamma Release Assays for Diagnosis of Latent Tuberculosis in HIV Patients: A Meta-Analysis

    PubMed Central

    Ayubi, Erfan; Doosti-Irani, Amin; Sanjari Moghaddam, Ali; Sani, Mohadeseh; Nazarzadeh, Milad; Mostafavi, Ehsan

    2016-01-01

    Background Accurate diagnosis of latent tuberculosis infection (LTBI) is becoming increasingly concerning due to the increasing the HIV epidemic, which have increased the risk for reactivation to active tuberculosis (TB) infection. LTBI is diagnosed by tuberculin skin test (TST) and interferon-gamma release assays (IGRAs). Objectives The aim of the present study was to conduct a meta-analysis of published papers on the agreement (kappa) between TST and QuantiFERON-TB Gold In-Tube (QFT-GIT) tests for diagnosis of LTBI in HIV patient. Methods Electronic databases including PubMed/Medline, Elsevier/Scopus and Embase/Ovid were reviewed up Jan. 2016. We performed a random effect model meta-analysis for estimation of pooled Kappa between the two methods of diagnosis. Meta regression was used for assessing potential heterogeneity and Egger’s test was used for assessing small study effect and publication bias. Results The initial search strategy produced 6744 records. Of them, 23 cross-sectional studies met the inclusion criteria and 20 studies entered in meta-analysis. The pooled kappa was and prevalence-adjusted and bias-adjusted kappa (PABAK) were 0.37 (95% CI: 0.28, 0.46) and 0.59 (0.49, 0.69). The discordance of TST-/QFT-GIT+ was more than TST+/QFT-GIT-. Kappa estimate between two tests was linearly associated with age and prevalence index and inversely associated with bias index. Conclusion Fair agreement between TST and QFT-GIT makes it difficult to know whether TST is as useful as the QFT-GIT in HIV-infected patients. The higher discordance of TST-/QFT-GIT+ in compared to TST+/QFT-GIT- can induce the higher sensitivity of QFT-GIT for diagnosis LTBI in HIV patients. Disagreement between two tests can be influenced by error in measurements and prevalence of HIV. PMID:27622293

  8. Key Role of Effector Memory CD4+ T Lymphocytes in a Short-Incubation Heparin-Binding Hemagglutinin Gamma Interferon Release Assay for the Detection of Latent Tuberculosis

    PubMed Central

    Wyndham-Thomas, Chloé; Corbière, Véronique; Dirix, Violette; Smits, Kaatje; Domont, Fanny; Libin, Myriam; Loyens, Marc; Locht, Camille

    2014-01-01

    The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing the Mycobacterium tuberculosis reservoir. Therefore, powerful LTBI screening tools are indispensable. A gamma interferon release assay (IGRA) in response to the stimulation of peripheral blood mononuclear cells by the latency antigen native heparin-binding hemagglutinin (nHBHA-IGRA) has proven its potential for this purpose. We have evaluated its possible optimization through a reduction of incubation time from 96 to 24 h, while compensating for this by adding interleukin 7 (IL-7) to the medium. We have also investigated the phenotypes of the gamma interferon (IFN-γ)-producing cells after both short and long incubation times. One hundred thirty-one nonimmunocompromised patients were recruited from 3 Brussels-based university hospitals. They were divided into 1 of 4 subgroups according to their M. tuberculosis infection status (LTBI, TB infection, undetermined M. tuberculosis infection status, and noninfected controls). The novel 24-h nHBHA-IGRA was performed for all subjects, and a simultaneous 96-h classical HBHA-IGRA was performed for 79 individuals. The results showed a good correlation between the two tests, and the novel 24-h nHBHA-IGRA maintained the principal advantages of the classical test, namely, a high specificity for LTBI diagnosis, an absence of interference of Mycobacterium bovis BCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remote M. tuberculosis infections, the 24-h nHBHA-IGRA appears to have comparable diagnostic powers for recent and remote LTBI. The IFN-γ detected by the 24-h nHBHA-IGRA was mainly secreted by effector memory CD4+ T lymphocytes, a finding suggestive of continuous HBHA presentation during latency. PMID:24391135

  9. Interferon Gamma Release Assay versus Tuberculin Skin Testing among Healthcare Workers of Highly Diverse Origin in a Moderate Tuberculosis Burden Country

    PubMed Central

    Al Hajoj, Sahal; Varghese, Bright; Datijan, Alria; Shoukri, Mohammed; Alzahrani, Ali; Alkhenizan, Abdallah; AlSaif, Abdulaziz; Althawadi, Sahar; Fernandez, Grace; Alrajhi, Abdulrahman

    2016-01-01

    Health care workers (HCW’s) are always at an increased risk of contracting tuberculosis (TB) infection. In Saudi Arabia, Interferon Gamma Release Assay (IGRA) has not been evaluated as a screening tool for latent TB infection (LTBI) among HCW’s considering their high demographic diversity. During February 2012 to January 2015 a cross sectional study has been conducted in a tertiary care center with maximum demographically diverse staff population in the capital city-Riyadh. After a short interview and consenting, all the candidates were subjected to tuberculin skin test (TST) and QuantiFERON TB gold In-tube test (QFT). A logistic regression analysis was carried out for establishing the associations between putative risk factors and the diagnostic tests. The candidates were classified according to geographical origin and a detailed analysis was conducted on the impact of their origin towards the results of TST and QFT. Of the 1595 candidates enrolled, 90.6% were BCG vaccinated, female (67.9%) and mainly nurses (53.2%). Candidates with high risk of suspected or confirmed TB patient exposure were 56.1% and 76.5% of them had <10 year’s work experience. TST positivity was observed in 503 (31.5%) candidates, while QFT was positive among 399 (25%). Majority of the candidates were non-Saudi (83%) and predominantly (52.4%) from Western Pacific region. Concordant results were obtained in 14.2% of positive cases and 57.7% negative cases. The disagreements between the two tests were relatively high (kappa co-efficient-0.312±0.026, p value- <0.00001) as TST positive/QFT negative discordance was 54.8% while TST negative/QFT positive discordance was 15.7%. Age of the candidates, BCG vaccination, and South East Asian origin were associated with TST positivity while Occupational TB exposure and geographical origin of the candidates were associated with QFT positivity. A regular follow up on recently TST converted candidates showed no progression to active TB. The putative

  10. The Significance of Sensitive Interferon Gamma Release Assays for Diagnosis of Latent Tuberculosis Infection in Patients Receiving Tumor Necrosis Factor-α Antagonist Therapy

    PubMed Central

    Jung, Yu Jung; Woo, Hye In; Jeon, Kyeongman; Koh, Won-Jung; Jang, Dong Kyoung; Cha, Hoon Suk; Koh, Eun Mi; Lee, Nam Yong; Kang, Eun-Suk

    2015-01-01

    Objective We compared two interferon gamma release assays (IGRAs), QuantiFERON-TB Gold In-Tube (QFT-GIT) and T-SPOT.TB, for diagnosis of latent tuberculosis infection (LTBI) in patients before and while receiving tumor necrosis factor (TNF)-α antagonist therapy. This study evaluated the significance of sensitive IGRAs for LTBI screening and monitoring. Methods Before starting TNF-α antagonist therapy, 156 consecutive patients with rheumatic diseases were screened for LTBI using QFT-GIT and T-SPOT.TB tests. According to our study protocol, QFT-GIT-positive patients received LTBI treatment. Patients positive by any IGRAs were subjected to follow-up IGRA tests after completing LTBI-treatment and/or during TNF-α antagonist therapy. Results At the initial LTBI screening, 45 (28.9%) and 70 (44.9%) patients were positive by QFT-GIT and T-SPOT.TB, respectively. The agreement rate between IGRA results was 78.8% (k = 0.56; 95% confidence interval [95% CI] = 0.43 to 0.68). Of 29 patients who were positive only by T-SPOT.TB in the initial screening, 83% (19/23) were persistently positive by T-SPOT.TB, while QFT-GIT testing showed that 36% (9/25) had conversion during TNF-α antagonist therapy. By the end of the follow-up period (218 to 1,264 days), four patients (4/137, 2.9%) developed active tuberculosis (TB) diseases during receiving TNF-α antagonist therapy. Among them, one was Q-T+, one was Q+T-, and the remaining two were Q-T- at the initial screening (Q, QuantiFERON-TB Gold In-Tube; T, T-SPOT.TB; +, positive; -, negative). Two (2/4, 50%) patients with TB reactivation had at least one prior risk factor consistent with previous TB infection. Conclusion This study demonstrated the need to capitalize on sensitive IGRAs to monitor for LTBI in at-risk patients for a more sensitive diagnosis in countries with an intermediate TB burden. PMID:26474294

  11. Screening for latent tuberculosis in Norwegian health care workers: high frequency of discordant tuberculin skin test positive and interferon-gamma release assay negative results

    PubMed Central

    2013-01-01

    Background Tuberculosis (TB) presents globally a significant health problem and health care workers (HCW) are at increased risk of contracting TB infection. There is no diagnostic gold standard for latent TB infection (LTBI), but both blood based interferon-gamma release assays (IGRA) and the tuberculin skin test (TST) are used. According to the national guidelines, HCW who have been exposed for TB should be screened and offered preventive anti-TB chemotherapy, but the role of IGRA in HCW screening is still unclear. Methods A total of 387 HCW working in clinical and laboratory departments in three major hospitals in the Western region of Norway with possible exposure to TB were included in a cross-sectional study. The HCW were asked for risk factors for TB and tested with TST and the QuantiFERON®TB Gold In-Tube test (QFT). A logistic regression model analyzed the associations between risk factors for TB and positive QFT or TST. Results A total of 13 (3.4%) demonstrated a persistent positive QFT, whereas 214 (55.3%) had a positive TST (≥ 6 mm) and 53 (13.7%) a TST ≥ 15 mm. Only ten (4.7%) of the HCW with a positive TST were QFT positive. Origin from a TB-endemic country was the only risk factor associated with a positive QFT (OR 14.13, 95% CI 1.37 - 145.38, p = 0.026), whereas there was no significant association between risk factors for TB and TST ≥ 15 mm. The five HCW with an initial positive QFT that retested negative all had low interferon-gamma (IFN-γ) responses below 0.70 IU/ml when first tested. Conclusions We demonstrate a low prevalence of LTBI in HCW working in hospitals with TB patients in our region. The “IGRA-only” seems like a desirable screening strategy despite its limitations in serial testing, due to the high numbers of discordant TST positive/IGRA negative results in HCW, probably caused by BCG vaccination or boosting due to repetitive TST testing. Thus, guidelines for TB screening in HCW should be updated in order to

  12. Comparison of Interferon-γ Release Assay to Two Cut-Off Points of Tuberculin Skin Test to Detect Latent Mycobacterium tuberculosis Infection in Primary Health Care Workers

    PubMed Central

    de Souza, Fernanda Mattos; do Prado, Thiago Nascimento; Pinheiro, Jair dos Santos; Peres, Renata Lyrio; Lacerda, Thamy Carvalho; Loureiro, Rafaela Borge; Carvalho, Jose Américo; Fregona, Geisa; Dias, Elias Santos; Cosme, Lorrayne Beliqui; Rodrigues, Rodrigo Ribeiro; Riley, Lee Wood; Maciel, Ethel Leonor Noia

    2014-01-01

    Background An interferon-γ release assay, QuantiFERON-TB (QFT) test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Here, we compared the performance of QFT with tuberculin skin test (TST) measured at two different cut-off points among primary health care work (HCW) in Brazil. Methods A cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points. Results We enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31), and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22). We investigated possible factors of discordance of TST vs QFT. Compared to the TST−/QFT− group, risk factors for discordance in the TST+/QFT− group with TST cut-off of ≥5 mm included age between 41–45 years [OR = 2.70; CI 95%: 1.32–5.51] and 46–64 years [OR = 2.04; CI 95%: 1.05–3.93], BCG scar [OR = 2.72; CI 95%: 1.40–5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09–4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03–4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01–2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05–2.62], were significantly associated with the TST+/QFT− group. No statistically significant associations were found among the TST−/QFT+ discordant group with either TST cut-off value. Conclusions Although we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed. PMID:25137040

  13. Utility of the Enzyme-Linked Immunospot Interferon-γ–Release Assay to Predict the Risk of Cytomegalovirus Infection in Hematopoietic Cell Transplant Recipients

    PubMed Central

    Nesher, Lior; Shah, Dimpy P.; Ariza-Heredia, Ella J.; Azzi, Jacques M.; Siddiqui, Hala K.; Ghantoji, Shasank S.; Marsh, Lisa Y.; Michailidis, Lamprinos; Makedonas, George; Rezvani, Katy; Shpall, Elizabeth J.; Chemaly, Roy F.

    2016-01-01

    The ability to distinguish allogeneic hematopoietic cell transplant (allo-HCT) recipients at risk for cytomegalovirus (CMV) reactivation from those who are not is central for optimal CMV management strategies. Interferon γ (IFN-γ) produced by CMV-challenged T cells may serve as an immune marker differentiating these 2 populations. We prospectively monitored 63 CMV-seropositive allo-HCT recipients with a CMV-specific enzyme-linked immunospot (ELISPOT) assay and for CMV infection from the period before transplantation to day 100 after transplantation. Assay results above certain thresholds (50 spots per 250 000 cells for immediate early 1 or 100 spots per 250 000 cells for phosphoprotein 65) identified patients who were protected against CMV infection as long as they had no graft-versus-host disease and/or were not receiving systemic corticosteroids. Based on the multivariable Cox proportional hazards regression model, the only significant factor for preventing CMV reactivation was a CMV-specific ELISPOT response above the determined thresholds (adjusted hazard ratio, 0.21; 95% confidence interval, .05–.97; P = .046). Use of this assay as an additional tool for managing allo-HCT recipients at risk for CMV reactivation needs further validation in future studies. Application of this new approach may reduce the duration and intensity of CMV monitoring and the duration of prophylaxis or treatment with antiviral agents in those who have achieved CMV-specific immune reconstitution. PMID:26908740

  14. Adsorption of inorganic and organic ions to polycarbophil as a means of sustained-release dosage formulation.

    PubMed

    See, N A; Russell, J; Connors, K A; Bass, P

    1987-06-01

    The adsorption and desorption of drugs and inorganic ions to and from polycarbophil (PC), a polymer, were investigated to determine if PC would be a suitable carrier for sustained-release dosage formulations. Both in vitro and in vivo experiments with a polycarbophil-atropine sulfate complex demonstrated the gradual-release properties of this system. Adsorbed Cr3+ ions, like atropine, are released slowly. In contrast, 51CrO4(2-) ions are predominantly bound in an irreversible manner. A third group of drugs minimally adsorbed to PC under the conditions studied. We conclude that PC under both in vitro and in vivo conditions is able to bind certain ions and drugs and then release them over a period of time in a predictable and repeatable manner.

  15. 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

  16. Comparison of two interferon-gamma release assays (QuantiFERON-TB Gold In-Tube and T-SPOT.TB) in testing for latent tuberculosis infection among HIV-infected adults.

    PubMed

    Sultan, B; Benn, P; Mahungu, T; Young, M; Mercey, D; Morris-Jones, S; Miller, R F

    2013-10-01

    There is currently no 'gold standard' for diagnosis of latent tuberculosis infection (LTBI), and both the tuberculin skin test and interferon-gamma release assays (IGRAs) are used for diagnosis; the latter have a higher sensitivity than tuberculin skin tests for diagnosis of LTBI in HIV-infected individuals with lower CD4 counts. No evidence base exists for selection of IGRA methodology to identify LTBI among human immunodeficiency virus-infected patients in the UK. We prospectively evaluated two commercially available IGRA methods (QuantiFERON-TB Gold In Tube [QFG] and T-SPOT.TB) for testing LTBI among HIV-infected patients potentially nosocomially exposed to an HIV-infected patient with 'smear-positive' pulmonary tuberculosis. Among the exposed patients median CD4 count was 550 cells/µL; 105 (90%) of 117 were receiving antiretroviral therapy, of who 104 (99%) had an undetectable plasma HIV load. IGRAs were positive in 12 patients (10.3%); QFG positive in 11 (9.4%) and T-SPOT.TB positive in six (5.1%); both IGRAs were positive in five patients (4.3%). There was one indeterminate QFG and one borderline T-SPOT.TB result. Concordance between the two IGRAs was moderate (κ = 0.56, 95% confidence interval = 0.27-0.85). IGRAs were positive in only 4 (29%) of 14 patients with previous culture-proven tuberculosis. No patient developed tuberculosis during 20 months of follow-up.

  17. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  18. Comparison between spontaneous gonadotropin concentration profiles and gonadotropin response to low-dose gonadotropin-releasing hormone in prepubertal and early pubertal boys and patients with hypogonadotropic hypogonadism: assessment by using ultrasensitive, time-resolved immunofluorometric assay.

    PubMed

    Goji, K; Tanikaze, S

    1992-05-01

    To assess whether nocturnal gonadotropin concentration profiles in children could be predicted by measurement of peak gonadotropin levels after gonadotropin-releasing hormone (GnRH) administration, we measured spontaneous gonadotropin levels every 20 min and the gonadotropin responses to low-dose GnRH using an ultrasensitive, time-resolved immunofluorometric assay in 61 boys with short stature and/or delayed puberty. Spontaneous nocturnal LH pulses were observed in 58 out of 61 patients. After GnRH administration in a dose of 25 ng/kg, all of the 61 patients had significant LH and FSH responses, and GnRH-stimulated peak LH and FSH levels were highly correlated with maximal spontaneous nocturnal LH and FSH levels, respectively (r = 0.83 for LH and r = 0.91 for FSH; p less than 0.00001). Analysis of individual subjects revealed that GnRH-stimulated peak LH levels were almost identical to maximal nocturnal LH levels in the subjects whose GnRH-stimulated peak LH levels were between 5 and 10 IU/L, whereas GnRH-stimulated peak LH levels tended to be higher than maximal nocturnal levels in the subjects whose GnRH-stimulated peak LH levels were 5 IU/L or lower. To determine if there were any parameters in the gonadotropin response to GnRH that might be useful in distinguishing early pubertal boys from prepubertal boys, we evaluated the gonadotropin response to GnRH in 44 prepubertal and 10 early pubertal normal short boys. Although maximal nocturnal LH levels did not overlap between prepubertal and pubertal groups, GnRH-stimulated LH peak levels overlapped considerably between the two groups. Even the GnRH-stimulated peak LH to peak FSH ratio overlapped between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Rate of tuberculosis infection in children and adolescents with household contact with adults with active pulmonary tuberculosis as assessed by tuberculin skin test and interferon-gamma release assays.

    PubMed

    Ferrarini, M A G; Spina, F G; Weckx, L Y; Lederman, H M; De Moraes-Pinto, M I

    2016-03-01

    Tuberculosis (TB) infection was evaluated in Brazilian immunocompetent children and adolescents exposed and unexposed (control group) to adults with active pulmonary TB. Both groups were analysed by clinical and radiological assessment, TST, QFT-IT and T-SPOT.TB. The three tests were repeated after 8 weeks in the TB-exposed group if results were initially negative. Individuals with latent tuberculosis infection (LTBI) were treated and tests were repeated after treatment. Fifty-nine TB-exposed and 42 controls were evaluated. Rate of infection was 69·5% and 9·5% for the exposed and control groups, respectively. The exposed group infection rate was 61% assessed by TST, 57·6% by T-SPOT.TB, and 59·3%, by QFT-IT. No active TB was diagnosed. Agreement between the three tests was 83·1% and 92·8% in the exposed and control groups, respectively. In the exposed group, T-SPOT.TB added four TB diagnoses [16%, 95% confidence interval (CI) 1·6-30·4] and QFT-IT added three TB diagnoses (12%, 95% CI 0-24·7) in 25 individuals with negative tuberculin skin test (TST). Risk factors associated to TB infection were contact with an adult with active TB [0-60 days: odds ratio (OR) 6·9; >60 days: OR 27·0] and sleeping in the same room as an adult with active TB (OR 5·2). In Brazilian immunocompetent children and adolescents, TST had a similar performance to interferon-gamma release assays and detected a high rate of LTBI.

  20. Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol.

    PubMed

    Kayacelebi, Arslan Arinc; Schauerte, Celina; Kling, Katharina; Herbers, Jan; Beckmann, Bibiana; Engeli, Stefan; Jordan, Jens; Zoerner, Alexander A; Tsikas, Dimitrios

    2017-03-15

    2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10μM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0-1μM) as the MAGL product. Unlabelled AA (d0-AA, 1μM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H](-)→[M-H - CO2](-), i.e., m/z 311→m/z 267 for d8-AA and m/z 303→m/z 259 for d0-AA. Prior to GC-MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB](-), i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1μM) of d8-AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r(2)=0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8

  1. Developmental toxicity, uptake and distribution of sodium chromate assayed by frog embryo teratogenesis assay-Xenopus(FETAX).

    PubMed

    Bosisio, Stefano; Fortaner, Salvador; Bellinetto, Sonia; Farina, Massimo; Del Torchio, Riccardo; Prati, Mariangela; Gornati, Rosalba; Bernardini, Giovanni; Sabbioni, Enrico

    2009-09-01

    The embryotoxicity and teratogenicity of Cr(VI) on the survival and morphology of the anuran Xenopus laevis have been assessed by frog embryo teratogenesis assay-Xenopus (FETAX). The lethal median (LC(50)) and teratogenic median (TC(50)) concentration values of Cr(VI) were 890 microM and 260 microM, respectively. The calculated teratogenic index (TI) value was 3.42, suggesting that hexavalent chromium has a teratogenic potential. Malformations of embryos included lifting of the body, coiling of the tail and body oedema. Furthermore, the chromium salt caused significant growth retardation at 25 microM exposure concentrations. The use of radiolabelled (51)Cr(VI) allowed the determination of the time course uptake of Cr in Xenopus exposed to concentrations ranging from 0.025 to 500 microM. The evaluation of its distribution into the body (head-abdomen-tail) was evaluated at different exposure times. Chromium is taken up at 24 h by Xenopus embryos for all concentrations tested. At 48 h post fertilization (stage of larva) the amount of Cr accumulated by the two-day-old larva ranged from 0.42 to 580 pg mg(-1) wet weight at 0.025 and 500 microM respectively. These amounts were lower than those at 24 h (2.77 to 11016 pg mg(-1) wet weight embryo) reaching values of the same order of magnitude at 120 h (five-days-old larva). Since at 48 h Xenopus development leads to a swimming embryo, the observed uptake at 24 h could be the result of the binding of Cr to jelly coat compounds surrounding the embryo body as confirmed by gel filtration experiments on (51)Cr-jelly coat. The interaction of Cr with jelly coat is in agreement with the role of jelly coat in protecting the embryo against pathogen and chemical toxins to ensure fertilization. This work further supports the hypothesis that Cr contamination of surface waters could contribute to explain the reported worldwide depletion of frog population.

  2. Contribution of Interferon gamma release assays testing to the diagnosis of latent tuberculosis infection in HIV-infected patients: A comparison of QuantiFERON-TB Gold In Tube, T-SPOT.TB and tuberculin skin test

    PubMed Central

    2012-01-01

    Background Diagnosis and treatment of latent tuberculosis infection (LTBI) is the most effective strategy to control tuberculosis (TB) among patients with HIV infection. The tuberculin skin test (TST) was the only available method to identify LTBI. The aim of the present work was to evaluate the usefulness of the interferon-gamma release assays (IGRAs): QuantiFERON-tuberculosis (TB) Gold-In-Tube test (QFG) and T-SPOT.TB for the diagnosis of LTBI in a diverse cohort of HIV-infected patients. Methods A prospective study was carried out in consecutive patients cared for in a single institution in Spain from January 2009 to October 2010. IGRAs and TST were performed simultaneously. TST induration ≥ 5 mm was considered positive. Results QFG, T-SPOT.TB and TST were performed in 373 subjects. Median CD4 cell count was 470/μl with a median nadir of 150/μl. TST, QFG and T-SPOT.TB were positive in 13.3%, 7.5% and 18.5% cases respectively. Among 277 patients with neither past or current TB nor previous treatment for LTBI and who had TST results, a positive TST result was obtained in 20 (7.2%) cases. When adding QFG results to TST, there were a total of 26 (8.6%) diagnoses of LTBI. When the results of both IGRAs were added, the number of diagnoses increased to 54 (17.9%) (incremental difference: 10.7% [95% confidence interval [CI]:5.3-16.2%] [p < 0.001]), and when both IGRAs were added, the number of diagnoses reached 56 (18.5%) (incremental difference: 11.3% [95% CI:5.7%–16.9%] [p < 0.001]). Patients with a CD4 cell count greater than 500 cells/μl and prior stay in prison were more likely to have a diagnosis of LTBI by TST and/or QFG and/or T-SPOT.TB (adjusted odds ratio [aOR]: 3.8; 95% CI, 1.4 – 9.9; and aOR: 3.3; 95% CI, 1.3 – 8.3, respectively). Conclusions IGRAs were more sensitive than TST for diagnosis of M. tuberculosis infection in HIV-infected patients. Dual sequential testing with TST and IGRAs may be the optimal approach for LTBI screening in this

  3. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  4. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  5. Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

    SciTech Connect

    Dannemann, B.R.; Morris, V.A.; Araujo, F.G.; Remington, J.S. )

    1989-10-15

    Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant. In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively, as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites.

  6. The Rabbit Corneal Pocket Assay.

    PubMed

    Morbidelli, Lucia; Ziche, Marina

    2016-01-01

    The rabbit corneal micropocket angiogenesis assay uses the avascular cornea as a substrate canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 1-2 weeks and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor and a synthetic polymer to allow slow release. A micropocket is surgically created in the rabbit cornea under anesthesia and a pellet implanted. On the days later, the angiogenic response is measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay are used to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter, the experimental details of the rabbit cornea assay and technical implementations to the original protocol are described.

  7. Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials.

    PubMed

    Martinelli, Elisa; Sarvothaman, Mahesh K; Galli, Giancarlo; Pettitt, Michala E; Callow, Maureen E; Callow, James A; Conlan, Sheelagh L; Clare, Anthony S; Sugiharto, Albert B; Davies, Cait; Williams, David

    2012-01-01

    Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700™. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.

  8. Optogenetic control of ATP release

    NASA Astrophysics Data System (ADS)

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

    2013-03-01

    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  9. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    SciTech Connect

    Hovi, T.; Roivainen, M.

    1989-04-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.

  10. Syndactyly Release.

    PubMed

    Braun, Tara L; Trost, Jeffrey G; Pederson, William C

    2016-11-01

    Syndactyly is one of the most common congenital hand anomalies treated by pediatric plastic surgeons. Established principles of syndactyly separation dictate the timing and order of syndactyly release, with the goals of surgery being the creation of an anatomically normal webspace, tension-free closure of soft tissue, and return of function to the fingers. Numerous surgical methods have been described, many of which involve the use of local flaps to reconstruct the commissure and full-thickness skin grafts for coverage of raw areas. Recently, reconstructive techniques without the use of skin grafts have been devised, which work well for certain indications. Special considerations are described for complete, complex, and syndromic syndactylies. Outcomes for simple syndactyly release are typically good when surgical principles are followed, whereas complex syndactyly release tends to have less-favorable outcomes and more complications.

  11. Insecticidal and sterilizing effect of Olyset Duo®, a permethrin and pyriproxyfen mixture net against pyrethroid-susceptible and -resistant strains of Anopheles gambiae s.s.: a release-recapture assay in experimental huts

    PubMed Central

    Djènontin, Armel; Ahoua Alou, Ludovic P.; Koffi, Alphonsine; Zogo, Barnabas; Duarte, Elves; N’Guessan, Raphael; Moiroux, Nicolas; Pennetier, Cédric

    2015-01-01

    In the context of the widespread distribution of pyrethroid resistance among malaria vectors, we did a release-recapture trial in experimental huts to investigate the insecticidal and sterilizing effects of a novel long-lasting net (LN), Olyset® Duo, incorporating a mixture of permethrin (PER) and the insect growth regulator (IGR), pyri-proxyfen (PPF). An LN containing PPF alone and a classic Olyset® Net were tested in parallel as positive controls. The effect of progressive number of holes (6, 30, or 150) that may accrue in nets over time was simulated. We used two laboratory Anopheles gambiae s.s. strains: the susceptible Kisumu strain and the pyrethroid-resistant VK-Per strain having solely kdr as resistance mechanism. The effect of these nets on the reproductive success of blood-fed females that survived the different LNs conditions was recorded. Regardless of the mosquito strain, the LNs containing PPF alone with as many as 30 holes drastically reduced the number of eggs laid by females succeeding in feeding, i.e. fecundity by 98% and egg hatching rate (fertility) by 93% relative to untreated control net. Very few of the resistant females blood fed and survived under the Olyset® Duo with similar number of holes (up to 30) but of these few, the inhibition of reproductive success was 100%. There was no evidence that the Olyset® Duo LN with 150 holes impacted fecundity or fertility of the resistant colony. The efficacy of Olyset® Duo is encouraging and clearly illustrates that this new net might be a promising tool for malaria transmission control and resistance management. PMID:26489479

  12. Toggle release

    NASA Technical Reports Server (NTRS)

    Graves, Thomas J. (Inventor); Yang, Robert A. (Inventor); Brown, Christopher W. (Inventor)

    1989-01-01

    A pyrotechnic actuated structural release device 10 which is mechanically two fault tolerant for release. The device 10 comprises a fastener plate 11 and fastener body 12, each attachable to a different one of a pair of structures to be joined. The fastener plate 11 and body 12 are fastenable by a toggle 13 supported at one end on the fastener plate and mounted for universal pivotal movement thereon. At its other end which is received in a central opening in the fastener body 12 and adapted for limited pivotal movement therein the toggle 13 is restrained by three retractable latching pins 61 symmetrically disposed in equiangular spacing about the axis of the toggle 13 and positionable in latching engagement with an end fitting on the toggle. Each pin 61 is individually retractable by combustion of a pyrotechnic charge 77, the expanding gases of which are applied to a pressure receiving face 67 on the latch pin 61 to effect its retraction from the toggle. While retraction of all three pins 62 releases the toggle, the fastener is mechanically two fault tolerant since the failure of any single one or pair of the latch pins to retract results in an asymmetrical loading on the toggle and its pivotal movement to effect a release. An annular bolt 18 is mounted on the fastener plate 11 as a support for the socket mounting 30, 37 of the toggle whereby its selective axial movement provides a means for preloading the toggle.

  13. 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.

  14. RAS - Screens & Assays

    Cancer.gov

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

  15. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  16. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  17. Evaluation of the Erythrocyte Malondialdehyde (MDA) Release Assay.

    DTIC Science & Technology

    1987-01-01

    a functional measure of vitamin E status. Clin Chim Acta 1985;151:169-176. 4. Haddad E, Blankenship J. Short term effect of a low fat diet on plasma...with increased plasma tocopherol accompanying hyperlipemia. Haddad placed hyperlipidemic men on low fat diets and found a decreased plasma tocopherol...Kitagawa et al (73,74). From another perspective, Haddad et al placed hyperlipidemic males on a short-term low fat diet and observed a decrease in the plasma

  18. The glucose oxidase-peroxidase assay for glucose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  19. Absolute nuclear material assay

    DOEpatents

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

    2012-05-15

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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  6. Tube-Forming Assays.

    PubMed

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

    2016-01-01

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

  7. Universal phosphatase-coupled glycosyltransferase assay.

    PubMed

    Wu, Zhengliang L; Ethen, Cheryl M; Prather, Brittany; Machacek, Miranda; Jiang, Weiping

    2011-06-01

    A nonradioactive glycosyltransferase assay is described here. This method takes advantage of specific phosphatases that can be added into glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving groups of glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite-based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multiwell plates and quantitated by a plate reader, thus making it amenable to high-throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases and sialyltransferases. As examples, we first assayed Clostridium difficile toxin B, a protein O-glucosyltransferase that specifically monoglucosylates and inactivates Rho family small GTPases; we then showed that human KTELC1, a homolog of Rumi from Drosophila, was able to hydrolyze UDP-Glc; and finally, we measured the kinetic parameters of human sialyltransferase ST6GAL1.

  8. 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.

  9. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  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.

  11. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  12. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  13. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  14. 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.

  15. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  16. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  17. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

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

  18. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  19. 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.

  20. Assays for calcitonin receptors

    SciTech Connect

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  1. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  2. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  3. 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.

  4. 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.

  5. Quantitative estimation of hemorrhage in chronic subdural hematoma using the /sup 51/Cr erythrocyte labeling method

    SciTech Connect

    Ito, H.; Yamamoto, S.; Saito, K.; Ikeda, K.; Hisada, K.

    1987-06-01

    Red cell survival studies using an infusion of chromium-51-labeled erythrocytes were performed to quantitatively estimate hemorrhage in the chronic subdural hematoma cavity of 50 patients. The amount of hemorrhage was determined during craniotomy. Between 6 and 24 hours after infusion of the labeled red cells, hemorrhage accounted for a mean of 6.7% of the hematoma content, indicating continuous or intermittent hemorrhage into the cavity. The clinical state of the patients and the density of the chronic subdural hematoma on computerized tomography scans were related to the amount of hemorrhage. Chronic subdural hematomas with a greater amount of hemorrhage frequently consisted of clots rather than fluid.

  6. 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.

  7. Specific cytotoxic T cells are found in the nonrejected kidneys of blood-transfused rats

    SciTech Connect

    Dallman, M.J.; Wood, K.J.; Morris, P.J.

    1987-02-01

    Preoperative, donor-specific blood transfusion leads to indefinite survival of rat renal allografts in the strain combinations used. /sup 51/Cr-release assays have shown that the level of specific cytotoxic effector activity in the grafts of transfused (nonrejected kidney) animals is very high and may equal or exceed that seen in the grafts of untreated (rejected kidney) recipients. Such cytotoxicity demonstrates specificity for the alloantigens of the kidney, is T cell-mediated, and may persist within the transplant.

  8. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  9. Cytotoxic activity of CD4+ T cells against autologous tumor cells.

    PubMed

    Konomi, Y; Sekine, T; Takayama, T; Fuji, M; Tanaka, T

    1995-09-01

    The 51Cr-release assay is mostly applied to detecting the cytotoxic activity of CD8+ T cells, and little is known about the activity of CD4+ T cells. Therefore, the correlation between the cytotoxic activity of CD4+ or CD8+ T cells and the incubation period with autologous tumor cells was analyzed by two methods. The incubation periods were 4 and 20 h (4 h and 20 h assay) for the 51Cr-release assay. Eight pairs of tumor cells and T cells were assayed. T cells were fractionated into CD4+ and CD8+ T cells by using magnetic beads and panning methods, and those cells were activated by culture with recombinant interleukin-2 and immobilized anti-CD3 monoclonal antibody. In 6 out of 8 cases, no cytotoxic activity of CD4+ T cells was detected by the 4 h assay, whereas cytotoxic activity was detected in all cases in the 20 h assay. The cytotoxic activities in 20 h assay of CD4+ T cells were increased 67-fold in comparison with the activities in 4 h assay (range: 5-197). In the case of CD8+ T cells, cytotoxic activities were detected in 6 out of 8 cases in the 4 h assay. The lytic unit ratio of CD4+ and CD8+ T cells was calculated as 1.5 in the 20 h assay (range: 0.2- > 7.2) versus 0.4 in the 4 h assay (range: < 0.1-1.3). Cytotoxic activities in colorimetric assay using Crystal Violet with a 24 h incubation were similar to those in the 20 h 51Cr-release assay in all eight cases. These results indicate that CD4+ T cells have cytotoxic activity as strong as that of CD8+ T cells towards autologous tumor cells.

  10. Delayed simultaneous release mechanism

    NASA Technical Reports Server (NTRS)

    Moyer, X. W.; Webb, J. B. (Inventor)

    1973-01-01

    The disclosed appendage release mechanism is particularly adapted for use with spacecraft operating with despin mechanisms and releasable appendages. It includes a flexible loop and a number of appendage releasing devices which are attached to the flexible loop. The appendage releasing devices are made up of piston-cams and ball latches which hold the appendages as long as the flexible loop is maintained in a taut condition, but which release the appendages upon relaxation of the flexible loop. The flexible loop remains taut as long as the despin weights remain attached, but relaxes when the despin weights are released.

  11. 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+.

  12. Growth cone collapse assay.

    PubMed

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

    2014-01-01

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

  13. 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+.

  14. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  15. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

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

  16. 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.

  17. [Lateral retinacular release].

    PubMed

    Verdonk, P; Bonte, F; Verdonk, R

    2008-09-01

    This overview of numerous studies discusses, based on short-term and long-term results, which diagnoses are indications for lateral retinacular release. No significant differences in outcome between arthroscopic and open lateral release could be documented. Isolated lateral release offers a good success rate for treating a stable patella with excessive lateral pressure. In patellar instability, the results are less favorable in long-term follow-up evaluation. Hyperlaxity with hypermobility of the patella is an absolute contraindication. Lateral release provides only temporary benefit for patellofemoral osteoarthritis. Proximal and/or distal realignment of the extensor mechanism gives better results than isolated lateral release.

  18. ELECTROMAGNETIC RELEASE MECHANISM

    DOEpatents

    Michelson, C.

    1960-09-13

    An electromagnetic release mechanism is offered that may be used, for example, for supporting a safety rod for a nuclear reactor. The release mechanism is designed to have a large excess holding force and a rapid, uniform, and dependable release. The fast release is accomplished by providing the electromagnet with slotttd polts separated by an insulating potting resin, and by constructing the poles with a ferro-nickel alloy. The combination of these two features materially reduces the eddy current power density whenever the magnetic field changes during a release operation. In addition to these features, the design of the armature is such as to provide ready entrance of fluid into any void that might tend to form during release of the armature. This also improves the release time for the mechanism. The large holding force for the mechanism is accomplished by providing a small, selected, uniform air gap between the inner pole piece and the armature.

  19. 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.

  20. Survival assays using Caenorhabditis elegans

    PubMed Central

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

    2017-01-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans. PMID:28241407

  1. Cell Proliferation and Cytotoxicity Assays.

    PubMed

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

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

  2. Coagulation assays and anticoagulant monitoring.

    PubMed

    Funk, Dorothy M Adcock

    2012-01-01

    Anticoagulant therapy, including conventional agents and a variety of new oral, fast-acting drugs, is prescribed for millions of patients annually. Each anticoagulant varies in its effect on routine and specialty coagulation assays and each drug may require distinct laboratory assay(s) to measure drug concentration or activity. This review provides an overview of the assorted assays that can measure anticoagulant drug concentration or activity and includes key assay interferences. The effect of these conventional and new anticoagulant agents on specialty coagulation assays used to evaluate for bleeding or clotting disorders, and whether this impact is physiological or factitious, is included. Also provided is a short review of superwarfarin poisoning and features distinguishing this from warfarin overdose. Knowledge of clinically significant pearls and pitfalls pertinent to coagulation assays in relation to anticoagulant therapy are important to optimize patient care.

  3. Multicomponent Implant Releasing Dexamethasone

    NASA Astrophysics Data System (ADS)

    Nikkola, L.; Vapalahti, K.; Ashammakhi, N.

    2008-02-01

    Several inflammatory conditions are usually treated with corticosteroids. There are various problems like side effects with traditional applications of steroids, e.g. topical, or systemic routes. Local drug delivery systems have been studied and developed to gain more efficient administration with fewer side effects. Earlier, we reported on developing Dexamethasone (DX) releasing biodegradable fibers. However, their drug release properties were not satisfactory in terms of onset of drug release. Thus, we assessed the development of multicomponent (MC) implant to enhance earlier drug release from such biodegradable fibers. Poly (lactide-co-glycolide) (PLGA) and 2 wt-% and 8 wt-% DX were compounded and extruded with twin-screw extruder to form of fibers. Some of the fibers were sterilized to obtain a change in drug release properties. Four different fiber classes were studied: 2 wt-%, 8 wt-%, sterilized 2 wt-%, and sterilized 8 wt-%. 3×4 different DX-releasing fibers were then heat-pressed to form one multicomponent rod. Half of the rods where sterilized. Drug release was measured from initial fibers and multicomponent rods using a UV/VIS spectrometer. Shear strength and changes in viscosity were also measured. Drug release studies showed that drug release commenced earlier from multicomponent rods than from component fibers. Drug release from multicomponent rods lasted from day 30 to day 70. The release period of sterilized rods extended from day 23 to day 57. When compared to the original component fibers, the drug release from MC rods commenced earlier. The initial shear strength of MC rods was 135 MPa and decreased to 105 MPa during four weeks of immersion in phosphate buffer solution. Accordingly, heat pressing has a positive effect on drug release. After four weeks in hydrolysis, no disintegration was observed.

  4. Biomolecular Interaction Assay

    SciTech Connect

    Bruckner-Lea, Cindy J.; Brown, L; Holman, David A.; Olson, Lydia; Grate, Jay W.

    2000-12-29

    Understanding the binding interactions of complexes of multiple proteins is an important area of medical research since many biological signaling pathways involve multiple protein complexes. A number of sensor technologies have been adapted to monitoring biomolecular interactions. Acoustic wave devices such as flexural plate wave devices, surface transverse waves, and quartz crystal microbalances detect the mass increase observed upon binding of a solution biomolecule to a surface bound biomolecule. However, these devices will also respond to changes in viscosity, temperature, liquid density, and viscoelastic effects, which may confound the interpretation of observed signals. Nonspecific binding is indistinguishable from specific binding. Several techniques for refractive index sensing, such as planar wave guides and surface plasmon resonance (SPR), can also be used to observe biomolecular interactions localized at a surface. Again, nonspecific binding is indistinguishable from specific binding. In addition, the derivatized surface must be very thin and uniform to obtain adequate sensitivity and reproducibility, and the technique is not suited for monitoring large multiple protein complexes since the measurement sensitivity decreases rapidly with distance from the sensor surface. All of these techniques use planar surfaces that are difficult to prepare and characterize, and must be prepared fresh for each assay.

  5. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

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

  6. Dendritic Release of Neurotransmitters.

    PubMed

    Ludwig, Mike; Apps, David; Menzies, John; Patel, Jyoti C; Rice, Margaret E

    2016-12-06

    Release of neuroactive substances by exocytosis from dendrites is surprisingly widespread and is not confined to a particular class of transmitters: it occurs in multiple brain regions, and includes a range of neuropeptides, classical neurotransmitters, and signaling molecules, such as nitric oxide, carbon monoxide, ATP, and arachidonic acid. This review is focused on hypothalamic neuroendocrine cells that release vasopressin and oxytocin and midbrain neurons that release dopamine. For these two model systems, the stimuli, mechanisms, and physiological functions of dendritic release have been explored in greater detail than is yet available for other neurons and neuroactive substances. © 2017 American Physiological Society. Compr Physiol 7:235-252, 2017.

  7. Release the Body, Release the Mind.

    ERIC Educational Resources Information Center

    Stoner, Martha Goff

    1998-01-01

    A college English teacher describes the anxiety and resentment of students during in-class writing assignments and the successful classroom use of meditation and body movement. Movement seemed to relax the students, change their attitudes, and release their creative impulses to write. Implications related to the body-mind connection are pondered.…

  8. Detection of calcium release via ryanodine receptors.

    PubMed

    Eu, Jerry P; Meissner, Gerhard

    2012-01-01

    The ryanodine receptor ion channels (RyRs) release Ca(2+) from the endo/sarcoplasmic reticulum in a variety of nonvertebrate and vertebrate species including flies, crustaceans, birds, fish, and amphibians. They are most abundant in skeletal and cardiac muscle, where in response to an action potential, the release of Ca(2+) ions from the sarcoplasmic reticulum through the RyRs into the cytoplasm leads to muscle contraction (i.e., excitation-contraction coupling). Here, we describe how to determine their cellular location using isoform-specific antibodies, their protein levels using an in vitro ((3)H)ryanodine-binding assay, and their cellular release of Ca(2+) using RyR-specific channel agonists and inhibitors.

  9. Cell migration and invasion assays.

    PubMed

    Moutasim, Karwan A; Nystrom, Maria L; Thomas, Gareth J

    2011-01-01

    A number of in vitro assays have been developed to study tumor cell motility. Historically, assays have been mainly monocellular, where carcinoma cells are studied in isolation. Scratch assays can be used to study the collective and directional movement of populations of cells, whereas two chamber assays lend themselves to the analysis of chemotactic/haptotactic migration and cell invasion. However, an inherent disadvantage of these assays is that they grossly oversimplify the complex process of invasion, lacking the tumor structural architecture and stromal components. Organotypic assays, where tumor cells are grown at an air/liquid interface on gels populated with stromal cells, are a more physiologically relevant method for studying 3-dimensional tumor invasion.

  10. Cardiac troponins and high-sensitivity cardiac troponin assays.

    PubMed

    Conrad, Michael J; Jarolim, Petr

    2014-03-01

    Measurement of circulating cardiac troponins I and T has become integral to the diagnosis of myocardial infarction. This article discusses the structure and function of the troponin complex and the release of cardiac troponin molecules from the injured cardiomyocyte into the circulation. An overview of current cardiac troponin assays and their classification according to sensitivity is presented. The diagnostic criteria, role, and usefulness of cardiac troponin for myocardial infarction are discussed. In addition, several examples are given of the usefulness of high-sensitivity cardiac troponin assays for short-term and long-term prediction of adverse events.

  11. From Antenna to Assay

    PubMed Central

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

    2009-01-01

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

  12. 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.

  13. Rad-Release

    SciTech Connect

    2011-01-01

    The R&D 100 Award winning Rad-Release Chemical Decontamination Technology is a highly effective (up to 99% removal rate), affordable, patented chemical-foam-clay decontamination process tailored to specific radiological and metal contaminants, which is applicable to a wide variety of substrates. For more information about this project, visit http://www.inl.gov/rd100/2011/rad-release/

  14. Rad-Release

    ScienceCinema

    None

    2016-07-12

    The R&D 100 Award winning Rad-Release Chemical Decontamination Technology is a highly effective (up to 99% removal rate), affordable, patented chemical-foam-clay decontamination process tailored to specific radiological and metal contaminants, which is applicable to a wide variety of substrates. For more information about this project, visit http://www.inl.gov/rd100/2011/rad-release/

  15. Controlled release of biologically active silver from nanosilver surfaces.

    PubMed

    Liu, Jingyu; Sonshine, David A; Shervani, Saira; Hurt, Robert H

    2010-11-23

    Major pathways in the antibacterial activity and eukaryotic toxicity of nanosilver involve the silver cation and its soluble complexes, which are well established thiol toxicants. Through these pathways, nanosilver behaves in analogy to a drug delivery system, in which the particle contains a concentrated inventory of an active species, the ion, which is transported to and released near biological target sites. Although the importance of silver ion in the biological response to nanosilver is widely recognized, the drug delivery paradigm has not been well developed for this system, and there is significant potential to improve nanosilver technologies through controlled release formulations. This article applies elements of the drug delivery paradigm to nanosilver dissolution and presents a systematic study of chemical concepts for controlled release. After presenting thermodynamic calculations of silver species partitioning in biological media, the rates of oxidative silver dissolution are measured for nanoparticles and macroscopic foils and used to derive unified area-based release kinetics. A variety of competing chemical approaches are demonstrated for controlling the ion release rate over 4 orders of magnitude. Release can be systematically slowed by thiol and citrate ligand binding, formation of sulfidic coatings, or the scavenging of peroxy-intermediates. Release can be accelerated by preoxidation or particle size reduction, while polymer coatings with complexation sites alter the release profile by storing and releasing inventories of surface-bound silver. Finally, the ability to tune biological activity is demonstrated through a bacterial inhibition zone assay carried out on selected formulations of controlled release nanosilver.

  16. Normal anti-Klebsiella lymphocytotoxicity in ankylosing spondylitis

    SciTech Connect

    Kinsella, T.D.; Fritzler, M.J.; Lewkonia, R.M.

    1986-03-01

    We compared in vitro lymphocytotoxicity (LCT) of peripheral blood lymphocytes (PBL), obtained from patients with ankylosing spondylitis (AS) and normal controls (NC). Assays were performed with antibacterial antisera prepared from AS- and NC-derived Klebsiella and coliforms Escherichia coli. LCT assessed by eosin staining was not significantly different in PBL of 12 AS patients and 28 controls when reacted with 3 Klebsiella and 1 E coli antisera. LCT assessed by /sup 51/Cr release was not significantly different for PBL of 20 age- and sex-matched pairs of AS patients and NC when reacted with 3 Klebsiella and 1 E coli antisera. Similarly, LCT-/sup 51/Cr of PBL of 15 matched AS and NC pairs was not significantly different for anti-K21, a serotype putatively implicated in Klebsiella-HLA-B27 antigenic cross-reactivity. Our results do not support the notion of molecular mimicry between Klebsiella and B27 in the pathogenesis of primary AS.

  17. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    PubMed Central

    Langel, Ülo

    2016-01-01

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies. PMID:27101307

  18. Solid waste transuranic storage and assay facility indoor air sampling

    SciTech Connect

    Pingel, L.A., Westinghouse Hanford

    1996-08-20

    The purpose of the study is to collect and analyze samples of the indoor air at the Transuranic Storage and Assay Facility (TRUSAF), Westinghouse Hanford. A modified US EPA TO-14 methodology, using gas chromatography/mass spectrography, may be used for the collection and analysis of the samples. The information obtained will be used to estimate the total release of volatile organic compounds from TRUSAF to determine the need for air emmission permits.

  19. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  20. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

  1. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  2. 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).

  3. Controlled-release microchips.

    PubMed

    Sharma, Sadhana; Nijdam, A Jasper; Sinha, Piyush M; Walczak, Robbie J; Liu, Xuewu; Cheng, Mark M-C; Ferrari, Mauro

    2006-05-01

    Efficient drug delivery remains an important challenge in medicine: continuous release of therapeutic agents over extended time periods in accordance with a predetermined temporal profile; local delivery at a constant rate to the tumour microenvironment to overcome much of the systemic toxicity and to improve antitumour efficacy; improved ease of administration, and increasing patient compliance required are some of the unmet needs of the present drug delivery technology. Microfabrication technology has enabled the development of novel controlled-release microchips with capabilities not present in the current treatment modalities. In this review, the current status and future prospects of different types of controlled-release microchips are summarised and analysed with reference to microneedle-based microchips, as well as providing an in-depth focus on microreservoir-based and nanoporous microchips.

  4. RAVEN Beta Release

    SciTech Connect

    Rabiti, Cristian; Alfonsi, Andrea; Cogliati, Joshua Joseph; Mandelli, Diego; Kinoshita, Robert Arthur; Wang, Congjian; Maljovec, Daniel Patrick; Talbot, Paul William

    2016-02-01

    This documents the release of the Risk Analysis Virtual Environment (RAVEN) code. A description of the RAVEN code is provided, and discussion of the release process for the M2LW-16IN0704045 milestone. The RAVEN code is a generic software framework to perform parametric and probabilistic analysis based on the response of complex system codes. RAVEN is capable of investigating the system response as well as the input space using Monte Carlo, Grid, or Latin Hyper Cube sampling schemes, but its strength is focused toward system feature discovery, such as limit surfaces, separating regions of the input space leading to system failure, using dynamic supervised learning techniques. RAVEN has now increased in maturity enough for the Beta 1.0 release.

  5. Altitude release mechanism

    DOEpatents

    Kulhanek, Frank C.

    1977-01-01

    An altitude release mechanism for releasing a radiosonde or other measuring instrument from a balloon carrying it up into the atmosphere includes a bottle partially filled with water, a tube sealed into the bottle having one end submerged in the water in the bottle and the free end extending above the top of the bottle and a strip of water-disintegrable paper held within the free end of the tube linking the balloon to the remainder of the package. As the balloon ascends, the lowered atmospheric air pressure causes the air in the bottle to expand, forcing the water in the bottle up the tubing to wet and disintegrate the paper, releasing the package from the balloon.

  6. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders.

    PubMed

    Mumford, Andrew D; Frelinger, Andrew L; Gachet, Christian; Gresele, Paolo; Noris, Patrizia; Harrison, Paul; Mezzano, Diego

    2015-07-01

    Measurement of platelet granule release to detect inherited platelet secretion disorders (IPSDs) is essential for the evaluation of patients with abnormal bleeding and is necessary to distinguish which granule sub-types are affected and whether there is abnormal granule bio-synthesis or secretion. The radioactive serotonin incorporation and release assay, described before 1970, is still considered the "gold standard" test to assess platelet δ-granule release, although is unsuitable for clinical diagnostic laboratories. Luciferin-based assays, such as lumiaggregometry, are the most widely performed alternatives, although these methods do not distinguish defects in δ-granule biosynthesis from defects in secretion. Platelet α-granule release is commonly evaluated using flow cytometry by measuring surface exposure of P-selectin after platelet activation. However, this assay has poor sensitivity for some α-granule disorders. Only few studies have been published with more recently developed assays and no critical reviews on these methods are available. In this review, we describe the rationale for developing robust and accurate laboratory tests of platelet granule release and describe the characteristics of the currently available tests. We identify an unmet need for further systematic evaluation of new assays and for standardisation of methodologies for clinical diagnostic laboratories.

  7. Method for quantifying alginate and determining release from a food vehicle in gastrointestinal digesta.

    PubMed

    Houghton, David; Wilcox, Matthew D; Brownlee, Iain A; Chater, Peter; Seal, Chris J; Pearson, Jeffrey P

    2014-05-15

    To assess the efficacy of alginate as a modifier of enzyme activity, a suitable method to quantify its release must be developed. This paper develops and assesses the ability of the Periodic Acid Schiffs (PAS) assay to quantify alginate, and its release from bread during digestion in a model gut. Control and alginate enriched (4% w/w wet dough) bread were used. A model gut replicating the mouth, stomach and small intestines was used. Standard curves were created for alginate in deionised H2O and model gut solutions using a modified PAS to remove interference. The PAS assay quantified alginate with excellent linearity (R(2)=0.99), and optical density range (0.02-0.5). There was a significant difference in alginate release at 180 min compared to 0 and 60 min. The data indicate the modified PAS assay is a simple method for quantifying alginate release and release rate from alginate enriched products.

  8. Monitoring ibuprofen enantiomers released from polymeric systems.

    PubMed

    Simó, C; Gallardo, A; Parejo, C; San Román, J; Barbas, C; Cifuentes, A

    2002-07-01

    Two methacrylic derivatives of ibuprofen (N-[4-[2-(4-isobutylphenyl)propionyloxy]phenyl] methacrylamide (MAI) and 2-[(4-isobutylphenyl)propionyloxy]ethyl methacrylate (MEI)) were used together with 2-hydroxyethyl methacrylate (HEMA) to synthesize four polymeric materials: two hydrophobic homopolymers, PMAI and PMEI, and two hydrophilic copolymers containing 70% (w/w) HEMA, MAI-HEMA 30 and MEI-HEMA 30. The enantiomeric determination of R- and S-IBU released from these four systems has been carried out by capillary electrophoresis. Release of R- and S-IBU was monitored during in vitro assays done at 37 degrees C at pH 7.4 and 10 in buffered solutions and rat plasma. There is a hydrolytical activation in plasma and at pH 10 compared to pH 7.4; moreover, the release rate from the copolymers is much higher than from the homopolymers as a consequence of the greater hydrophilic character. A slight excess of the S-enantiomer of IBU is observed in all the experiments, being more relevant at higher release rates, i.e. copolymers at pH 10.

  9. A fluorogenic assay for methylglyoxal.

    PubMed

    Shaheen, Fozia; Shmygol, Anatoly; Rabbani, Naila; Thornalley, Paul J

    2014-04-01

    MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

  10. Detection of platelet alloimmunity with a platelet-associated IgG assay

    SciTech Connect

    Myers, T.J.; Kim, B.K.; Steiner, M.; Bishop, J.; Baldini, M.G.

    1981-06-01

    A quantitative immunofluorescence PA-IgG assay was used to detect alloimmunity to platelets. The assay identified serum alloantibodies in 10 out of 14 multitransfused patients and for two of three infants with neonatal thrombocytopenia. The correct separation of all multitransfused patients into alloimmune and nonalloimmune groups by the PA-IgG assay was substantiated with chromium-51-labeled platelet survival studies. The allogeneic nature of the serum antibodies was demonstrated by progressive absorption of the antibody with increasing numbers of allogeneic platelets but not with autologous platelets. The sensitivity of the PA-IgG assay for detection of serum alloantibodies was superior to that of platelet aggregation, platelet serotonin release, and lymphocytotoxicity testing. In dilution experiments with alloimmune serum, elevated levels of serum PA-IgG could still be detected on donor platelets when platelet aggregation and serotonin release tests became negative. Platelet survival studies with selected platelets performed in the 10 alloimmunized, multitransfused patients confirmed the results of the PA-IgG assays, predicting alloimmunity to the donor platelets. In contrast, platelet aggregation, platelet serotonin release, and lymphocytotoxicity testing indicated alloimmunity for 50% or less of the patients. Reduced platelet survival times were also seen with HLA A- and HLA B-matched donor platelets when donor-recipient incompatibility was demonstrated by the PA-IgG assay. Thus the PA-IgG assay provides a sensitive method to detect serum platelet alloantibodies and may offer a technique in platelet crossmatching.

  11. Decreased NK killing in patients with multiple sclerosis: An analysis on the level of the single effector cell in peripheral blood and cerebrospinal fluid in relation to the activity of the disease

    PubMed Central

    Merrill, Jean; Jondal, M.; Seeley, Janet; Ullberg, M.; Sidén, Å.

    1982-01-01

    Natural killer cell activity has earlier been shown to be depressed in patients with multiple sclerosis (MS) (Benczur et al., 1980). In the present study, this defect was more clearly characterized in different stages of the disease. By using a single-cell cytotoxicity assay in agarose (Grimm & Bonavida, 1979), in combination with the conventional 51Cr-release, the number of target-binding cells (TBCs) and the fraction of active killer cells therein could be compared with the radioisotope release in the different patient groups. It was found that patients with active and chronic MS showed lower natural killer (NK) activity in the 51Cr-release assay as compared with age and sex-matched controls, in contrast to stable MS patients who were comparable with their control group. The single cell cytotoxicity assay demonstrated that acute MS patients had a decreased number of TBCs in peripheral blood and that they also had a decreased percentage of active NK cells in their TBC fractions. Patients with chronic MS were normal in the single-cell cytotoxicity assay. When cells present in CSF were analysed in acute and chronic MS, few cells were found with target binding capacity and only in two instances out of 13 could any cytotoxicity at all be detected. Patients with other neurological diseases (OND) were found to have detectable NK activity in CSF in six cases out of ten in the single-cell assay. OND patients as a group also had higher peripheral NK activity in the 51Cr-release assay as compared with the control group. When peripheral and CSF cells from MS patients and OND patients were treated with interferon, no increase in TBCs or fraction of killer cells in TBCs was found. In the 51Cr-release assay, comparable increases in cytotoxicity were found in all groups. One possible explanation for the stage-related NK suppression seen in the present investigation may be a decreased interferon production combined with immune-complex induced, macrophage-produced prostaglandins

  12. Barcoded microchips for biomolecular assays.

    PubMed

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

    2015-01-20

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

  13. Releasable Asbestos Field Sampler

    EPA Science Inventory

    Asbestos aerosolization (or releasability) is the potential for fibrous asbestos structures that are present in a material or on a solid surface to become airborne when the source is disturbed by human activities or natural forces. In turn, the magnitude of the airborne concentra...

  14. Release of OLe peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OLe is a high oleic Spanish-type peanut that has excellent yield and enhanced Sclerotinia blight and pod rot resistance when compared to other high oleic Spanish cultivars. The purpose for releasing OLe is to provide peanut producers with a true Spanish peanut that is high oleic and has enhanced yi...

  15. Hydraulic release oil tool

    SciTech Connect

    Mims, M.G.; Mueller, M.D.; Ehlinger, J.C.

    1992-03-11

    This patent describes a hydraulic release tool. It comprises a setting assembly; a coupling member for coupling to drill string or petroleum production components, the coupling member being a plurality of sockets for receiving the dogs in the extended position and attaching the coupling member the setting assembly; whereby the setting assembly couples to the coupling member by engagement of the dogs in the sockets of releases from and disengages the coupling member in movement of the piston from its setting to its reposition in response to a pressure in the body in exceeding the predetermined pressure; and a relief port from outside the body into its bore and means to prevent communication between the relief port and the bore of the body axially of the piston when the piston is in the setting position and to establish such communication upon movement of the piston from the setting position to the release position and reduce the pressure in the body bore axially of the piston, whereby the reduction of the pressure signals that the tool has released the coupling member.

  16. Data Release Summary Statement

    Atmospheric Science Data Center

    2016-11-14

    ... 3 CALIOP Level 1 data are released with a product maturity classification of Validated Stage1 , indicating that initial validation of ... users for evaluation and to provide feedback to the CALIOP algorithm development team. Users should carefully read the section of the Data ...

  17. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    PubMed

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  18. Antioxidant assays for plant and food components.

    PubMed

    Moon, Joon-Kwan; Shibamoto, Takayuki

    2009-03-11

    Recently, research on natural antioxidants has become increasingly active in various fields. Accordingly, numerous articles on natural antioxidants, including polyphenols, flavonoids, vitamins, and volatile chemicals, have been published. Assays developed to evaluate the antioxidant activity of plants and food constituents vary. Therefore, to investigate the antioxidant activity of chemical(s), choosing an adequate assay based on the chemical(s) of interest is critical. There are two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidations, including the thiobarbituric acid assay (TBA), malonaldehyde/high-performance liquid chromatography (MA/HPLC) assay, malonaldehyde/gas chromatography (MA/GC) assay, beta-carotene bleaching assay, and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/carboxylic acid (ACA) assay. In this review, assays used recently were selected for extended discussion, including discussion of the mechanisms underlying each assay and its application to various plants and foods.

  19. A homogeneous biochemiluminescent assay for detection of influenza

    NASA Astrophysics Data System (ADS)

    Hui, Kwok Min; Li, Xiao Jing; Pan, Lu; Li, X. J.

    2015-05-01

    Current methods of rapid detection of influenza are based on detection of the nucleic acids or antigens of influenza viruses. Since influenza viruses constantly mutate leading to appearance of new strains or variants of viruses, these detection methods are susceptible to genetic changes in influenza viruses. Type A and B influenza viruses contain neuraminidase, an essential enzyme for virus replication which enables progeny influenza viruses leave the host cells to infect new cells. Here we describe an assay method, the homogeneous biochemiluminescent assay (HBA), for rapid detection of influenza by detecting viral neuraminidase activity. The assay mimics the light production process of a firefly: a viral neuraminidase specific substrate containing a luciferin moiety is cleaved in the presence of influenza virus to release luciferin, which becomes a substrate to firefly luciferase in a light production system. All reagents can be formulated in a single reaction mix so that the assay involves only one manual step, i.e., sample addition. Presence of Type A or B influenza virus in the sample leads to production of strong, stable and easily detectable light signal, which lasts for hours. Thus, this influenza virus assay is suitable for use in point-of-care settings.

  20. Release Fraction Evaluation

    SciTech Connect

    Bamberger, Judith A.; Glissmeyer, John A.

    2004-01-01

    This document presents results of experiments conducted to measure release fractions during certain tank retrieval processes. The tests were performed in a 1/4 scale model of a waste storage tank. The retrieval processes simulated were: (1) Discharging liquid or slurry from the mouth of a vertically oriented two-in. Schedule 40 pipe. The discharging material was in free-fall from the mouth of the pipe near the top of the tank into a liquid or slurry pool at the bottom of the tank. (2) The jet from a 9/16-in.-diameter nozzle transferring liquid or slurry waste from one side of the tank to the other. The discharging liquid was aimed at the opposite side of the tank from the nozzle and either impacted the tank wall or fell into a liquid or slurry pool in the bottom of the tank. (3) A high pressure fan jet of liquid striking a steel plate or simulated waste from a stand-off distance of a few inches. For each process, a water-soluble fluorescent dye was added to the liquid fraction as a tracer. Kaolin clay was used to represent the solids. The tank was covered and there was no forced ventilation in the tank during the tests. Six air samples were collected during each test. The air samples were collected at fixed positions in the tank. The air sample filters were dried and weighed to determine the solids collection. The fluorescent dye was then leached from each filter and quantified with a fluorometer to determine the collection of liquid. Samples of the slurry and liquid simulants were also collected to determine the quantities of simulant used in each test. To calculate the release fraction, the quantity collected on each air sample was adjusted for the fraction of the tank volume sampled and divided by the quantity of material exposed in the simulation. The method was not as sensitive for the solids content as it was for the liquid content, but in those instances where a solids release fraction was determined, it was in relatively good agreement with that of the

  1. 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.

  2. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  3. 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.

  4. 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.

  5. 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.

  6. Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

    PubMed Central

    Farris, M. Heath; Ford, Kara A.; Doyle, Richard C.

    2016-01-01

    Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest. PMID:27166738

  7. Releasable locking mechanisms

    NASA Technical Reports Server (NTRS)

    Ahmed, Rafiq (Inventor); Wingate, Robert J. (Inventor)

    2005-01-01

    In the aerospace field spacecraft components are held together by separation systems until a specific time when they must be separated or deployed. Customarily a threaded joining bolt engages one of the components to be joined, and a threaded nut is placed on that bolt against the other component so they can be drawn together by a releasable locking assembly. The releasable locking assembly herein includes a plunger having one end coupled to one end of a plunger bolt. The other end is flanged to abut and compress a coil spring when the plunger is advanced toward the interface plane between the two components. When the plunger is so advanced toward the interface plane, the end of the plunger bolt can be connected to the joining bolt. Thus during retraction the joining bolt is drawn to one side of the interface plane by the force of the expanding spring.

  8. Cryogenic hydrogen release research.

    SciTech Connect

    LaFleur, Angela Christine

    2015-12-01

    The objective of this project was to devolop a plan for modifying the Turbulent Combustion Laboratory (TCL) with the necessary infrastructure to produce a cold (near liquid temperature) hydrogen jet. The necessary infrastructure has been specified and laboratory modifications are currently underway. Once complete, experiments from this platform will be used to develop and validate models that inform codes and standards which specify protection criteria for unintended releases from liquid hydrogen storage, transport, and delivery infrastructure.

  9. Sudden releases of gases

    NASA Astrophysics Data System (ADS)

    Chaloupecká, Hana; Jaňour, Zbyněk; Jurčáková, Klára; Kukačka, Libor; Nosek, Štěpán

    2014-03-01

    Conurbations all over the world have enlarged for numberless years. The accidental or intentional releases of gases become more frequent. Therefore, these crises situations have to be studied. The aim of this paper is to describe experiments examining these processes that were carried out in the laboratory of Environmental Aerodynamics of the Institute of Thermomechanics AS CR in Nový Knín. Results show huge puff variability from replica to replica.

  10. Slow-release fertilizer

    NASA Technical Reports Server (NTRS)

    Ming, Douglas W. (Inventor); Golden, D. C. (Inventor)

    1992-01-01

    A synthetic apatite containing agronutrients and a method for making the apatite are disclosed. The apatite comprises crystalline calcium phosphate having agronutrients dispersed in the crystalline structure. The agronutrients can comprise potassium, magnesium, sulfur, iron, manganese, molybdenum, chlorine, boron, copper and zinc in amounts suited for plant growth. The apatite can optionally comprise a carbonate and/or silicon solubility control agent. The agronutrients are released slowly as the apatite dissolves.

  11. Slow-release fertilizer

    NASA Technical Reports Server (NTRS)

    Ming, Douglas W. (Inventor); Golden, Dadigamuwage C. (Inventor)

    1995-01-01

    A synthetic apatite containing agronutrients and a method for making the apatite are disclosed. The apatite comprises crystalline calcium phosphate having agronutrients dispersed in the crystalline structure. The agronutrients can comprise potassium, magnesium, sulfur, iron, manganese, molybdenum, chlorine, boron, copper and zinc in amounts suited for plant growth. The apatite can optionally comprise a carbonate and/or silicon solubility control agent. The agronutrients are released slowly as the apatite dissolves.

  12. EIA new releases

    SciTech Connect

    Not Available

    1994-12-01

    This report was prepared by the Energy Information Administration. It contains news releases on items of interest to the petroleum, coal, nuclear, electric and alternate fuels industries ranging from economic outlooks to environmental concerns. There is also a listing of reports by industry and an energy education resource listing containing sources for free or low-cost energy-related educational materials for educators and primary and secondary students.

  13. Slow-release fertilizer

    NASA Astrophysics Data System (ADS)

    Ming, Douglas W.; Golden, D. C.

    1992-10-01

    A synthetic apatite containing agronutrients and a method for making the apatite are disclosed. The apatite comprises crystalline calcium phosphate having agronutrients dispersed in the crystalline structure. The agronutrients can comprise potassium, magnesium, sulfur, iron, manganese, molybdenum, chlorine, boron, copper and zinc in amounts suited for plant growth. The apatite can optionally comprise a carbonate and/or silicon solubility control agent. The agronutrients are released slowly as the apatite dissolves.

  14. Clinton releases oceans report

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    U.S. President Bill Clinton is trying to beat the clock on the January 20 close of his administration by maintaining a flurry of activity on resource and conservation issues.During a December 4 speech in Washington, D.C., he released a broad-ranging report by the Presidents Panel on Ocean Exploration, entitled “Discovering Earth's Final Frontier: A U.S. Strategy for Ocean Exploration.”

  15. Contact: Releasing the news

    NASA Astrophysics Data System (ADS)

    Pinotti, Roberto

    The problem of mass behavior after man's future contacts with other intelligences in the universe is not only a challenge for social scientists and political leaders all over the world, but also a cultural time bomb as well. In fact, since the impact of CETI (Contact with Extraterrestrial Intelligence) on human civilization, with its different cultures, might cause a serious socio-anthropological shock, a common and predetermined worldwide strategy is necessary in releasing the news after the contact, in order to keep possible manifestations of fear, panic and hysteria under control. An analysis of past studies in this field and of parallel historical situations as analogs suggests a definite "authority crisis" in the public as a direct consequence of an unexpected release of the news, involving a devastating "chain reaction" process (from both the psychological and sociological viewpoints) of anomie and maybe the collapse of today's society. The only way to prevent all this is to prepare the world's public opinion concerning contact before releasing the news, and to develop a long-term strategy through the combined efforts of scientists, political leaders, intelligence agencies and the mass media, in order to create the cultural conditions in which a confrontation with ETI won't affect mankind in a traumatic way. Definite roles and tasks in this multi-level model are suggested.

  16. Assay strategies and methods for phospholipases

    SciTech Connect

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is {approximately} as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous.

  17. Conformationally selective biophysical assay for influenza vaccine potency determination.

    PubMed

    Wen, Yingxia; Han, Liqun; Palladino, Giuseppe; Ferrari, Annette; Xie, Yuhong; Carfi, Andrea; Dormitzer, Philip R; Settembre, Ethan C

    2015-10-05

    Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as reduced pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines.

  18. 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...

  19. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  20. 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...

  1. 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...

  2. Triggered Release from Polymer Capsules

    SciTech Connect

    Esser-Kahn, Aaron P.; Odom, Susan A.; Sottos, Nancy R.; White, Scott R.; Moore, Jeffrey S.

    2011-07-06

    Stimuli-responsive capsules are of interest in drug delivery, fragrance release, food preservation, and self-healing materials. Many methods are used to trigger the release of encapsulated contents. Here we highlight mechanisms for the controlled release of encapsulated cargo that utilize chemical reactions occurring in solid polymeric shell walls. Triggering mechanisms responsible for covalent bond cleavage that result in the release of capsule contents include chemical, biological, light, thermal, magnetic, and electrical stimuli. We present methods for encapsulation and release, triggering methods, and mechanisms and conclude with our opinions on interesting obstacles for chemically induced activation with relevance for controlled release.

  3. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  4. Recommended for release on recognizance: factors affecting pretrial release recommendations.

    PubMed

    Petee, T A

    1994-06-01

    Researchers have acknowledged the influence of pretrial release agencies in judicial decision making regarding bail; however, few researchers have focused on the process used by the pretrial release agencies to make bail-bond recommendations. In this study I sought to establish which factors were most salient in making the decision to recommend a defendant for release on recognizance. I found that both officially sanctioned release criteria and "extralegal" variables were predictive of this decision.

  5. Sustained-release, extended-release, and other time-release formulations in neuropsychiatry.

    PubMed

    Andrade, Chittaranjan

    2015-08-01

    Pills and capsules may release their contents within minutes of ingestion; these are immediate-release formulations. Pills and capsules may also release their contents after a time lag, or a little at a time, or in some other predetermined way; these are time-release formulations. Many drugs in psychiatry have been time-release formulated to reduce their local adverse effects in the gastrointestinal tract, to reduce adverse effects associated with peak blood levels, or to artificially extend their half-life. Time-release formulations are associated with the added advantages of convenience of dosing, improved compliance, and less fluctuation in blood levels across the course of the day. A disadvantage of time-release formulations is that they may be incompletely absorbed; this is a serious issue in patients with acute or chronic intestinal hurry disorders, such as gastroenteritis or irritable bowel syndrome. Time-release formulations may also be more expensive than immediate-release formulations.

  6. Severe microvascular injury induced by lysosomal releasates of human polymorphonuclear leukocytes. Increase in vasopermeability, hemorrhage, and microthrombosis due to degradation of subendothelial and perivascular matrices.

    PubMed Central

    Movat, H. Z.; Wasi, S.

    1985-01-01

    The purpose of this study was to assess the nature of the lesions in the microcirculation of the dermis of rabbits induced with lysosomal releasates of human polymorphonuclear leukocytes (PMNs). No attempt was made in the studies presented in this publication to deal with the offending agent in the releasate. Four parameters of microvascular injury were quantitated: increase in vascular permeability with 125I-labeled serum albumin, hemorrhage with 59Fe-labeled erythrocytes, accumulation (aggregation) of platelets with 111In-labeled platelets. In one experiment accumulation of 51Cr-PMNs was investigated. The lysosomal releasate induced a rapid increase in vasopermeability, but both hemorrhage and exudate formation peaked 1 hour after intradermal injection. Platelet accumulation was also demonstrable in these lesions, and microthrombosis was a very prominent feature. The microvascular injury, including microthrombosis, could be elicited also in animals rendered leukopenic with nitrogen mustard. Simultaneous injection of prostaglandin E2 with the releasate enhanced the microvascular injury. The morphologic changes in the microcirculation of the rabbit's dermis were assessed in lesions 5 minutes to 5 hours old. Several changes were encountered, primarily in the wall of venules and small veins and to a lesser degree in small arteries and capillaries. Ultrastructurally very early lesions (up to 15 minutes) had gaps or spaces in the endothelium, resembling those induced by mediators such as histamine or bradykinin. Older lesions were different, quite characteristic, and represent the hallmark of these lesions. Lysis and disappearance of vascular basement membrane, of perivascular collagen, and of the internal elastic lamina were a frequent finding, best demonstrable when microthrombi did not abut on vessel walls. Cellular components of vessels (endothelium, pericytes, smooth muscle) showed fragmentation, leading to complete disappearance of cellular elements. These

  7. 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.

  8. 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.

  9. 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.

  10. [Visible spectrophotometric assay of ranitidine].

    PubMed

    Apostu, M; Dorneanu, V; Bibire, Nela

    2003-01-01

    Ranitidine, belonging to H2-antagonist group, is a compound containing a furanic moiety and is used in peptic ulcer therapy. This paper debates the possibility of developing a new visible spectrophotometric assessment by using the reaction between ranitidine and eosine. We carried out our determinations at 505 nm, where the absorbency of ranitidine-eosine complex is maximal, and we have established the optimal reaction conditions. This method was successfully applied for ranitidine assay from pharmaceutical dosage forms.

  11. 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.

  12. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  13. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, (/sup 14/C)-NaHCO/sub 3/, and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30/sup 0/. The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number.

  14. Optical fiber hybridization assay fluorosensor

    NASA Astrophysics Data System (ADS)

    Pilevar, Saeed; Davis, Christopher C.; Hodzic, Vildana; Portugal, Frank

    1999-04-01

    The present work describes an all-fiber hybridization assay sensor that relies on the evanescent field excitation of fluorescence from surface-bound fluorophores. The evanescent field is made accessible through the use of a long adiabatically tapered single-mode fiber probe. A semiconductor laser operating at 785 nm wavelength is used in a pulsed mode to excite fluorescence in the tapered region of a fiber probe using the near-infrared fluorophore IRD 41. We have carried out real-time hybridization tests for IRD 41-labeled oligonucleotide at various probe concentrations binding to complementary oligonucleotide cross-linked to the tapered fiber surface. Short oligonucleotides (20-mer) bound to the fiber surface have been used to detect near-infrared dye labeled complementary sequences at sub-nanomolar levels. Sandwich assays with total RNA were conducted to examine the capability of the biosensor for detecting bacterial cells using rRNA as the target. The results indicate that this fluorosensor is capable of detecting H. pylori in a sandwich assay at picomolar concentrations.

  15. Effect of Quaternary Ammonium Carboxymethylchitosan on Release Rate In-vitro of Aspirin Sustained-release Matrix Tablets

    PubMed Central

    Meng, Lingbin; Teng, Zhongqiu; Zheng, Nannan; Meng, Weiwei; Dai, Rongji; Deng, Yulin

    2013-01-01

    The aim of this study was to develop a derivative of chitosan as pharmaceutical excipient used in sustained-release matrix tablets of poorly soluble drugs. A water-soluble quaternary ammonium carboxymethylchitosan was synthesized by a two-step reaction with carboxymethylchitosan (CMCTS), decylalkyl dimethyl ammonium and epichlorohydrin. The elemental analysis showed that the target product with 10.27% of the maximum grafting degree was obtained. To assess the preliminary safety of this biopolymer, cell toxicity assay was employed. In order to further investigate quaternary ammonium carboxymethylchitosan application as pharmaceutical excipient, aspirin was chosen as model drug. The effect of quaternary ammonium CMCTS on aspirin release rate from sustained-release matrix tablets was examined by in-vitro dissolution experiments. The results showed that this biopolymer had a great potential in increasing the dissolution of poorly soluble drug. With the addition of CMCTS-CEDA, the final cumulative release rate of drug rose up to 90%. After 12 h, at the grade of 10, 20 and 50 cps, the drug release rate increased from 58.1 to 90.7%, from 64.1 to 93.9%, from 69.3 to 96.1%, respectively. At the same time, aspirin release rate from sustainedrelease model was found to be related to the amount of quaternary ammonium CMCTS employed. With the increase of CMCTS-CEDA content, the accumulated release rate increased from 69.1% to 86.7%. The mechanism of aspirin release from sustained-release matrix tablets was also preliminary studied to be Fick diffusion. These data demonstrated that the chitosan derivative has positive effect on drug release from sustained-release matrix tablets. PMID:24250627

  16. Gas releases from salt

    SciTech Connect

    Ehgartner, B.; Neal, J.; Hinkebein, T.

    1998-06-01

    The occurrence of gas in salt mines and caverns has presented some serious problems to facility operators. Salt mines have long experienced sudden, usually unexpected expulsions of gas and salt from a production face, commonly known as outbursts. Outbursts can release over one million cubic feet of methane and fractured salt, and are responsible for the lives of numerous miners and explosions. Equipment, production time, and even entire mines have been lost due to outbursts. An outburst creates a cornucopian shaped hole that can reach heights of several hundred feet. The potential occurrence of outbursts must be factored into mine design and mining methods. In caverns, the occurrence of outbursts and steady infiltration of gas into stored product can effect the quality of the product, particularly over the long-term, and in some cases renders the product unusable as is or difficult to transport. Gas has also been known to collect in the roof traps of caverns resulting in safety and operational concerns. The intent of this paper is to summarize the existing knowledge on gas releases from salt. The compiled information can provide a better understanding of the phenomena and gain insight into the causative mechanisms that, once established, can help mitigate the variety of problems associated with gas releases from salt. Outbursts, as documented in mines, are discussed first. This is followed by a discussion of the relatively slow gas infiltration into stored crude oil, as observed and modeled in the caverns of the US Strategic Petroleum Reserve. A model that predicts outburst pressure kicks in caverns is also discussed.

  17. Hydrogen release behavior.

    SciTech Connect

    LaChance, Jeffrey L.; Dedrick, Daniel E.; Keller, Jay O.; Evans, Gregory Herbert; Houf, William G.; Winters, William Stanley, Jr.; Ruggles, A.; Zhang, J.

    2010-04-01

    The summary of this presentation is: (1) Barrier walls are used to reduce setbacks by factor of 2; (2) We found no ignition-timing vs. over-pressure sensitivities for jet flow obstructed by barrier walls; (3) Cryogenic vapor cloud model indicates hazard length scales exceed the room-temperature release; validation experiments are required to confirm; (4) Light-up maps developed for lean limit ignition; flammability factor model provides good indication of ignition probability; and (5) Auto-ignition is enhanced by blunt-body obstructions - increases gas temperature and promotes fuel/air mixing.

  18. Radioreceptor assay analysis of tamsulosin and terazosin pharmacokinetics

    PubMed Central

    Taguchi, Katsunari; Schäfers, Rafael F; Michel, Martin C

    1998-01-01

    Aims A radioreceptor assay has been developed for α1-adrenoceptor subtypes and applied to a pharmacokinetic analysis of tamsulosin and terazosin. Methods Young, male, healthy volunteers received 0.4 mg tamsulosin (as Omnic® modified release capsules) or 5 mg terazosin (as Flotrin® tablets) in a randomized, cross-over design. Before and after 1, 3, 5, 7, 10 and 23.5 h plasma was analyzed by radioreceptor assay using cloned human α1A-, α1B- and α1D-adrenoceptors and specific h.p.l.c. analysis. Results Following ingestion of tamsulosin median peak plasma levels of 16 ng ml−1 were reached after 5 h and declined to 2 ng ml−1 at 23.5 h. The time course in the radioreceptor assay was similar, and at most time points binding to α1A-adrenoceptors was significantly greater than to α1B- and α1D-adrenoceptors. Following ingestion of terazosin median peak plasma levels of 91 ng ml−1 were reached after 1 h and declined to 11 ng ml−1 at 23.5 h. In the radioreceptor assay binding also peaked at 1 h and declined thereafter, but even after 23.5 h considerable binding activity remained detectable at all three subtypes. At most time points binding to the α1A- and α1D-adrenoceptor was significantly greater than to the α1B-adrenoceptor. Conclusions We conclude that α1-adrenoceptor antagonist pharmacokinetics can be monitored by radioreceptor assays in a subtype-selective manner. Tamsulosin and terazosin exhibit subtype selective receptor binding ex vivo. The discordance between terazosin blood levels as determined by h.p.l.c. and radioreceptor assay at late time points indicates the possible involvement of metabolites in in vivo terazosin effects. PMID:9489594

  19. Implementation of a Portable HPGe for Field Contamination Assay.

    PubMed

    Hayes, Robert Bruce

    2016-06-01

    Using MCNP to construct a detector model based initially on x-ray images of a portable high purity germanium (HPGe) detector followed by normalizing covering material values to also agree with check source responses, a validation of the model was attained. By calibrating the detector parameters using large count spectra, rigorous reproducibility is attained for high activity measurements but does not prevent deviations from normality in error distributions at the very low count events where spectral peaks are not always identifiable. The resulting model was created to allow operational assay of contamination over large areal distributions that could not otherwise be measured, such as the exhaust shaft at the Waste Isolation Pilot Plant (WIPP). Results indicate that contamination levels of activity in the exhaust shaft can be assayed to within a factor of 2. Detection limits are evaluated to be well below the contamination levels, which would constitute a legal environmental release if unfiltered ventilation of the underground facility were used.

  20. Investigation on photorespiration with a sensitive C-assay.

    PubMed

    Zelitch, I

    1968-11-01

    A leaf disk assay for photorespiration has been developed based on the rate of release of recently fixed (14)CO(2) in light in a rapid stream of CO(2)-free air at 30 degrees to 35 degrees . In tobacco leaves (Havana Seed) photorespiration with this assay is 3 to 5 times greater than the (14)CO(2) output in the dark. In maize, photorespiration is only 2% of that in tobacco.The importance of open leaf stomata, rapid flow rates of CO(2)-free air, elevated temperatures, and oxygen in the atmosphere in order to obtain release into the air of a larger portion of the (14)CO(2) evolved within the tissue in the light was established in tobacco. Photorespiration, but not dark respiration, was inhibited by alpha-hydroxy-2-pyridinemethanesulfonic acid, an inhibitor of glycolate oxidase, and by 3-(4-chlorophenyl)-1,1-dimethylurea (CMU), an inhibitor of photosynthetic electron transport, under conditions which did not affect the stomata. These experiments show that the substrates of photorespiration and dark respiration differ and also provide additional support for the role of glycolate as a major substrate of photorespiration. It was also shown that at 35 degrees the quantity of (14)CO(2) released in the assay may represent only 33% of the gross (14)CO(2) evolved in the light, the remainder being recycled within the tissue.It was concluded that maize does not evolve appreciable quantities of CO(2) in the light and that this largely accounts for the greater efficiency of net photosynthesis exhibited by maize. Hence low rates of photorespiration may be expected to be correlated with a high rate of CO(2) uptake at the normal concentrations of CO(2) found in air and at higher light intensities.

  1. Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

    PubMed

    Jose, D Amilan; Elstner, Martin; Schiller, Alexander

    2013-10-18

    Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples.

  2. Posttransfusion Survival and Distribution of 51-CR Labeled Fresh and Liquid Preserved Syngeneic Mouse Red Blood Cells

    DTIC Science & Technology

    2007-11-02

    LABORATORY BOSTON UNIVERSITY SCHOOL OF MEDICINE 615 ALBANY STREET BOSTON, MA 02118 CO 3 JUNE 1991 ZQ CO o Reproduction in whole or in part is...Distribution unlimited. 17. DISTRIBUTION STATEMENT (ol Cha abotract ontotod In Block 20, II dlltoranl bom Report) 18. SUPPLEMENTARY NOTES 19. KEY WORDS...transfusion of nonviable red blood cells (RBC) inhibits the reticuloendothelial system. The study reported here examines the in vivo distribution of

  3. Plasma-mediated release of morphine from synthesized prodrugs.

    PubMed

    Thomas, Thommey P; Huang, Baohua; Desai, Ankur; Zong, Hong; Cheng, Xue-Min; Kotlyar, Alina; Leroueil, Pascale R; Dunham, Thomas; van der Spek, Abraham; Ward, Brent B; Baker, James R

    2010-11-01

    Two morphine prodrugs ('PDA' and 'PDB') were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5-10min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse>rat>guinea pig>human). Morphine's release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.

  4. Potency tests of diphtheria, tetanus and combined vaccines. Suggestion for a simplified potency assay.

    PubMed

    Aggerbeck, H; Heron, I

    1996-01-01

    Two diphtheria-tetanus vaccines (DT), adsorbed to either aluminium hydroxide or calcium phosphate but identical with respect to toxoid origin and amounts, were compared in full potency assays in mice according to the European Pharmacopoeia (EP) and in a reduced potency assay in guinea-pigs using a double dose immunization schedule. The efficacy of the vaccines was compared in a clinical trial with revaccination of 313 military recruits. The reduced potency assay gave a better reflection of the efficacy of the two vaccines in humans than the required assays of the EP. For release of combined, final vaccine formulations the reduced potency assay suggested will reduce the number of animals in quality control.

  5. Controlled Release of Biologically Active Silver from Nanosilver Surfaces

    PubMed Central

    Liu, Jingyu; Sonshine, David A.; Shervani, Saira; Hurt, Robert H.

    2010-01-01

    Major pathways in the antibacterial activity and eukaryotic toxicity of nano-silver involve the silver cation and its soluble complexes, which are well established thiol toxicants. Through these pathways, nano-silver behaves in analogy to a drug delivery system, in which the particle contains a concentrated inventory of an active species, the ion, which is transported to and released near biological target sites. Although the importance of silver ion in the biological response to nano-silver is widely recognized, the drug delivery paradigm has not been well developed for this system, and there is significant potential to improve nano-silver technologies through controlled release formulations. This article applies elements of the drug delivery paradigm to nano-silver dissolution and presents a systematic study of chemical concepts for controlled release. After presenting thermodynamic calculations of silver species partitioning in biological media, the rates of oxidative silver dissolution are measured for nanoparticles and macroscopic foils and used to derive unified area-based release kinetics. A variety of competing chemical approaches are demonstrated for controlling the ion release rate over four orders of magnitude. Release can be systematically slowed by thiol and citrate ligand binding, formation of sulfidic coatings, or the scavenging of peroxy-intermediates. Release can be accelerated by pre-oxidation or particle size reduction, while polymer coatings with complexation sites alter the release profile by storing and release inventories of surface-bound silver. Finally, the ability to tune biological activity is demonstrated through bacterial inhibition zone assay carried out on selected formulations of controlled release nano-silver. PMID:20968290

  6. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  7. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  8. Radioenzymatic assay for quinolinic acid

    SciTech Connect

    Foster, A.C.; Okuno, E.; Brougher, D.S.; Schwarcz, R.

    1986-10-01

    A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing (/sup 3/H)ATP, further to (/sup 3/H) deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

  9. [Compound erythromycin sustained release preparation and its in vitro release].

    PubMed

    Chen, Hai-xia; Chen, Zhi-peng; Wang, Qi-rong; Liu, Ze-kun; Ma, Quan-long

    2011-11-01

    Using the weight-average molecular weight 50 000 polylactic acid (PLA) as a carrier, and a certain proportion of erythromycin (EM) and prednisone acetate (PNA) to mixed prepare the compound erythromycin sustained release preparation (sustained-release tablets). Using ultraviolet spectrophotometry and high performance liquid chromatography (HPLC) to detect separately the release amount of EM and PNA in vitro medium. The sustained-release tablets release for about 21 days, the average content of EM is 99.7 mg/table, RSD = 0.82%; and the average content of PNA is 10.03 mg/table, RSD = 0.93%. Within 21 days, the cumulative releases of EM and PNA are 86.1% and 78.3%, respectively. The drug release is steady and slow after 5 days, the burst release phenomenon in early stage is more significant. The results showed that the sustained-release tablet preparation method is feasible, the release performance is good and the clinical efficacy is significant.

  10. QUICK RELEASABLE DRIVE

    DOEpatents

    Dickson, J.J.

    1958-07-01

    A quick releasable mechanical drive system suitable for use in a nuclear reactor is described. A small reversible motor positions a control rod by means of a worm and gear speed reducer, a magnetic torque clutch, and a bell crank. As the control rod is raised to the operating position, a heavy coil spring is compressed. In the event of an emergency indicated by either a''scram'' signal or a power failure, the current to the magnetic clutch is cut off, thereby freeing the coil spring and the bell crank positioner from the motor and speed reduction gearing. The coil spring will immediately act upon the bell crank to cause the insertion of the control rod. This arrangement will allow the slow, accurate positioning of the control rod during reactor operation, while providing an independent force to rapidly insert the rod in the event of an emergency.

  11. Quick release engine cylinder

    DOEpatents

    Sunnarborg, Duane A.

    2000-01-01

    A quick release engine cylinder allows optical access to an essentially unaltered combustion chamber, is suitable for use with actual combustion processes, and is amenable to rapid and repeated disassembly and cleaning. A cylinder member, adapted to constrain a piston to a defined path through the cylinder member, sealingly engages a cylinder head to provide a production-like combustion chamber. A support member mounts with the cylinder member. The support-to-cylinder mounting allows two relationships therebetween. In the first mounting relationship, the support engages the cylinder member and restrains the cylinder against the head. In the second mounting relationship, the cylinder member can pass through the support member, moving away from the head and providing access to the piston-top and head.

  12. SITELLE's Data Release 1

    NASA Astrophysics Data System (ADS)

    Martin, T. B.; Drissen, L.

    2016-12-01

    Installed at the Canada-France-Hawaii Telescope (CFHT) since August 2015, SITELLE is an Imaging Fourier Transform Spectrometer (IFTS) with an 11×11 field of view. After its prototype SpIOMM, installed at Mont Mégantic (Québec, Canada), it is the second IFTS in the world operating in the visible band (350-1000 nm). It delivers hyperspectral data cubes of 4 million spectra at R˜1500-5000 with a spatial sampling of 0.32" and a filling factor of 100 %. A suite of softwares has been designed to reduce (ORBS) and analyze (ORCS) the data. Based on commissioning data obtained in August 2015, a first stable version has been released in March 2016 which is capable of reducing all the data. In this paper the quality of the calibration is discussed.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways

    EPA Science Inventory

    The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. “Toxicity Testin...

  19. Complete Genome Sequences for 35 Biothreat Assay-Relevant Bacillus Species

    DOE PAGES

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; ...

    2015-04-30

    In 2011, the Association of Analytical Communities (AOAC) International released a list of Bacillus strains relevant to biothreat molecular detection assays. Presented in this document are the complete and annotated genome assemblies for the 15 strains listed on the inclusivity panel, as well as the 20 strains listed on the exclusivity panel.

  20. Abuse-related effects of dual dopamine/serotonin releasers with varying potency to release norepinephrine in male rats and rhesus monkeys.

    PubMed

    Banks, Matthew L; Bauer, Clayton T; Blough, Bruce E; Rothman, Richard B; Partilla, John S; Baumann, Michael H; Negus, S Stevens

    2014-06-01

    d-Amphetamine selectively promotes release of both dopamine (DA) and norepinephrine (NE) versus serotonin (5HT), and chronic d-amphetamine treatment decreases cocaine-taking behavior in rats, nonhuman primates, and humans. However, abuse liability limits the clinical utility of amphetamine maintenance for treating cocaine abuse. One strategy to improve safety and efficacy of monoamine releasers as candidate anticocaine medications has been to develop dual DA/5HT releasers like 1-napthyl-2-aminopropane (PAL-287), but the pharmacology of this class of compounds has not been extensively examined. In particular, PAL-287 has similar potencies to release DA, 5HT, and NE, and the role of manipulating NE release potency on abuse-related or anticocaine effects of dual DA/5HT releasers is not known. To address this issue, the present study compared effects of four novel DA/5HT releasers that varied >800-fold in their selectivities to release DA/5HT versus NE: [1-(5-chloro-1H-indol-3-yl)propan-2-amine (PAL-542), 1-(5-fluoro-1H-indol-3-yl)propan-2-amine (PAL-544), 1-(1H-indol-5-yl)propan-2-amine (PAL-571), and (R)-1-(1H-indol-1-yl)propain-2-amine (PAL-569). Abuse-related effects of all four compounds were evaluated in assays of intracranial self-stimulation (ICSS) in rats and cocaine discrimination in rats and monkeys, and none of the compounds reliably facilitated ICSS or substituted for cocaine. Anticocaine effects of the compound with highest selectivity to release DA/5HT versus NE (PAL-542) were tested in an assay of cocaine versus food choice in rhesus monkeys, and PAL-542 failed to reduce cocaine choice. These results suggests that potency to release NE has minimal influence on abuse liability of dual DA/5HT releasers, and reducing relative potency to release NE versus DA/5HT does not improve anticocaine efficacy.

  1. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  2. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  3. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  4. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  5. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  6. A colorimetric assay for cytokinin oxidase.

    PubMed

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  7. 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.

  8. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  9. Steroid assays in paediatric endocrinology.

    PubMed

    Honour, John W

    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.

  10. 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

  11. Optimized microturbidimetric assay for fibrinogen.

    PubMed

    Macart, M; Koffi, A; Henocque, G; Mathieu, J F; Guilbaud, J C

    1989-02-01

    In this assay we measure the turbidity produced by precipitation of plasma fibrinogen with a reagent composed of ammonium sulfate, EDTA, and guanidine hydrochloride. The two-step reagent addition, and use of fixed reaction times, eliminates interference from bilirubin, hemoglobin, and chylomicrons. We checked 135 monoclonal proteins for interference, finding the probability of encountering major interference in samples from adults to be very low, P = 0.0002. The method is calibrated with purified fibrinogen and the response is linear over the range 0-10 g/L. Within-run precision (CV) is less than 2% from 1 to 10 g/L. Correlations with the immunoturbidimetric (r = 0.99), chronometric (r = 0.99), and clotting (r = 0.97) methods were extremely high.

  12. Human Arterial Ring Angiogenesis Assay.

    PubMed

    Seano, Giorgio; Primo, Luca

    2016-01-01

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

  13. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  14. Assay of methylglyoxal-derived protein and nucleotide AGEs.

    PubMed

    Rabbani, Naila; Shaheen, Fozia; Anwar, Attia; Masania, Jinit; Thornalley, Paul J

    2014-04-01

    Glyoxalase- and methylglyoxal-related research has required the development of quantitative and reliable techniques for the measurement of methylglyoxal-derived glycation adducts of protein and DNA. There are also other glycation adducts, oxidation adducts and nitration adducts of proteins and oxidation adducts of DNA. Proteolysis of protein releases glycation, oxidation and nitration free adducts (glycated, oxidized and nitrated amino acids) in plasma and nuclease digestion of DNA releases glycated and oxidized nucleosides into plasma and other body fluids for excretion in urine. The gold standard method for quantifying these adducts is stable isotopic dilution analysis LC-MS/MS. Protein and DNA adduct residues are determined by assay of enzymatic hydrolysates of protein and DNA extracts prepared using cocktails of proteases and nucleases respectively. Free adducts are determined by analysis of ultrafiltrates of plasma, urine and other physiological fluids. Protein damage markers (13 glycation adducts, five oxidation adducts and 3-nitrotyrosine) and DNA damage markers (three glycation adducts and one oxidation adduct) are quantified using 25 μg of protein, 10 μg of DNA or 5 μl of physiological fluid. Protein and nucleotide AGE (advanced glycation end-product) assay protocols resistant to interferences is described.

  15. Predictive assay for cancer targets

    NASA Astrophysics Data System (ADS)

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

    2005-11-01

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

  16. 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.

  17. Comparative assessment of in vitro release kinetics of calcitonin polypeptide from biodegradable microspheres.

    PubMed

    Prabhu, Sunil; Sullivan, Jennifer L; Betageri, Guru V

    2002-01-01

    The objective of our study was to compare the in vitro release kinetics of a sustained-release injectable microsphere formulation of the polypeptide drug, calcitonin (CT), to optimize the characteristics of drug release from poly-(lactide-co-glycolide) (PLGA) copolymer biodegradable microspheres. A modified solvent evaporation and double emulsion technique was used to prepare the microspheres. Release kinetic studies were carried out in silanized tubes and dialysis bags, whereby microspheres were suspended and incubated in phosphate buffered saline, sampled at fixed intervals, and analyzed for drug content using a modified Lowry protein assay procedure. An initial burst was observed whereby about 50% of the total dose of the drug was released from the microspheres within 24 hr and 75% within 3 days. This was followed by a period of slow release over a period of 3 weeks in which another 10-15% of drug was released. Drug release from the dialysis bags was more gradual, and 50% CT was released only after 4 days and 75% after 12 days of release. Scanning electron micrographs revealed spherical particles with channel-like structures and a porous surface after being suspended in an aqueous solution for 5 days. Differential scanning calorimetric studies revealed that CT was present as a mix of amorphous and crystalline forms within the microspheres. Overall, these studies demonstrated that sustained release of CT from PLGA microspheres over a 3-week period is feasible and that release of drug from dialysis bags was more predictable than from tubes.

  18. Re-examining how complexin inhibits neurotransmitter release

    PubMed Central

    Trimbuch, Thorsten; Xu, Junjie; Flaherty, David; Tomchick, Diana R; Rizo, Josep; Rosenmund, Christian

    2014-01-01

    Complexins play activating and inhibitory functions in neurotransmitter release. The complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by ‘superclamp’ and ‘poor-clamp’ mutations that enhanced or decreased the complexin-I inhibitory activity in cell–cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes. DOI: http://dx.doi.org/10.7554/eLife.02391.001 PMID:24842998

  19. External Dentin Stimulation Induces ATP Release in Human Teeth.

    PubMed

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

  20. Deficient natural killer cell function in preeclampsia

    SciTech Connect

    Alanen, A.; Lassila, O.

    1982-11-01

    Natural killer cell activity of peripheral blood lymphocytes was measured against K-562 target cells with a 4-hour /sup 51/Cr release assay in 15 primigravid women with preeclamptic symptoms. Nineteen primigravid women with an uncomplicated pregnancy and 18 nonpregnant women served as controls. The natural killer cell activity of preeclamptic women was observed to be significantly lower than that of both control groups. Natural killer cells in preeclamptic women responded normally to augmentation caused by interferon. These findings give further evidence for the participation of the maternal immune system in this pregnancy disorder.

  1. Microdroplet chain array for cell migration assays.

    PubMed

    Ma, Yan; Pan, Jian-Zhang; Zhao, Shi-Ping; Lou, Qi; Zhu, Ying; Fang, Qun

    2016-11-29

    Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

  2. A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate.

    PubMed

    Tran, Thuy Thi; Hatti-Kaul, Rajni; Dalsgaard, Søren; Yu, Shukun

    2011-03-15

    Phytase (EC 3.1.3.-) hydrolyzes phytate (IP(6)) present in cereals and grains to release inorganic phosphate (P(i)), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P(i) liberated from IP(6). This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP(6)-lysozyme as a substrate. The assay is based on the principle that IP(6) forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP(6) in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P(i) from IP(6). This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl(2), and phosphate, showed a concentration-dependent interference.

  3. Development of forensic assay signatures for ebolaviruses.

    PubMed

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

    2015-03-01

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

  4. 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.

  5. Imaging renin content and release in the living kidney.

    PubMed

    Toma, Ildikó; Kang, Jung Julie; Peti-Peterdi, János

    2006-01-01

    Renin release is the first, and at least initially, the rate-limiting step in the activation of the renin-angiotensin system, which helps to maintain body salt and water balance. Recent advances in our understanding of pathophysiology have generated a renewed interest in the multiple roles of renin and prorenin as a hormone, enzyme, and signaling molecule. The assays available to measure renin content, release and tissue activity are complex, indirect and work with significant internal errors. We developed an imaging approach to directly visualize renin content and study the dynamics of both the release and tissue activity of renin. Our experimental model uses multiphoton fluorescence microscopy, which is ideal for deep optical sectioning of the living renal tissue. Here we review the application of this renin imaging approach to the dissected, in vitro microperfused glomerulus as well as in the intact kidney in vivo.

  6. A Bioluminescent Cytotoxicity Assay for Assessment of Membrane Integrity Using a Proteolytic Biomarker

    PubMed Central

    Cho, Ming-Hsuang; Niles, Andrew; Huang, Ruili; Inglese, James; Austin, Christopher P.; Riss, Terry; Xia, Menghang

    2008-01-01

    Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic “release” assays which measure the extracellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known toxins from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity. PMID:18400464

  7. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination.

    PubMed

    Field, Anjalie; Field, Jeffrey

    2010-08-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives.

  8. Multiplexed assays by high-content imaging for assessment of GPCR activity.

    PubMed

    Ross, D A; Lee, S; Reiser, V; Xue, J; Alves, K; Vaidya, S; Kreamer, A; Mull, R; Hudak, E; Hare, T; Detmers, P A; Lingham, R; Ferrer, M; Strulovici, B; Santini, F

    2008-07-01

    G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor imaging assay is based on redistribution of cytosolic beta-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor alone has limitations: relying on a single mechanistic step of beta-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multiplexed the Transfluor assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization.

  9. The "kynurenate test", a biochemical assay for putative cognition enhancers.

    PubMed

    Pittaluga, A; Vaccari, D; Raiteri, M

    1997-10-01

    Some putative cognition enhancers (oxiracetam, aniracetam and D-cycloserine) were previously shown to prevent the kynurenic acid antagonism of the N-methyl-D-aspartate (NMDA)-evoked norepinephrine (NE) release in rat hippocampal slices. This functional in vitro assay was further characterized in the present work. D-Serine, a glutamate coagonist at the NMDA receptor glycine site, concentration-dependently (EC50 approximately 0.1 microM) prevented the kynurenate (100 microM) block of the NMDA (100 microM)-evoked [3H]NE release. L-Serine was ineffective up to 10 microM. The gamma-aminobutyric acidB (GABA[B]) receptor antagonist CGP 36742, reported to improve cognitive performance, potently prevented the kynurenate antagonism. The activity of CGP 36742 (1 microM) appeared to be unaffected by 10 microM (-)-baclofen, a GABA(B) receptor agonist; furthermore, CGP 52432, a GABA(B) antagonist more potent than CGP 36742, but reportedly devoid of nootropic properties, was inactive in the "kynurenate test." The novel putative cognition enhancer CR2249, but not its enantiomer CR2361, also potently prevented the kynurenate antagonism. In contrast, linopirdine, nicotine and tacrine were inactive. In rat hippocampal synaptosomes glycine and D-cycloserine enhanced the NMDA-evoked [3H]NE release, whereas oxiracetam and CR2249 did not. These four compounds were all similarly effective in preventing kynurenate antagonism, both in slices and in synaptosomes. The NMDA potentiation caused by glycine (0.1-100 microM) was not affected by 100 microM oxiracetam, which suggested that drugs active in the "kynurenate test" may bind to sites different from the glycine site of the NMDA receptor. To conclude, the "kynurenate test" is an in vitro assay useful in the identification and characterization of putative cognition enhancers acting via NMDA receptors.

  10. Controlled Release Applications of Organometals.

    ERIC Educational Resources Information Center

    Thayer, John S.

    1981-01-01

    Reviews two classes of controlled release organometals: (1) distributional, to distribute bioactive materials to control a certain target organism; and (2) protective, to protect surface or interior of some structure from attach by organisms. Specific examples are given including a discussion of controlled release for schistosomiasis. (SK)

  11. Kepler Data Release 4 Notes

    NASA Technical Reports Server (NTRS)

    Van Cleve, Jeffrey (Editor); Jenkins, Jon; Caldwell, Doug; Allen, Christopher L.; Batalha, Natalie; Bryson, Stephen T.; Chandrasekaran, Hema; Clarke, Bruce D.; Cote, Miles T.; Dotson, Jessie L.; Gilliland, Ron; Girouard, Forrest; Haas, Michael R.; Hall, Jennifer; Ibrahim, Khadeejah; Klaus, Todd; Kolodziejczak, Jeff; Li, Jie; McCauliff, Sean D.; Middour, Christopher K.; Pletcher, David L.; Quintana, Elisa V.; Tenenbaum, Peter G.; Twicken, Joe; Uddin, Akm Kamal

    2010-01-01

    The Data Analysis Working Group have released long and short cadence materials, including FFIs and Dropped Targets for the Public. The Kepler Science Office considers Data Release 4 to provide "browse quality" data. These notes have been prepared to give Kepler users of the Multimission Archive at STScl (MAST) a summary of how the data were collected and prepared, and how well the data processing pipeline is functioning on flight data. They will be updated for each release of data to the public archive and placed on MAST along with other Kepler documentation, at http://archive.stsci.edu/kepler/documents.html. Data release 3 is meant to give users the opportunity to examine the data for possibly interesting science and to involve the users in improving the pipeline for future data releases. To perform the latter service, users are encouraged to notice and document artifacts, either in the raw or processed data, and report them to the Science Office.

  12. Improving techniques for clonogenic assays.

    PubMed

    Eliason, J F; Fekete, A; Odartchenko, N

    1984-01-01

    A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.

  13. Nitrogen release during coal combustion

    SciTech Connect

    Baxter, L.L.; Mitchell, R.E.; Fletcher, T.H.; Hurt, R.H.

    1995-02-01

    Experiments in entrained flow reactors at combustion temperatures are performed to resolve the rank dependence of nitrogen release on an elemental basis for a suite of 15 U.S. coals ranging from lignite to low-volatile bituminous. Data were obtained as a function of particle conversion, with overall mass loss up to 99% on a dry, ash-free basis. Nitrogen release rates are presented relative to both carbon loss and overall mass loss. During devolatilization, fractional nitrogen release from low-rank coals is much slower than fractional mass release and noticeably slower than fractional carbon release. As coal rank increases, fractional nitrogen release rate relative to that of carbon and mass increases, with fractional nitrogen release rates exceeding fractional mass and fractional carbon release rates during devolatilization for high-rank (low-volatile bituminous) coals. At the onset of combustion, nitrogen release rates increase significantly. For all coals investigated, cumulative fractional nitrogen loss rates relative to those of mass and carbon passes through a maximum during the earliest stages of oxidation. The mechanism for generating this maximum is postulated to involve nascent thermal rupture of nitrogen-containing compounds and possible preferential oxidation of nitrogen sites. During later stages of oxidation, the cumulative fractional loss of nitrogen approaches that of carbon for all coals. Changes in the relative release rates of nitrogen compared to those of both overall mass and carbon during all stages of combustion are attributed to a combination of the chemical structure of coals, temperature histories during combustion, and char chemistry.

  14. Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

    PubMed

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Inscore, Frank; Sengupta, Atanu; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-12-21

    A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

  15. [Review on characteristics and detecting assay of bacterial endotoxin contamination in water environment].

    PubMed

    Zhang, Can; Liu, Wen-Jun; Zhang, Ming-Lu; Tian, Fang; Yang, Yi; An, Dai-Zhi

    2014-04-01

    Endotoxins, also known as lipopolysaccharide complexes, are anchored in the outer membrane cell wall of most Gram-negative bacteria and some cyanobacteria. They are continuously released to environment during cell decay. Being common pyrogens and highly immunogenic molecules, endotoxins are related to many human diseases. Due to the tolerances and thermo-stability of endotoxin molecules, they were hard to be removed by common methods. The health risk caused by the endotoxin contamination in drinking water and water environment by various exposure pathways have attracted more and more attention in recent years. In this paper, the physical and chemical properties, biological activities and detection assay of the endotoxin contamination were reviewed, and interfere factors of the main assay, the LAL/TAL (Limulus amebocyte lysate/Tachypleus amebocyte lysate) assay, for detecting endotoxin in water sample were investigated, and the development tendency of the endotoxin detection assay was analyzed.

  16. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    SciTech Connect

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-02-20

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  17. Stabilization of spontaneous neurotransmitter release at ribbon synapses by ribbon-specific subtypes of complexin.

    PubMed

    Vaithianathan, Thirumalini; Zanazzi, George; Henry, Diane; Akmentin, Wendy; Matthews, Gary

    2013-05-08

    Ribbon synapses of tonically releasing sensory neurons must provide a large pool of releasable vesicles for sustained release, while minimizing spontaneous release in the absence of stimulation. Complexins are presynaptic proteins that may accomplish this dual task at conventional synapses by interacting with the molecular machinery of synaptic vesicle fusion at the active zone to retard spontaneous vesicle exocytosis yet facilitate release evoked by depolarization. However, ribbon synapses of photoreceptor cells and bipolar neurons in the retina express distinct complexin subtypes, perhaps reflecting the special requirements of these synapses for tonic release. To investigate the role of ribbon-specific complexins in transmitter release, we combined presynaptic voltage clamp, fluorescence imaging, electron microscopy, and behavioral assays of photoreceptive function in zebrafish. Acute interference with complexin function using a peptide derived from the SNARE-binding domain increased spontaneous synaptic vesicle fusion at ribbon synapses of retinal bipolar neurons without affecting release triggered by depolarization. Knockdown of complexin by injection of an antisense morpholino into zebrafish embryos prevented photoreceptor-driven migration of pigment in skin melanophores and caused the pigment distribution to remain in the dark-adapted state even when embryos were exposed to light. This suggests that loss of complexin function elevated spontaneous release in illuminated photoreceptors sufficiently to mimic the higher release rate normally associated with darkness, thus interfering with visual signaling. We conclude that visual system-specific complexins are required for proper illumination-dependent modulation of the rate of neurotransmitter release at visual system ribbon synapses.

  18. Stabilization of spontaneous neurotransmitter release at ribbon synapses by ribbon-specific subtypes of Complexin

    PubMed Central

    Vaithianathan, Thirumalini; Zanazzi, George; Henry, Diane; Akmentin, Wendy; Matthews, Gary

    2013-01-01

    Ribbon synapses of tonically releasing sensory neurons must provide a large pool of releasable vesicles for sustained release, while minimizing spontaneous release in the absence of stimulation. Complexins are presynaptic proteins that may accomplish this dual task at conventional synapses, by interacting with the molecular machinery of synaptic vesicle fusion at the active zone to retard spontaneous vesicle exocytosis yet facilitate release evoked by depolarization. However, ribbon synapses of photoreceptor cells and bipolar neurons in the retina express distinct Complexin subtypes, perhaps reflecting the special requirements of these synapses for tonic release. To investigate the role of ribbon-specific Complexins in transmitter release, we combined presynaptic voltage-clamp, fluorescence imaging, electron microscopy, and behavioral assays of photoreceptive function in zebrafish. Acute interference with Complexin function using a peptide derived from the SNARE-binding domain increased spontaneous synaptic vesicle fusion at ribbon synapses of retinal bipolar neurons without affecting release triggered by depolarization. Knockdown of Complexin by injection of an antisense morpholino into zebrafish embryos prevented photoreceptor-driven migration of pigment in skin melanophores and caused the pigment distribution to remain in the dark-adapted state even when embryos were exposed to light. This suggests that loss of Complexin function elevated spontaneous release in illuminated photoreceptors sufficiently to mimic the higher release rate normally associated with darkness, thus interfering with visual signaling. We conclude that visual system-specific Complexins are required for proper illumination-dependent modulation of the rate of neurotransmitter release at visual system ribbon synapses. PMID:23658160

  19. Design, synthesis and pharmacological evaluation of novel NO-releasing benzimidazole hybrids as potential antihypertensive candidate.

    PubMed

    Zhang, Yanchun; Xu, Jinyi; Li, Yunman; Yao, Hequan; Wu, Xiaoming

    2015-05-01

    Two series of novel NO-releasing benzimidazole derivatives (8a-e, 9a-g) were designed and synthesized by coupling nitro ester and furoxan NO-donor moieties with benzimidazole biphenyl skeleton. The NO-releasing assay indicated that all the target compounds had different level of NO-releasing ability. Furthermore, the isolated organ assay (rat aortic strips) was used to evaluate the antagonism of Ang II-induced vasoconstriction ability. It was observed that the pA2 values of compounds 8e and 9e were better than that of lead compound 6. Moreover, the pharmacological investigation showed that the antagonism of Ang II-induced pressure response by oral administration of compound 8e was obviously superior to that of lead compound 6, and comparable to that of the positive control losartan. These results suggested that NO-releasing hybrids may provide a promising approach for the discovery of novel antihypertensive agents.

  20. 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.

  1. Sensitive field assays for water analysis

    SciTech Connect

    Douglas, W.L.

    1984-08-01

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

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

    PubMed

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

    2016-01-01

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

  3. Magnetoresistive Sensors in Biological Assays

    NASA Astrophysics Data System (ADS)

    Tondra, Mark

    2010-03-01

    Magnetic beads or nanoparticles can be used as ``labels'' in biochemical assays by attaching the beads to the biospecies of interest using a bio-specific attachment. Once the labels are attached, they can be used to manipulate, capture, and detect the species to be analyzed. Magnetoresistive (MR) sensors may be used to detect and count these labels, and thus make an inference about the concentration of the species of interest. MR technology is especially promising for biosensor applications where making the detector small and integrated with related sample handling tools to form a ``lab-on-a-chip'' miniaturized system. The function of the MR sensors is to detect stray magnetic fields from the beads while they are exposed to a magnetic excitation field. Generally, the stray fields from beads and clusters of beads are complicated functions of geometry, so some care is required to relate the detected magnetic signal to the number and location of the bead labels. This presentation will begin with a broad overview of results from many groups working in this area. For convenience, the applications are divided into three categories, detection of: flowing magnetic beads, immobilized beads, and scanned samples. Next will be some discussion of how the choice of spintronic sensor technology might affect detection capabilities (AMR, GMR, TMR, Hall effect, etc). Then, challenges relating to integration of MR sensors into microfluidic products will be discussed. This is the focus of the presenter's current day-to-day work on developing and producing MR-based biosensors. And finally, a description of possible future avenues of study and development will be presented.

  4. Toxics Release Inventory indicates big increases in releases

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2012-01-01

    Nearly 4 billion pounds of tracked toxic chemicals were released into the environment throughout the United States during 2010, according to an analysis by the U.S. Environmental Protection Agency (EPA) of the Toxics Release Inventory (TRI), the agency announced on 5 January. This is a 16% increase above 2009. The agency said the increase is mainly due to changes in the metal-mining sector, where differences in the chemical composition of ore being mined can result in significant changes in the amount of toxic chemicals. The chemical and primary metals industries were other sectors with increases in toxic releases in 2010, the latest year for which data collection is complete. EPA also noted that although releases in 2010 were higher than during the previous 2 years, they were lower than in 2007 and in prior years.

  5. Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-vargeted luciferase from neuronal cells.

    PubMed

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Genz, Lara; Püchel, Gerhard P

    2015-01-01

    Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

  6. Mast cell release of superoxide

    SciTech Connect

    Dileepan, K.N.; Simpson, K.M.; Stechschulte, D.J.

    1987-05-01

    The ability of rat serosal mast cells (MC) to release superoxide (O/sub 2//sup -/) upon activation by immunologic and nonimmunologic stimuli was investigated. Purified MC (90-95%) were either sensitized with monoclonal IgE reactive against dinitrophenyl bovine serum albumin (DNP-BSA) and challenged with DNP-BSA, or naive MC were treated with compound 48/80 or ionophore A23187. O/sub 2//sup -/ release was measured by O/sub 2//sup -/ dismutase (SOD)-sensitive reduction of cytochrome C and MC activation was assessed by the release of histamine or (/sup 14/C)5-hydroxytryptamine (5HT). The results reveal that activation of MC by 48/80 or immunologic challenge does not release O/sub 2//sup -/, although these stimuli induce substantial release of histamine and 5HT (40-70%). In contrast, A23187 released O/sub 2//sup -/ (3-6 nMols/10/sup 6/ MC) and histamine (40-80%). In mixed cell preparations containing MC and macrophages (M0), activation of MC with 48/80 resulted in inhibition of M0 O/sub 2//sup -/ release. The MC-mediated inhibition of O/sub 2//sup -/ production was not due to histamine or 5HT, but was due to MC-granule SOD. MC contain abundant quantities of SOD and, therefore, release O/sub 2//sup -/ only when its production exceeds the intracellular SOD threshold following activation with selective stimuli. In addition, the apparent differences in the mode and site of action of various stimuli on MC may contribute to the discriminative release of O/sub 2//sup -/.

  7. Controlled release liquid dosage formulation

    DOEpatents

    Benton, Ben F.; Gardner, David L.

    1989-01-01

    A liquid dual coated dosage formulation sustained release pharmaceutic having substantial shelf life prior to ingestion is disclosed. A dual coating is applied over controlled release cores to form dosage forms and the coatings comprise fats melting at less than approximately 101.degree. F. overcoated with cellulose acetate phthalate or zein. The dual coated dosage forms are dispersed in a sugar based acidic liquid carrier such as high fructose corn syrup and display a shelf life of up to approximately at least 45 days while still retaining their release profiles following ingestion. Cellulose acetate phthalate coated dosage form cores can in addition be dispersed in aqueous liquids of pH <5.

  8. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

    PubMed

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

    2017-03-04

    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1(-/-) mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  9. Highly Efficient Thermoresponsive Nanocomposite for Controlled Release Applications

    NASA Astrophysics Data System (ADS)

    Yassine, Omar; Zaher, Amir; Li, Er Qiang; Alfadhel, Ahmed; Perez, Jose E.; Kavaldzhiev, Mincho; Contreras, Maria F.; Thoroddsen, Sigurdur T.; Khashab, Niveen M.; Kosel, Jurgen

    2016-06-01

    Highly efficient magnetic release from nanocomposite microparticles is shown, which are made of Poly (N-isopropylacrylamide) hydrogel with embedded iron nanowires. A simple microfluidic technique was adopted to fabricate the microparticles with a high control of the nanowire concentration and in a relatively short time compared to chemical synthesis methods. The thermoresponsive microparticles were used for the remotely triggered release of Rhodamine (B). With a magnetic field of only 1 mT and 20 kHz a drug release of 6.5% and 70% was achieved in the continuous and pulsatile modes, respectively. Those release values are similar to the ones commonly obtained using superparamagnetic beads but accomplished with a magnetic field of five orders of magnitude lower power. The high efficiency is a result of the high remanent magnetization of the nanowires, which produce a large torque when exposed to a magnetic field. This causes the nanowires to vibrate, resulting in friction losses and heating. For comparison, microparticles with superparamagnetic beads were also fabricated and tested; while those worked at 73 mT and 600 kHz, no release was observed at the low field conditions. Cytotoxicity assays showed similar and high cell viability for microparticles with nanowires and beads.

  10. Highly Efficient Thermoresponsive Nanocomposite for Controlled Release Applications

    PubMed Central

    Yassine, Omar; Zaher, Amir; Li, Er Qiang; Alfadhel, Ahmed; Perez, Jose E.; Kavaldzhiev, Mincho; Contreras, Maria F.; Thoroddsen, Sigurdur T.; Khashab, Niveen M.; Kosel, Jurgen

    2016-01-01

    Highly efficient magnetic release from nanocomposite microparticles is shown, which are made of Poly (N-isopropylacrylamide) hydrogel with embedded iron nanowires. A simple microfluidic technique was adopted to fabricate the microparticles with a high control of the nanowire concentration and in a relatively short time compared to chemical synthesis methods. The thermoresponsive microparticles were used for the remotely triggered release of Rhodamine (B). With a magnetic field of only 1 mT and 20 kHz a drug release of 6.5% and 70% was achieved in the continuous and pulsatile modes, respectively. Those release values are similar to the ones commonly obtained using superparamagnetic beads but accomplished with a magnetic field of five orders of magnitude lower power. The high efficiency is a result of the high remanent magnetization of the nanowires, which produce a large torque when exposed to a magnetic field. This causes the nanowires to vibrate, resulting in friction losses and heating. For comparison, microparticles with superparamagnetic beads were also fabricated and tested; while those worked at 73 mT and 600 kHz, no release was observed at the low field conditions. Cytotoxicity assays showed similar and high cell viability for microparticles with nanowires and beads. PMID:27335342

  11. Quantification of intracellular payload release from polymersome nanoparticles

    PubMed Central

    Scarpa, Edoardo; Bailey, Joanne L.; Janeczek, Agnieszka A.; Stumpf, Patrick S.; Johnston, Alexander H.; Oreffo, Richard O. C.; Woo, Yin L.; Cheong, Ying C.; Evans, Nicholas D.; Newman, Tracey A.

    2016-01-01

    Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology. PMID:27404770

  12. Radio-synthesized polyacrylamide hydrogels for proteins release

    NASA Astrophysics Data System (ADS)

    Ferraz, Caroline C.; Varca, Gustavo H. C.; Lopes, Patricia S.; Mathor, Monica B.; Lugão, Ademar B.

    2014-01-01

    The use of hydrogels for biomedical purposes has been extensively investigated. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release, using papain and bovine serum albumin as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using γ-radiation under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density and evaluated according to protein release, bioactivity and cytotoxicity. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-5% (w/v) polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 h), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay it was also adequate for biomedical purposes, however this method was not able to generate a matrix with controlled pore sizes.

  13. Pulsatile Release of Parathyroid Hormone from an Implantable Delivery System

    PubMed Central

    Liu, Xiaohua; Pettway, Glenda J.; McCauley, Laurie K.; Ma, Peter X.

    2007-01-01

    Intermittent (pulsatile) administration of parathyroid hormone (PTH) is known to improve bone micro-architecture, mineral density and strength. Therefore, daily injection of PTH has been clinically used for the treatment of osteoporosis. However, this regimen of administration is not convenient and is not a favorable choice of patients. In this study, an implantable delivery system has been developed to achieve pulsatile release of PTH. A well-defined cylindrical device was first fabricated with a biodegradable polymer, poly(lactic acid) (PLLA), using a reverse solid free form fabrication technique. Three-component polyanhydrides composed of sebacic acid, 1,3-bis(p-carboxyphenoxy) propane and poly(ethylene glycol) were synthesized and used as isolation layers. The polyanhydride isolation layers and PTH-loaded alginate layers were then stacked alternately within the delivery device. The gap between the stacked PTH-releasing core and the device frame was filled with PLLA to seal. Multi-pulse PTH release was achieved using the implantable device. The lag time between two adjacent pulses were modulated by the composition and the film thickness of the polyanhydride. The released PTH was demonstrated to be biologically active using an in vitro assay. Timed sequential release of multiple drugs has also been demonstrated. The implantable device holds promise for both systemic and local therapies. PMID:17576005

  14. Quantification of intracellular payload release from polymersome nanoparticles

    NASA Astrophysics Data System (ADS)

    Scarpa, Edoardo; Bailey, Joanne L.; Janeczek, Agnieszka A.; Stumpf, Patrick S.; Johnston, Alexander H.; Oreffo, Richard O. C.; Woo, Yin L.; Cheong, Ying C.; Evans, Nicholas D.; Newman, Tracey A.

    2016-07-01

    Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

  15. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  16. Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

    PubMed

    Llewellyn, David; Miura, Kazutoyo; Fay, Michael P; Williams, Andrew R; Murungi, Linda M; Shi, Jianguo; Hodgson, Susanne H; Douglas, Alexander D; Osier, Faith H; Fairhurst, Rick M; Diakite, Mahamadou; Pleass, Richard J; Long, Carole A; Draper, Simon J

    2015-09-16

    The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.

  17. Particle-induced artifacts in the MTT and LDH viability assays.

    PubMed

    Holder, Amara L; Goth-Goldstein, Regine; Lucas, Donald; Koshland, Catherine P

    2012-09-17

    In vitro testing is a common first step in assessing combustion-generated and engineered nanoparticle-related health hazards. Commercially available viability assays are frequently used to compare the toxicity of different particle types and to generate dose-response data. Nanoparticles, well-known for having large surface areas and chemically active surfaces, may interfere with viability assays, producing a false assessment of toxicity and making it difficult to compare toxicity data. The objective of this study is to measure the extent of particle interference in two common viability assays, the MTT reduction and the lactate dehydrogenase (LDH) release assays. Diesel particles, activated carbon, flame soot, oxidized flame soot, and titanium dioxide particles are assessed for interactions with the MTT and LDH assay under cell-free conditions. Diesel particles, at concentrations as low as 0.05 μg/mL, reduce MTT. Other particle types reduce MTT only at a concentration of 50 μg/mL and higher. The activated carbon, soot, and oxidized soot particles bind LDH to varying extents, reducing the concentration measured in the LDH assay. The interfering effects of the particles explain in part the different toxicities measured in human bronchial epithelial cells (16HBE14o). We conclude that valid particle toxicity assessments can only be assured after first performing controls to verify that the particles under investigation do not interfere with a specific assay at the expected concentrations.

  18. Particle-induced artifacts in the MTT and LDH viability assays

    PubMed Central

    Holder, Amara L.; Goth-Goldstein, Regine; Lucas, Donald; Koshland, Catherine P.

    2012-01-01

    In vitro testing is a common first step in assessing combustion generated and engineered nanoparticle related health hazards. Commercially available viability assays are frequently used to compare the toxicity of different particle types and to generate dose response data. Nanoparticles, well known for having large surface areas and chemically active surfaces, may interfere with viability assays, producing a false assessment of toxicity and making it difficult to compare toxicity data. The objective of this study is to measure the extent of particle interference in two common viability assays, the MTT reduction and the lactate dehydrogenase (LDH) release assays. Diesel particles, activated carbon, flame soot, oxidized flame soot, and titanium dioxide particles are assessed for interactions with the MTT and LDH assay under cell-free conditions. Diesel particles, at concentrations as low as 0.05 μg/ml, reduce MTT. Other particle types reduce MTT only at a concentration of 50 μg/ml and higher. The activated carbon, soot, and oxidized soot particles bind LDH to varying extents, reducing the concentration measured in the LDH assay. The interfering effects of the particles explain in part the different toxicities measured in human bronchial epithelial cells (16HBE14o). We conclude that valid particle toxicity assessments can only be assured after first performing controls to verify that the particles under investigation do not interfere with a specific assay at the expected concentrations. PMID:22799765

  19. Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays.

    PubMed

    Prather, Brittany; Ethen, Cheryl M; Machacek, Miranda; Wu, Zhengliang L

    2012-04-01

    Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(1), to various acceptor substrates, generating 3'-phosphoadenosine-5'-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3'-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3'-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis-Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.

  20. Automated saccharification assay for determination of digestibility in plant materials

    PubMed Central

    2010-01-01

    Background Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. Results We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. Conclusions The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered

  1. Salt release from potato crisps.

    PubMed

    Tian, Xing; Fisk, Ian D

    2012-04-01

    The rate of salt release in-mouth from salted potato crisps was evaluated. It was hypothesised that a slow steady release of sodium would occur on chewing and hydration; to test this a crisp was chewed and held in the oral cavity without swallowing for 60 s. Sodium release was measured over the entire holding period, after 20-30 s a peak in salivary sodium levels was recorded. A similar trend was observed with sensory perceived saltiness by trained panellists. The results suggest that a significant proportion of the crisp's salt flavouring is released in a pulse-type mechanism which would not be encountered when the crisp is exposed to normal eating patterns and would result in the consumption of a large proportion of unperceived sodium.

  2. Tyrosine - Effects on catecholamine release

    NASA Technical Reports Server (NTRS)

    Acworth, Ian N.; During, Matthew J.; Wurtman, Richard J.

    1988-01-01

    Tyrosine administration elevates striatal levels of dopamine metabolites in animals given treatments that accelerate nigrostriatal firing, but not in untreated rats. We examined the possibility that the amino acid might actually enhance dopamine release in untreated animals, but that the technique of measuring striatal dopamine metabolism was too insensitive to demonstrate such an effect. Dopamine release was assessed directly, using brain microdialysis of striatal extracellular fluid. Tyrosine administration (50-200 mg/kg IP) did indeed cause a dose related increase in extracellular fluid dopamine levels with minor elevations in levels of DOPAC and HVA, its major metabolites, which were not dose-related. The rise in dopamine was short-lived, suggesting that receptor-mediated feedback mechanisms responded to the increased dopamine release by diminishing neuronal firing or sensitivity to tyrosine. These observations indicate that measurement of changes in striatal DOPAC and HVA, if negative, need not rule out increases in nigrostriatal dopamine release.

  3. Birth control - slow release methods

    MedlinePlus

    Contraception - slow-release hormonal methods; Progestin implants; Progestin injections; Skin patch; Vaginal ring ... implants while breastfeeding. Progestin implants work better than birth control pills to prevent pregnancy. Very few women who ...

  4. SELF-RELEASING GRAPPLING DEVICE

    DOEpatents

    Hoover, D.A. Sr.

    1963-11-01

    >A self-releasing grappling device that lifts by virtue of engagement between clamping jaws and the undercut lower side of a conical head of a lifting lug attached to the object to be lifted and employs a releasing sleeve on the lug to free the jaws from the lug is presented. When the jaws are to be released, they are dropped over the releasing sleeve, which is located well below lug head. When the jaws are lifted, they engage a conical surface on the sleeve and lift it up to the head of the lifting lug. In this position of the sleeve, the lower side of the lug head is covered by the sleeve and so cannot be engaged by the jaws, which move past before clearing the sleeve. (AEC)

  5. A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism.

    PubMed

    Sharma, D K; Vidugiriene, J; Bangs, J D; Menon, A K

    1999-06-04

    We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPI-anchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time- and temperature-dependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl

  6. Energy release in solar flares

    NASA Technical Reports Server (NTRS)

    Brown, John C.; Correia, Emilia; Farnik, Frantisek; Garcia, Howard; Henoux, Jean-Claude; La Rosa, Ted N.; Machado, Marcos E. (Compiler); Nakajima, Hiroshi; Priest, Eric R.

    1994-01-01

    Team 2 of the Ottawa Flares 22 Workshop dealt with observational and theoretical aspects of the characteristics and processes of energy release in flares. Main results summarized in this article stress the global character of the flaring phenomenon in active regions, the importance of discontinuities in magnetic connectivity, the role of field-aligned currents in free energy storage, and the fragmentation of energy release in time and space.

  7. Oxidative calcium release from catechol.

    PubMed

    Riley, Patrick A; Stratford, Michael R L

    2015-04-01

    Oxidation of 4-methylcatechol previously exposed to aqueous calcium chloride was shown by ion chromatography to be associated with release of calcium ions. The catechol was oxidised to the corresponding orthoquinone by the use of tyrosinase from Agaricus bisporus. The oxidative release of calcium from the catechol is ascribed to the diminution of the available hydroxyl functions able to act as chelating groups. Our results suggest that the redox status of melanin may regulate calcium binding and influence calcium levels in pigmented cells.

  8. 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....

  9. 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....

  10. 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....

  11. 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....

  12. 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....

  13. 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....

  14. Statistical inference for serial dilution assay data.

    PubMed

    Lee, M L; Whitmore, G A

    1999-12-01

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

  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 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....

  17. A lateral electrophoretic flow diagnostic assay.

    PubMed

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

    2015-03-21

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

  18. 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... circulation. These assays are quantitative clotting time procedures using the effect of heparin on...

  19. 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... circulation. These assays are quantitative clotting time procedures using the effect of heparin on...

  20. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    SciTech Connect

    Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

    1988-07-01

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

  1. Exosomes Released from Breast Cancer Carcinomas Stimulate Cell Movement

    PubMed Central

    Harris, Dinari A.; Patel, Sajni H.; Gucek, Marjan; Hendrix, An; Westbroek, Wendy; Taraska, Justin W.

    2015-01-01

    For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis. PMID:25798887

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

    PubMed

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

    2012-01-01

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

  3. Paper disk assay for glycosaminoglycan sulfotransferases

    SciTech Connect

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

    1987-11-01

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

  4. 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.

  5. Added release time in diffusion/dissolution coupled release.

    PubMed

    Nuxoll, Eric

    2015-10-15

    While increasingly sophisticated models have been developed to more accurately predict dispersed solute release from complex systems, distillation of their results into quantitative trends has been difficult. Here, the numerically calculated release profiles of coupled diffusion/dissolution systems are quantified by their cumulative release time (CRT) and compared against corresponding diffusion-controlled limits. The increase in CRT due to a finite dissolution rate was found to vary inversely with the second Damköhler number across several orders of magnitude, and also vary linearly with the amount of solid drug loaded in the system. The analytical nature of the relationship provides new physical insights into the system and appears to be indifferent to the form of the secondary rate-limiting step. This work provides a simple analytical expression with which one can not only predict the mean release time for a given set of parameter values, but understand precisely how each parameter value will affect it. The simplicity of the correlation and the lack of apparent limits to its validity also suggest the existence of an analytical pathway for its derivation, which may yield additional insights into the effect of secondary rate processes on controlled release.

  6. [Detection of influenza virus (RT-PCR assay and others)].

    PubMed

    Matsuzaki, Yoko

    2003-11-01

    Viral isolation is the conventional method for influenza virus diagnosis but it is less useful for immediate patient management. RT-PCR is the sensitive and rapid assay for the detection of respiratory viruses. Single step and multiplex RT-PCR is able to detect several viruses simultaneously in a single reaction. Real time PCR(TaqMan method) is able to detect the amplicon directly by release of a fluorescent reporter of the probe during the amplification reactions. This procedure can save time since it eliminates post-PCR processing steps. These RT-PCR methods should be useful for the accurate and rapid diagnosis of influenza virus infection, especially severe cases such as pneumonia and encephalopathy.

  7. 1H NMR investigation of thermally triggered insulin release from poly(N-isopropylacrylamide) microgels.

    PubMed

    Nolan, Christine M; Gelbaum, Leslie T; Lyon, L Andrew

    2006-10-01

    We describe investigations of insulin release from thermoresponsive microgels using variable temperature (1)H NMR. Microgel particles composed of poly(N-isopropylacrylamide) were loaded with the peptide via a swelling technique, and this method was compared to simple equilibrium partitioning. Variable temperature (1)H NMR studies suggest that the swelling loading method results in enhanced entrapment of the peptide versus equilibrium partitioning. A centrifugation-loading assay supports this finding. Pseudo-temperature jump (1)H NMR measurements suggest that the insulin release rate is partially decoupled from microgel collapse. These types of direct release investigations could prove to be useful methods in the future design of controlled macromolecule drug delivery devices.

  8. Cholecystokinin facilitates glutamate release by increasing the number of readily releasable vesicles and releasing probability.

    PubMed

    Deng, Pan-Yue; Xiao, Zhaoyang; Jha, Archana; Ramonet, David; Matsui, Toshimitsu; Leitges, Michael; Shin, Hee-Sup; Porter, James E; Geiger, Jonathan D; Lei, Saobo

    2010-04-14

    Cholecystokinin (CCK), a neuropeptide originally discovered in the gastrointestinal tract, is abundantly distributed in the mammalian brains including the hippocampus. Whereas CCK has been shown to increase glutamate concentration in the perfusate of hippocampal slices and in purified rat hippocampal synaptosomes, the cellular and molecular mechanisms whereby CCK modulates glutamatergic function remain unexplored. Here, we examined the effects of CCK on glutamatergic transmission in the hippocampus using whole-cell recordings from hippocampal slices. Application of CCK increased AMPA receptor-mediated EPSCs at perforant path-dentate gyrus granule cell, CA3-CA3 and Schaffer collateral-CA1 synapses without effects at mossy fiber-CA3 synapses. CCK-induced increases in AMPA EPSCs were mediated by CCK-2 receptors and were not modulated developmentally and transcriptionally. CCK reduced the coefficient of variation and paired-pulse ratio of AMPA EPSCs suggesting that CCK facilitates presynaptic glutamate release. CCK increased the release probability and the number of readily releasable vesicles with no effects on the rate of recovery from vesicle depletion. CCK-mediated increases in glutamate release required the functions of phospholipase C, intracellular Ca(2+) release and protein kinase Cgamma. CCK released endogenously from hippocampal interneurons facilitated glutamatergic transmission. Our results provide a cellular and molecular mechanism to explain the roles of CCK in the brain.

  9. 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

  10. Simultaneous quantification of drug release and erosion from hypromellose hydrophilic matrices.

    PubMed

    Ghori, Muhammad U; Ginting, Gidion; Smith, Alan M; Conway, Barbara R

    2014-04-25

    Hypromellose, HPMC, is frequently used to control drug release from matrix tablet formulations. Drug is released by a combination of diffusion through and erosion of, the matrix and is usually measured invitro by separate dissolution and swelling/erosion studies. The present study was designed to measure matrix erosion, polymer dissolution and drug release kinetics and their inter-relationship in a single experiment using a phenol-sulphuric acid assay to quantify dissolved HPMC alongside spectrophotometrical analysis of drug release. HPMC-based matrix tablets were manufactured containing two drugs at various drug:HPMC ratios. Drug release was determined and the degree of erosion was calculated by gravimetry. Results showed the matrix erosion rate and drug release were dependent on HPMC content and drug solubility, as expected. It was also apparent that the erosion rate was directly related to the drug release kinetics and comparative analysis of both matrix erosion techniques showed a high level of correlation. The findings show that a simple and inexpensive assay can be utilised not only to quantify HPMC but can also be used to calculate the degree of erosion of tablet matrices, negating the need for a separate study and providing a simplified practical approach that may be of use during product optimization.

  11. 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.

  12. 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.

  13. Assuring reliable performance of antibiotic assay media.

    PubMed

    Freeman, K A; Johnson, D P; Garth, M A

    1977-11-01

    The Microbiological Assay Branch of the National Center for Antibiotics Analysis assays over 100,000 samples of antibiotic products annually, using more than 1000 Ib dehydrated media. The media must be consistently dependable to produce accurate, reliable test results. To assure that the supply of media will meet the established requirements, each lot before purchase is subjected to a series of trials designed to examine growth support, sensitivity, and behavioral and physical factors. Actual antibiotic assays are conducted with the test medium, and performance is rated against a control medium. Controls on the system reduce the variables to allow appraisal of the medium itself.

  14. Suprachiasmatic nuclei may regulate the rhythm of leptin hormone release in Syrian hamsters (Mesocricetus auratus).

    PubMed

    Karakas, Alper; Gündüz, Bülent

    2006-01-01

    The suprachiasmatic nuclei (SCN) generate the circadian rhythm of many hormones. The hormone leptin is a metabolic signal that informs the brain about fat and energy stores of the body. We investigated whether the rhythm of leptin hormone release in Syrian hamsters is directly controlled by the SCN. Three experiments were performed: in the first, hamsters were SCN-lesioned; in the second, hamsters were exposed to different feeding regimes; and in the third, hamsters were adrenalectomized and implanted with cortisol capsules to maintain constant glucocorticoid release. Blood samples were collected before and after the experiments at different clock times and examined for leptin levels by enzyme-linked immunosorbant assay (ELISA). Different feeding regimes and constant glucocorticoid release did not alter the rhythm of leptin release; whereas, SCN lesions abolished the rhythm. The results of the present study suggest the rhythm in leptin release in Syrian hamsters may be controlled by the SCN.

  15. Controlled Release Formulations of Auxinic Herbicides

    NASA Astrophysics Data System (ADS)

    Kowalski, Witold J.; Siłowiecki, Andrzej.; Romanowska, Iwona; Glazek, Mariola; Bajor, Justyna; Cieciwa, Katarzyna; Rychter, Piotr

    2013-04-01

    Controlled release formulations are applied extensively for the release of active ingredients such as plant protection agents and fertilizers in response to growing concern for ecological problems associated with increased use of plant protection chemicals required for intensive agricultural practices [1]. We synthesized oligomeric mixtures of (R,S)-3-hydroxy butyric acid chemically bonded with 2,4-D, Dicamba and MCPA herbicides (HBA) respectively, and determined their molecular structure and molecular weight dispersion by the size exclusion chromatography, proton magnetic resonance spectrometry and electro-spray ionization mass spectrometry. Further we carried out bioassays of herbicidal effectiveness of the HBA herbicides vs. series of dicotyledonous weeds and crop injury tests [2, 3, 4]. Field bioassays were accomplished according to the EPPO standards [5]. Groups of representative weeds (the development stages in the BCCH scale: 10 - 30) were selected as targets. Statistical variabilities were assessed by the Fisher LSD test for plants treated with the studied herbicides in form of HBA oligomers, the reference herbicides in form of dimethyl ammonium salts (DMA), and untreated plants. No statistically significant differences in the crop injuries caused by the HBA vs. the DMA reference formulation were observed. The effectiveness of the HBA herbicides was lower through the initial period (ca. 2 weeks) relative to the DMA salts, but a significant increase in the effectiveness of the HBA systems followed during the remaining fraction of each assay. After 6 weeks all observed efficiencies approached 100%. The death of weeds treated with the HBA herbicides was delayed when compared with the DMA reference herbicides. The delayed uptake observed for the HBA oligomers relative to the DMA salts was due to controlled release phenomena. In case of the DMA salts the total amount of active ingredients was available at the target site. By contrast, the amount of an active

  16. Tests for oil/dispersant toxicity: In situ laboratory assays

    SciTech Connect

    Wright, D.A.; Coelho, G.M.; Aurand, D.V.

    1995-12-31

    As part of its readiness program in oil spill response, the Marine Pollution Control Unit (MPCU), Department of Transport, U.K. conducts annual field trials in the North Sea, approximately 30 nautical miles from the southeast coast of England. The trials take the form of controlled releases of crude oil or Medium Fuel/Gas Oil mix (MFO), with and without the application of Corexit 9527 dispersant. In 1994 and 1995 the authors conducted a series of in situ toxicity bioassays in association with these spills with included 48h LC50 tests for turbot (Scophthalmus maximus) and oyster (Crassostrea gigas) larvae, a 48 h oyster (C. gigas) embryonic development test and two full life-cycle assays using the copepods Acartia tonsa and Tisbe battagliai. Tests were also conducted in the Chesapeake Bay laboratory using estuarine species including the copepod Eurytemora affinis and the inland silverside Menidia beryllina. Here, the authors report on the results of these assays, together with 1996 in situ toxicity data resulting from Norwegian field trials in the northern North Sea.

  17. Lipase assay in soils by copper soap colorimetry.

    PubMed

    Saisuburamaniyan, N; Krithika, L; Dileena, K P; Sivasubramanian, S; Puvanakrishnan, R

    2004-07-01

    A simple and sensitive method for the estimation of lipase activity in soils is reported. In this method, 50mg of soil is incubated with emulsified substrate, the fatty acids liberated are treated with cupric acetate-pyridine reagent, and the color developed is measured at 715 nm. Use of olive oil in this protocol leads to an estimation of true lipase activity in soils. The problem of released fatty acids getting adsorbed onto the soil colloids is obviated by the use of isooctane, and separate standards for different soils need not be developed. Among the various surfactants used for emulsification, polyvinyl alcohol is found to be the most effective. Incubation time of 20 min, soil concentration of 50 mg, pH 6.5, and incubation temperature of 37 degrees C were found to be the most suitable conditions for this assay. During the process of enrichment of the soils with oil, interference by the added oil is avoided by the maintenance of a suitable control, wherein 50 mg of soil is added after stopping the reaction. This assay is sensitive and it could be adopted to screen for lipase producers from enriched soils and oil-contaminated soils before resorting to isolation of the microbes by classical screening methods.

  18. Complex enzyme hydrolysis releases antioxidative phenolics from rice bran.

    PubMed

    Liu, Lei; Wen, Wei; Zhang, Ruifen; Wei, Zhencheng; Deng, Yuanyuan; Xiao, Juan; Zhang, Mingwei

    2017-01-01

    In this study, phenolic profiles and antioxidant activity of rice bran were analyzed following successive treatment by gelatinization, liquefaction and complex enzyme hydrolysis. Compared with gelatinization alone, liquefaction slightly increased the total amount of phenolics and antioxidant activity as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Complex enzyme hydrolysis significantly increased the total phenolics, flavonoids, FRAP and ORAC by 46.24%, 79.13%, 159.14% and 41.98%, respectively, compared to gelatinization alone. Furthermore, ten individual phenolics present in free or soluble conjugate forms were also analyzed following enzymatic processing. Ferulic acid experienced the largest release, followed by protocatechuic acid and then quercetin. Interestingly, a major proportion of phenolics existed as soluble conjugates, rather than free form. Overall, complex enzyme hydrolysis releases phenolics, thus increasing the antioxidant activity of rice bran extract. This study provides useful information for processing rice bran into functional beverage rich in phenolics.

  19. Modified drug release using atmospheric pressure plasma deposited siloxane coatings

    NASA Astrophysics Data System (ADS)

    Dowling, D. P.; Maher, S.; Law, V. J.; Ardhaoui, M.; Stallard, C.; Keenan, A.

    2016-09-01

    This pilot study evaluates the potential of atmospheric plasma polymerised coatings to modify the rate of drug release from polymeric substrates. The antibiotic rifampicin was deposited in a prototype multi-layer drug delivery system, consisting of a nebulized layer of active drug between a base layer of TEOS deposited on a plastic substrate (polystyrene) and an overlying layer of plasma polymerised PDMS. The polymerised TEOS and PDMS layers were deposited using a helium atmospheric plasma jet system. Elution of rifampicin was measured using UV-VIS spectroscopy, in addition to a antimicrobial well diffusion assay with an established indicator organism. The multi-layered plasma deposited coatings significantly extended the duration of release of the rifampicin from 24 h for the uncoated polymer to 144 h for the coated polymer.

  20. Optical control of insulin release using a photoswitchable sulfonylurea

    PubMed Central

    Broichhagen, Johannes; Schönberger, Matthias; Cork, Simon C.; Frank, James A.; Marchetti, Piero; Bugliani, Marco; Shapiro, A. M. James; Trapp, Stefan; Rutter, Guy A.; Hodson, David J.; Trauner, Dirk

    2014-01-01

    Sulfonylureas are widely prescribed for the treatment of type 2 diabetes mellitus (T2DM). Through their actions on ATP-sensitive potassium (KATP) channels, sulfonylureas boost insulin release from the pancreatic beta cell mass to restore glucose homeostasis. A limitation of these compounds is the elevated risk of developing hypoglycemia and cardiovascular disease, both potentially fatal complications. Here, we describe the design and development of a photoswitchable sulfonylurea, JB253, which reversibly and repeatedly blocks KATP channel activity following exposure to violet-blue light. Using in situ imaging and hormone assays, we further show that JB253 bestows light sensitivity upon rodent and human pancreatic beta cell function. Thus, JB253 enables the optical control of insulin release and may offer a valuable research tool for the interrogation of KATP channel function in health and T2DM. PMID:25311795

  1. Optical control of insulin release using a photoswitchable sulfonylurea.

    PubMed

    Broichhagen, Johannes; Schönberger, Matthias; Cork, Simon C; Frank, James A; Marchetti, Piero; Bugliani, Marco; Shapiro, A M James; Trapp, Stefan; Rutter, Guy A; Hodson, David J; Trauner, Dirk

    2014-10-14

    Sulfonylureas are widely prescribed for the treatment of type 2 diabetes mellitus (T2DM). Through their actions on ATP-sensitive potassium (KATP) channels, sulfonylureas boost insulin release from the pancreatic beta cell mass to restore glucose homeostasis. A limitation of these compounds is the elevated risk of developing hypoglycemia and cardiovascular disease, both potentially fatal complications. Here, we describe the design and development of a photoswitchable sulfonylurea, JB253, which reversibly and repeatedly blocks KATP channel activity following exposure to violet-blue light. Using in situ imaging and hormone assays, we further show that JB253 bestows light sensitivity upon rodent and human pancreatic beta cell function. Thus, JB253 enables the optical control of insulin release and may offer a valuable research tool for the interrogation of KATP channel function in health and T2DM.

  2. A liposome-PCR assay for the ultrasensitive detection of biological toxins.

    PubMed

    Mason, Jeffrey T; Xu, Lixin; Sheng, Zong-mei; O'Leary, Timothy J

    2006-05-01

    We describe an ultrasensitive immunoassay for detecting biotoxins that uses liposomes with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as a detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time PCR. Assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods.

  3. Controlled Release from Recombinant Polymers

    PubMed Central

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  4. Payload holddown and release mechanism

    NASA Technical Reports Server (NTRS)

    Chaput, Dale; Visconti, Mark; Edwards, Michael; Moran, Tom

    1994-01-01

    A payload holddown and release mechanism, designated the Model 1172, was designed and built at G&H Technology during the winter of 1992/1993. The mechanism is able to restrain and release a 45-pound payload with minimal tipoff. The payload is held in place by a stainless steel band and released using electrically triggered non-explosive actuators. These actuators provide reliable operation with negligible shock and no special handling requirements. The performance of the mechanism was demonstrated in two flight tests. Data showed pitch and yaw tipoff rates of less than 0.07 radian (4 degree) per second. The Model 1172 design is an efficient replacement for conventional payload deployment devices, especially where low transmitted shock is required.

  5. Controlled release from recombinant polymers.

    PubMed

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed.

  6. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  7. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters

    PubMed Central

    Dold, S. -M.; Zimmermann, S.; Hamacher, K.; Schmitz, K.; Rudat, J.

    2016-01-01

    β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods—namely, the classical Z’-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening. PMID:26730596

  8. Nanostructured Diclofenac Sodium Releasing Material

    NASA Astrophysics Data System (ADS)

    Nikkola, L.; Vapalahti, K.; Harlin, A.; Seppälä, J.; Ashammakhi, N.

    2008-02-01

    Various techniques have been developed to produce second generation biomaterials for tissue repair. These include extrusion, molding, salt leaching, spinning etc, but success in regenerating tissues has been limited. It is important to develop porous material, yet with a fibrous structure for it to be biomimetic. To mimic biological tissues, the extra-cellular matrix usually contains fibers in nano scale. To produce nanostructures, self-assembly or electrospinning can be used. Adding a drug release function to such a material may advance applications further for use in controlled tissue repair. This turns the resulting device into a multifunctional porous, fibrous structure to support cells and drug releasing properties in order to control tissue reactions. A bioabsorbable poly(ɛ-caprolactone-co-D,L lactide) 95/5 (PCL) was made into diluted solution using a solvent, to which was added 2w-% of diclofenac sodium (DS). Nano-fibers were made by electrospinning onto substrate. Microstructure of the resulting nanomat was studied using SEM and drug release profiles with UV/VIS spectroscopy. Thickness of the electrospun nanomat was about 2 mm. SEM analysis showed that polymeric nano-fibers containing drug particles form a highly interconnected porous nano structure. Average diameter of the nano-fibers was 130 nm. There was a high burst peak in drug release, which decreased to low levels after one day. The used polymer has slow a degradation rate and though the nanomat was highly porous with a large surface area, drug release rate is slow. It is feasible to develop a nano-fibrous porous structure of bioabsorbable polymer, which is loaded with test drug. Drug release is targeted at improving the properties of biomaterial for use in controlled tissue repair and regeneration.

  9. Blood histamine release: A new allergy blood test

    SciTech Connect

    Faraj, B.A.; Gottlieb, G.R.; Camp, V.M.; Lollies, P.

    1985-05-01

    Allergen-mediated histamine release from human leukocytes represents an important model for in vitro studies of allergic reactions. The purpose of this study was to determine whether the measurement of histamine released in allergic patients (pts) by radioenzymatic assay following mixing of their blood with common allergens represents a reliable index for diagnosis of atopic allergy. Three categories of allergies were used: (1) housedust and mite; (2) cat and dog dander; (3) trees and grasses and ragweed mixture. The presence of allergy was established by intradermal skin testing in the study group of 82 pts. Significant atopy was defined as greater than or equal to 3+ (overall range 0-4 +, negative to maximum) on skin testing. The test was carried out in tubes with 0.5 ml heparinized blood, 0.5 ml tris albumin buffer, and one of the allergens (60-100 PNU/ml). In 20 controls without allergy, there always was less than or equal to 4% histamine release (normal response). A significant allergen-mediated histamine release, ranging from 12 to 30% of the total blood histamine content, was observed in 96% of the pts with skin test sensitivity of greater than or equal to 3+. There was good agreement between skin testing and histamine release in terms of the allergen causing the response. Thus, measurement of histamine release in blood in response to allergen challenge represents a clinically useful in vitro test for the diagnosis of atopic allergy. Because data can be obtained from a single sample and are highly quantitative, this new method should have application to the longitudinal study of allergic pts and to the assessment of interventions.

  10. Hydrocarbon release investigations in Missouri

    SciTech Connect

    Fels, J.B.

    1996-09-01

    Hydrocarbon releases are among the most common environmental problems in Missouri, as well as across the country. Old, unprotected underground storage tanks and buried piping from the tanks to pumps are notorious sources of petroleum contamination at LUST (leaking underground storage tank) sites. Missouri has an estimated 5000 LUST sites across the state with the majority being simple spills into clay-rich soils or into a shallow perched water system. However, in the southern half of the state, where residual soils and karst bedrock are not conducive to trapping such releases, significant groundwater supplies are at risk. This article discusses the process used to identify the source of contamination.

  11. Counterion release and electrostatic adsorption

    PubMed

    Sens; Joanny

    2000-05-22

    The effective charge of a rigid polyelectrolyte (PE) approaching an oppositely charged surface is studied. The cases of a weak (annealed) and strongly charged PE with condensed counterions (such as DNA) are discussed. In the most interesting case of the adsorption onto a substrate of low dielectric constant (such as a lipid membrane or a mica sheet) the condensed counterions are not always released as the PE approaches the substrate, because of the major importance of the image-charge effect. For the adsorption onto a surface with freely moving charges, the image-charge effect becomes less important and full release is often expected.

  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. FACS binding assay for analysing GDNF interactions.

    PubMed

    Quintino, Luís; Baudet, Aurélie; Larsson, Jonas; Lundberg, Cecilia

    2013-08-15

    Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

  14. Linearization of the bradford protein assay.

    PubMed

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  15. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

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

    2016-01-01

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

  16. 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. ...

  17. 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

  18. Aptamer-based liposomes improve specific drug loading and release.

    PubMed

    Plourde, Kevin; Derbali, Rabeb Mouna; Desrosiers, Arnaud; Dubath, Céline; Vallée-Bélisle, Alexis; Leblond, Jeanne

    2017-04-10

    Aptamer technology has shown much promise in cancer therapeutics for its targeting abilities. However, its potential to improve drug loading and release from nanocarriers has not been thoroughly explored. In this study, we employed drug-binding aptamers to actively load drugs into liposomes. We designed a series of DNA aptamer sequences specific to doxorubicin, displaying multiple binding sites and various binding affinities. The binding ability of aptamers was preserved when incorporated into cationic liposomes, binding up to 15equivalents of doxorubicin per aptamer, therefore drawing the drug into liposomes. Optimization of the charge and drug/aptamer ratios resulted in ≥80% encapsulation efficiency of doxorubicin, ten times higher than classical passively-encapsulating liposomal formulations and similar to a pH-gradient active loading strategy. In addition, kinetic release profiles and cytotoxicity assay on HeLa cells demonstrated that the release and therapeutic efficacy of liposomal doxorubicin could be controlled by the aptamer's structure. Our results suggest that the aptamer exhibiting a specific intermediate affinity is the best suited to achieve high drug loading while maintaining efficient drug release and therapeutic activity. This strategy was successfully applied to tobramycin, a hydrophilic drug suffering from low encapsulation into liposomes, where its loading was improved six-fold using aptamers. Overall, we demonstrate that aptamers could act, in addition to their targeting properties, as multifunctional excipients for liposomal formulations.

  19. Immunomodulatory Drugs Regulate HMGB1 Release from Activated Human Monocytes

    PubMed Central

    Schierbeck, Hanna; Wähämaa, Heidi; Andersson, Ulf; Harris, Helena Erlandsson

    2010-01-01

    Several HMGB1-specific antagonists have provided beneficial results in multiple models of inflammatory disease–preclinical trials including arthritis. Since no HMGB1-specific targeted therapy has yet reached the clinic, we have performed in vitro studies to investigate whether any of a selection of well-established antirheumatic drugs inhibit HMGB1 release as part of its mode of action. Freshly purified peripheral blood monocytes from healthy donors were stimulated in cultures with LPS and IFNγ to cause HMGB1 and TNF release detected in ELISPOT assays. Effects on the secretion were assessed in cultures supplemented with dexamethasone, cortisone, chloroquine, gold sodium thiomalate, methotrexate, colchicine, etanercept or anakinra. Pharmacologically relevant doses of dexamethasone, gold sodium thiomalate and chloroquine inhibited the extracellular release of HMGB1 in a dose-dependent mode. Immunostaining demonstrated that dexamethasone caused intracellular HMGB1 retention. No effects on HMGB1 secretion were observed in cultures with activated monocytes by any of the other studied agents. TNF production in LPS/IFNγ-activated monocytes was readily downregulated by dexamethasone and, to some extent, by chloroquine and etanercept. We conclude that dexamethasone, gold sodium thiomalate and chloroquine share a capacity to inhibit HMGB1 release from activated monocytes. PMID:20386869

  20. Triggered release of therapeutic antibodies from nanodiamond complexes.

    PubMed

    Smith, Adrienne H; Robinson, Erik M; Zhang, Xue-Qing; Chow, Edward K; Lin, Yang; Osawa, Eiji; Xi, Jianzhong; Ho, Dean

    2011-07-01

    Recent reports have revealed that detonation nanodiamonds (NDs) can serve as efficient, biocompatible, and versatile drug delivery platforms. Consequently, further investigations exploring additional therapeutic applications are warranted. Current limitations associated with the non-specific nature of intravenous drugs limit the potential of certain pharmacological agents. One such treatment that could benefit from a stable delivery platform is antibody (Ab) therapy. Determination of Ab adsorption and desorption to a ND surface was subsequently examined using the transforming growth factor β (TGF-β) antibody as a model therapeutic. ND-Ab complexes were found to be stable in water through enzyme-linked immunosorbent assays (ELISAs), UV-vis spectroscopy and TEM, with no Ab released after ten days. Released Abs were detected in extreme pH solutions (3.5), DMEM (+) serum with pH levels ranging from 4 to 10.5, and inorganic saline solutions. Preserved activity of Abs released in DMEM (+) serum was confirmed using an ELISA. These results suggest ND-Ab complexes are synthesized and stabilized in water and are triggered to release active Abs upon exposure to physiological conditions.

  1. Lithium stimulates the release of human parathyroid hormone in vitro.

    PubMed

    Birnbaum, J; Klandorf, H; Giuliano, A; Van Herle, A

    1988-06-01

    The effect of lithium on PTH release from human parathyroid tissue was studied using a perifusion system and an immunoradiometric assay for intact human PTH. Tissue was obtained from three patients undergoing surgery for thyroid disease, three patients with secondary hyperparathyroidism due to chronic renal insufficiency, and four patients with primary hyperparathyroidism due to a parathyroid adenoma. Addition of lithium in concentrations equivalent to the therapeutic serum levels normally attained in man (1.3 mmol/L) resulted in a significant (P less than 0.05) increase in PTH release under normocalcemic (1.15 mmol/L) conditions from normal and hyperplastic tissues. The magnitude of the lithium-induced response of PTH release ranged from a 1.4- to 5.3-fold increase above basal levels (perifusion with 1.15 mmol/L calcium alone) and was comparable to the response during a low calcium (0.42 mmol/L) perifusion. Although the response to lithium was delayed compared to that of hypocalcemia, PTH returned to basal levels immediately after removal of either stimulator. In contrast, parathyroid adenomas did not respond to either lithium or hypocalcemia in a characteristic manner, but, rather, functioned in an autonomous fashion with repeated pulsatile bursts of PTH release that were not suppressible even under hypercalcemic (1.70 mmol/L) conditions. These in vitro studies suggest that lithium therapy may elevate serum PTH levels in some patients and could, thus, be responsible for hypercalcemia in them.

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

    SciTech Connect

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

    1984-01-01

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

  3. Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

    PubMed Central

    Nascimento, Fabio D.; Souza, Daianne S. P.; Araujo, Mariana S.; Souza, Sinval E. G.; Sampaio, Misako U.; Nader, Helena B.; Tersariol, Ivarne L. S.; Motta, Guacyara

    2015-01-01

    Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present

  4. 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.

  5. Optical assay for biotechnology and clinical diagnosis.

    PubMed

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  6. Organic Phosphorus Characterisation in Agricultural Soils by Enzyme Addition Assays

    NASA Astrophysics Data System (ADS)

    Jarosch, Klaus; Frossard, Emmanuel; Bünemann, Else K.

    2013-04-01

    Phosphorus (P) is a non-renewable resource and it is a building block of many molecules indispensable for life. Up to 80 per cent of total soil P can be in organic form. Hydrolysability and thereby availability to plants and microorganisms differ strongly among the multitude of chemical forms of soil organic P. A recent approach to characterise organic P classes is the addition of specific enzymes which hydrolyse organic P to inorganic orthophosphate, making it detectable by colorimetry. Based on the substrate specificity of the added enzymes, conclusions about the hydrolysed forms of organic P can then be made. The aim of this study was to determine the applicability of enzyme addition assays for the characterisation of organic P species in soil:water suspensions of soils with differing properties. To this end, ten different soil samples originating from four continents, with variable pH (in water) values (4.2-8.0), land management (grassland or cropped land) and P fertilization intensity were analysed. Three different enzymes were used (acid phosphatase, nuclease and phytase). Acid phosphatase alone or in combination with nuclease was applied to determine the content of P in simple monoesters (monoester-like P) and P in DNA (DNA-like P), while P hydrolysed from myo-inositol hexakisphosphate (Ins6P-like P) was calculated from P release after incubation with phytase minus P release by acid phosphatase. To reduce sorption of inorganic P on soil particles of the suspension, especially in highly weathered soils, soil specific EDTA additions were determined in extensive pre-tests. The results of these pre-tests showed that recoveries of at least 30 per cent could be achieved in all soils. Thus, detection of even small organic P pools, such as DNA-like P, was possible in all soils if a suitable EDTA concentration was chosen. The enzyme addition assays provided information about the hydrolysable quantities of the different classes of soil organic P compounds as affected

  7. Development and Testing of a Colorimetric 96 Well Plate Assay for the Determination of HD Hydrolysis Rate in Various Formulations

    DTIC Science & Technology

    2005-01-01

    Cold Foam 1% ethylene glycol 1% benzalkonium chloride 1% polyvinylpolypyrrolidone 1% Brij 58 1% Benzyldimethyl tetradecyl ammonium chloride dihydrate...LITERA TURE CITED ..................................................................................... 15 5 FIGURES 1. Rates of Chloride Release in...Ground, MD). The HD was stabilized with tributylamine and was approximately 90% pure. Quantitative assays were conducted with a chloride electrode

  8. In vitro cell migration and invasion assays.

    PubMed

    Kramer, Nina; Walzl, Angelika; Unger, Christine; Rosner, Margit; Krupitza, Georg; Hengstschläger, Markus; Dolznig, Helmut

    2013-01-01

    Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.

  9. 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

  10. 96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

    PubMed

    Koshy, P J; Rowan, A D; Life, P F; Cawston, T E

    1999-11-15

    We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.

  11. Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay.

    PubMed

    Brawner, Andrew; Hinrichs, Steven H; Larson, Marilynn A; Lockridge, Oksana

    2016-04-05

    The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min(-1) and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 °C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.

  12. Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

    PubMed Central

    Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E.; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A.; Freyberg, Zachary

    2016-01-01

    Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

  13. Surface Bacterial-Spore Assay Using Tb3+/DPA Luminescence

    NASA Technical Reports Server (NTRS)

    Ponce, Adrian

    2007-01-01

    Equipment and a method for rapidly assaying solid surfaces for contamination by bacterial spores are undergoing development. The method would yield a total (nonviable plus viable) spore count of a surface within minutes and a viable-spore count in about one hour. In this method, spores would be collected from a surface by use of a transparent polymeric tape coated on one side with a polymeric adhesive that would be permeated with one or more reagent(s) for detection of spores by use of visible luminescence. The sticky side of the tape would be pressed against a surface to be assayed, then the tape with captured spores would be placed in a reader that illuminates the sample with ultraviolet light and counts the green luminescence spots under a microscope to quantify the number of bacterial spores per unit area. The visible luminescence spots seen through the microscope would be counted to determine the concentration of spores on the surface. This method is based on the chemical and physical principles of methods described in several prior NASA Tech Briefs articles, including Live/Dead Spore Assay Using DPA-Triggered Tb Luminescence (NPO-30444), Vol. 27, No. 3 (March 2003), page 7a. To recapitulate: The basic idea is to exploit the observations that (1) dipicolinic acid (DPA) is present naturally only in bacterial spores; and (2) when bound to Tb3+ ions, DPA triggers intense green luminescence of the ions under ultraviolet excitation; (3) DPA can be released from the viable spores by using L-alanine to make them germinate; and (4) by autoclaving, microwaving, or sonicating the sample, one can cause all the spores (non-viable as well as viable) to release their DPA. One candidate material for use as the adhesive in the present method is polydimethysiloxane (PDMS). In one variant of the method for obtaining counts of all (viable and nonviable) spores the PDMS would be doped with TbCl3. After collection of a sample, the spores immobilized on the sticky tape surface

  14. Spatial release from informational masking

    NASA Astrophysics Data System (ADS)

    Rakerd, Brad; Aaronson, Neil L.

    2001-05-01

    A new method for investigating spatial release from informational masking was developed and employed in two experiments. The new method is computer controlled and efficient. It employs the versatile coordinate response measure speech stimulus set [Bolia et al., J. Acoust. Soc. Am. 107, 1065 (2000)]. The experiments were conducted in an anechoic room, with a primary loudspeaker in front of the listener and a secondary loudspeaker at 60 deg to the right. Target messages were presented from the primary speaker only. For a standard, distractor messages, simultaneous with the target, were also presented from the primary speaker only. Spatial release was measured by presenting the distractors from both primary and secondary speakers with a temporal offset. Experiment 1 fixed the offset (secondary leading, +4 ms) and varied the number of distractors (1 to 3) and the target-to-distractor ratio (-12 to +4 dB). Masking release, sometimes as large as 10 dB, was found for all combinations of these variables. Experiment 2 varied the offset over a wide range of values. Substantial release from masking was found for both positive and negative offsets, but only in the range in which speech echoes are suppressed (<50 ms). [Work supported by NIDCD grant DC 00181.

  15. Very low shock release pyromechanisms

    NASA Astrophysics Data System (ADS)

    Soulier, Grégory; Gaechter, J. Pierre

    2003-09-01

    Pyromechanisms have long been used in space for launchers and satellites applications, particularly for release or separation purposes, such as bolt cutters, release nuts, pyrovalves, etc. They offer a great variety of uses, a high potential between the power supplied and the weight on board with, at the same time, a high reliability. However, they also feature a drawback due to the high dynamics generated by their functioning. Pyroshocks levels may damage adjacent sensible equipments (eg electronic boxes, reaction wheels,...) and require to design damping systems or to remove those equipments from the shock source. In a mechanism using standard pyrodevices, shock generation comes from three sources: 1. Pyrotechnic reaction. 2. Energy from internal parts in motion. 3. The release of structural constraints. Devices developed by E. LACROIX have the objectives to avoid the two last ones by: Using heat and gas generated by pyrotechnic effects. Reducing speed of parts in motion. Reducing release speed of mechanical constraints. In this paper, LACROIX presents two products named "PYROSOFT" and "VIROSOFT " designed by LACROIX and supported by CNES Toulouse (French Space Agency). R&T contracts.

  16. 2014 Pee Dee germplasm releases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PD 05035, PD 05041, PD 05064, PD 05069, PD 05070, PD 05071, PD 06001, and PD 06078 are noncommercial breeding lines of cotton jointly released by the Agricultural Research Service, United States Department of Agriculture, Clemson University Experiment Station, and Cotton Incorporated in 2014. These ...

  17. 7 CFR 550.29 - Press releases.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 6 2010-01-01 2010-01-01 false Press releases. 550.29 Section 550.29 Agriculture... Program Management § 550.29 Press releases. Press releases or other forms of public notification will be... opportunity to review, in advance, all written press releases and any other written information to be...

  18. 28 CFR 2.83 - Release planning.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Release planning. 2.83 Section 2.83... Release planning. (a) All grants of parole shall be conditioned on the development of a suitable release... parole date for purposes of release planning for up to 120 days without a hearing. If efforts...

  19. Index to NASA News Releases 1995

    NASA Technical Reports Server (NTRS)

    1996-01-01

    This issue of the index to NASA News Releases contains a listing of news releases distributed by the Office of Public Affairs, NASA Headquarters, during 1995. The index is arranged in six sections: Subject index, Personal name index, News release number index, Accession number index, Speeches, and News releases.

  20. Factors Affecting Release of Heat-Labile Enterotoxin by Enterotoxigenic Escherichia coli

    PubMed Central

    Kunkel, Steven L.; Robertson, Donald C.

    1979-01-01

    Various conditions affecting the release of heat-labile enterotoxin (LT) by enterotoxigenic Escherichia coli have been examined. The pH of a defined medium containing three amino acids, M-9 salts, and 0.5% glucose decreased to less than 7.0 in early log phase of growth, and no extracellular LT was detected. Adjustment of the pH at 8 h from 6.0 to 8.0 resulted in a concomitant increase in LT activity in culture supernatants. The release of cell-associated LT was significantly reduced by preincubation with protease inhibitors and increased by preincubation with trypsin. Cell-associated LT was not released by pH adjustment of cells grown at 21°C; however, polymyxin B treatment released a toxin species active in only the pigeon erythrocyte lysate (PEL) assay system. As the growth temperature was increased, polymyxin B released toxin species which exhibited both PEL and Y-1 adrenal tumor cell activity. Polymyxin B extracts of enterotoxigenic E. coli in early log phase grown at 37°C possessed only PEL activity, whereas extracts from cells in late-log and stationary phases had biological activity in both assay systems. Also, LT released by pH adjustment from mid-log to stationary phase was active in both PEL and Y-1 adrenal tumor cell assays. Gel electrophoresis of polymyxin B extracts revealed at least three molecular weight species active in either the PEL (22,000 daltons and 30,000 daltons) or both the PEL and the Y-1 adrenal tumor cell assay (72,000 daltons), depending on the growth temperature. These observations may help to explain the chemical and biological heterogeneity of most LT preparations and facilitate purification of LT by increasing the yield of enterotoxin. PMID:37162

  1. Modelling and simulations of controlled release fertilizer

    NASA Astrophysics Data System (ADS)

    Irfan, Sayed Ameenuddin; Razali, Radzuan; Shaari, Ku Zilati Ku; Mansor, Nurlidia

    2016-11-01

    The recent advancement in controlled release fertilizer has provided an alternative solution to the conventional urea, controlled release fertilizer has a good plant nutrient uptake they are environment friendly. To have an optimum plant intake of nutrients from controlled release fertilizer it is very essential to understand the release characteristics. A mathematical model is developed to predict the release characteristics from polymer coated granule. Numerical simulations are performed by varying the parameters radius of granule, soil water content and soil porosity to study their effect on fertilizer release. Understanding these parameters helps in the better design and improve the efficiency of controlled release fertilizer.

  2. Kepler Data Release 3 Notes

    NASA Technical Reports Server (NTRS)

    Cleve, Jeffrey E.

    2010-01-01

    This describes the collection of data and the processing done on it so when researchers around the world get the Kepler data sets (which are a set of pixels from the telescope of a particular target (star, galaxy or whatever) over a 3 month period) they can adjust their algorithms fro things that were done (like subtracting all of one particular wavelength for example). This is used to calibrate their own algorithms so that they know what it is they are starting with. It is posted so that whoever is accessing the publicly available data (not all of it is made public) can understand it .. (most of the Kepler data is under restriction for 1 - 4 years and is not available, but the handbook is for everyone (public and restricted) The Data Analysis Working Group have released long and short cadence materials, including FFls and Dropped Targets for the Public. The Kepler Science Office considers Data Release 3 to provide "browse quality" data. These notes have been prepared to give Kepler users of the Multimission Archive at STScl (MAST) a summary of how the data were collected and prepared, and how well the data processing pipeline is functioning on flight data. They will be updated for each release of data to the public archive and placed on MAST along with other Kepler documentation, at http:// archive.stsci.edu/kepler/documents.html .Data release 3 is meant to give users the opportunity to examine the data for possibly interesting science and to involve the users in improving the pipeline for future data releases. To perform the latter service, users are encouraged to notice and document artifacts, either in the raw or processed data, and report them to the Science Office.

  3. Menadione-catalyzed luminol luminescent assay as a novel evaluation method of ethanol tolerance in yeast cells.

    PubMed

    Yamashoji, Shiro

    2009-02-01

    In this study, ethanol inhibited the growth and glucose-induced proton release of yeast cells in a dose-dependent manner. On the other hand, ethanol tolerance of menadione-catalyzed luminol luminescence by yeast cells increased with increasing ethanol concentrations in the growth medium. The intracellular reduced-form nicotinamide adenine dinucleotide (NADH) concentration also increased with increasing ethanol concentrations in the medium and was enough to maintain constant menadione-catalyzed luminol luminescence. These facts suggest that the menadione-catalyzed luminol luminescent assay depending on a NADH:quinone reductase and NADH generation system is useful as a new evaluation assay for assessing the vitality of ethanol-stressed yeast cells, whereas the glucose-induced proton release assay is expected to be useful for the evaluation of cell growth under ethanol stress.

  4. Selection and Separation of Viable Cells Based on a Cell-Lethal Assay

    PubMed Central

    Xu, Wei; Herman, Annadele; Phillips, Colleen; Pai, Jeng-Hao; Sims, Christopher E.; Allbritton, Nancy L.

    2010-01-01

    A method to select and separate viable cells based on the results of a cell-lethal assay was developed. Cells were plated on an array of culture sites with each site composed of closely spaced, releasable micropallets. Clonal colonies spanning multiple micropallets on individual culture sites were established within 72 h of plating. Adjacent sites were widely spaced with 100% of the colonies remaining sequestered on a single culture site during expansion. A laser-based method mechanically released a micropallet underlying a colony to segment the colony into two genetically identical colonies. One portion of the segmented colony was collected with 90% efficiency while viability of both fractions was 100%. The segmented colonies released from the array were fixed and subjected to immunofluorescence staining of intracellular phospho-ERK kinase to identify colonies that were highly resistant or sensitive to phorbol ester-induced activation of ERK. These resistant and sensitive cells were then matched to the corresponding viable colonies on the array. Sensitive and resistant colonies on the array were released and cultured. When these cultured cells were reanalyzed for phorbol ester-induced ERK activity, the cells retained the sensitive or resistant phenotype of the originally screened subcolony. Thus cells were separated and collected based using the result of a cell-lethal assay as selection criteria. These microarrays enabling clonal colony segmentation permitted sampling and manipulation of the colonies at very early times and at small cell numbers to reduce reagent, time and manpower requirements. PMID:21142138

  5. 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

  6. Gelatin-based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering.

    PubMed

    Gnavi, S; di Blasio, L; Tonda-Turo, C; Mancardi, A; Primo, L; Ciardelli, G; Gambarotta, G; Geuna, S; Perroteau, I

    2017-02-01

    Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics 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; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.

    2016-02-12

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

  8. An epidermal equivalent assay for identification and ranking potency of contact sensitizers

    SciTech Connect

    Gibbs, Susan; Corsini, Emanuela; Spiekstra, Sander W.; Galbiati, Valentina; Fuchs, Horst W.; DeGeorge, George; Troese, Matthew; Hayden, Patrick; Deng, Wei; Roggen, Erwin

    2013-10-15

    The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24 h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens. A putative prediction model is proposed from data obtained from two laboratories yielding 95% accuracy. Correlating the in vitro EE sensitizer potency data, which assesses the chemical concentration which results in 50% cytotoxicity (EE-EC{sub 50}) with human and animal data showed a superior correlation with human DSA{sub 05} (μg/cm{sup 2}) data (Spearman r = 0.8500; P value (two-tailed) = 0.0061) compared to LLNA data (Spearman r = 0.5968; P value (two-tailed) = 0.0542). DSA{sub 05} = induction dose per skin area that produces a positive response in 5% of the tested population Also a good correlation was observed for release of IL-18 (SI-2) into culture supernatants with human DSA{sub 05} data (Spearman r = 0.8333; P value (two-tailed) = 0.0154). This easily transferable human in vitro assay appears to be very promising, but additional testing of a larger chemical set with the different EE models is required to fully evaluate the utility of this assay and to establish a definitive prediction model. - Highlights: • A potential epidermal equivalent assay to label and classify sensitizers • Il-18 release distinguishes sensitizers from non sensitizers • IL-18 release can rank sensitizer potency

  9. Fouling-release performance of silicone oil-modified siloxane-polyurethane coatings.

    PubMed

    Galhenage, Teluka P; Hoffman, Dylan; Silbert, Samantha D; Stafslien, Shane J; Daniels, Justin; Miljkovic, Tatjana; Finlay, John A; Franco, Sofia C; Clare, Anthony S; Nedved, Brian T; Hadfield, Michael G; Wendt, Dean E; Waltz, Grant; Brewer, Lenora; Teo, Serena Lay-Ming; Lim, Chin-Sing; Webster, Dean C

    2016-10-03

    The effect of incorporation of silicone oils into a siloxane-polyurethane fouling-release coatings system was explored. Incorporation of phenylmethyl silicone oil has been shown to improve the fouling-release performance of silicone-based fouling-release coatings through increased interfacial slippage. The extent of improvement is highly dependent upon the type and composition of silicone oil used. The siloxane-polyurethane (SiPU) coating system is a tough fouling-release solution, which combines the mechanical durability of polyurethane while maintaining comparable fouling-release performance with regard to commercial standards. To further improve the fouling-release performance of the siloxane-PU coating system, the use of phenylmethyl silicones oils was studied. Coatings formulations were prepared incorporating phenyl-methyl silicone oils having a range of compositions and viscosities. Contact angle and surface energy measurements were conducted to evaluate the surface wettability of the coatings. X-ray photoelectron spectroscopy (XPS) depth profiling experiments demonstrated self-stratification of silicone oil along with siloxane to the coating-air interface. Several coating formulations displayed improved or comparable fouling-release performance to commercial standards during laboratory biological assay tests for microalgae (Navicula incerta), macroalgae (Ulva linza), adult barnacles (Balanus amphitrite syn. Amphibalanus amphitrite) and mussels (Geukensia demissa). Selected silicone oil-modified siloxane-PU coatings also demonstrated comparable fouling-release performance in field immersion trials. In general, modifying the siloxane-PU fouling-release coatings with a small amount (1-5% wt basis) of phenylmethyl silicone oil resulted in improved performance in several laboratory biological assays and in long-term field immersion assessments.

  10. Nickel release from stainless steels.

    PubMed

    Haudrechy, P; Mantout, B; Frappaz, A; Rousseau, D; Chabeau, G; Faure, M; Claudy, A

    1997-09-01

    In 1994, a study of nickel release and allergic contact dermatitis from nickel-plated metals and stainless steels was published in this journal. It was shown that low-sulfur stainless steel grades like AISI 304, 316L or 430 (S < or = 0.007%) release less than 0.03 microgram/cm2/week of nickel in acid artificial sweat and elicit no reactions in patients already sensitized to nickel. In contrast, nickel-plated samples release around 100 micrograms/cm2/week of Ni and high-sulfur stainless steel (AISI 303-S approximately 0.3%) releases about 1.5 micrograms/cm2/week in this acid artificial sweat. Applied on patients sensitized to nickel, these metals elicit positive reactions in 96% and 14%, respectively, of the patients. The main conclusion was that low-sulfur stainless steels like AISI 304, 316L or 430, even when containing Ni, should not elicit nickel contact dermatitis, while metals having a mean corrosion resistance like a high-sulfur stainless steel (AISI 303) or nickel-plated steel should be avoided. The determining characteristic was in fact the corrosion resistance in chloride media, which, for stainless steels, is connected, among other factors, to the sulfur content. Thus, a question remained concerning the grades with an intermediate sulfur content, around 0.03%, which were not studied. They are the object of the study presented in this paper. 3 tests were performed: leaching experiments, dimethylglyoxime and HNO3 spot tests, and clinical patch tests; however, only stainless steels were tested: a low-sulfur AISI 304 and AISI 303 as references and 3 grades with a sulfur content around 0.03%: AISI 304L, AISI 304L added with Ca, AISI 304L+Cu. Leaching experiments showed that the 4 non-resulfurised grades released less than 0.5 microgram/cm2/week in acid sweat while the reulfurized AISI 303 released around or more than 0.5 microgram/cm2/week. This is explained by the poorer corrosion resistance of the resulfurized grade. Yet all these grades had the same

  11. Research highlights: digital assays on chip.

    PubMed

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

    2015-01-07

    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.

  12. Oxidant-mediated ciliary dysfunction. Possible role in airway disease

    SciTech Connect

    Burman, W.J.; Martin, W.J. 2d.

    1986-03-01

    The effects of reactive species of oxygen on the airway are not well known. This study examined the effects of hydrogen peroxide (H2O2) on the structure and function of the airway epithelium. Tracheal rings were prepared from 200 g male rats. Damage to the airway epithelium was assayed by monitoring the ciliary beat frequency, the release of 51Cr, and histology. H2O2 at concentrations of 1.0 mM and above caused a very rapid decrease in ciliary beat frequency. After ten minutes' exposure to 1.0 mM, the ciliary beat frequency was 72 +/- 20 percent of control. Release of 51Cr was a less sensitive measure with significant release occurring after four hours of exposure to ciliotoxic concentrations of H2O2. Histologic changes were not evident within the experimental time period. All toxic effects of H2O2 were completely blocked by catalase. This study shows that H2O2 causes a rapid decline in ciliary activity and suggests that oxidant-mediated ciliary dysfunction could play a role in the pathogenesis of airway disease. The ciliary beat frequency provides a sensitive, physiologically relevant parameter for the in vitro study of these diseases.

  13. Controlled Release Pulmonary Administration of Curcumin Using Swellable Biocompatible Microparticles

    PubMed Central

    El-Sherbiny, Ibrahim M.; Smyth, Hugh D. C.

    2012-01-01

    This study involves a promising approach to achieve sustained pulmonary drug delivery. Dry powder particulate carriers were engineered to allow simultaneous aerosol lung delivery, evasion of macrophage uptake, and sustained drug release through a controlled polymeric architecture. Chitosan grafted with PEG was synthesized and characterized (FTIR, EA, DSC and 2D-XRD). Then, a series of respirable amphiphilic hydrogel microparticles were developed via spray drying of curcumin-loaded PLGA nanoparticles with chitosan-grafted-PEG or chitosan. The nano and microparticles were fully characterized using an array of physicochemical analytical methods including particle size, surface morphology, dynamic swelling, density, moisture content and biodegradation rates. The PLGA nanoparticles and the hydrogel microspheres encapsulating the curcumin-loaded PLGA nanoparticles showed average size of (221-243 nm) and (3.1-3.9 μm), respectively. The developed carriers attained high swelling within a few minutes, showed low moisture content as dry powders (0.9-1.8%), desirable biodegradation rates, high drug loading (up to 97%), and good sustained release. An aerosolization study was conducted using a next generation impactor and promising aerosolization characteristics were shown. In vitro macrophage uptake studies, cytotoxicity and in-vitro TNF-α assays were performed for the investigated particles. These assays revealed promising bio-interactions for the respirable/swellable nano-micro particles developed in this study as potential carriers for sustained pulmonary drug delivery. PMID:22136259

  14. Small molecule glutaminase inhibitors block glutamate release from stimulated microglia.

    PubMed

    Thomas, Ajit G; O'Driscoll, Cliona M; Bressler, Joseph; Kaufmann, Walter; Rojas, Camilo J; Slusher, Barbara S

    2014-01-03

    Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.

  15. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.

    2016-05-01

    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

  16. Limited incision carpal tunnel release

    PubMed Central

    Gaba, Sunil; Bhogesha, Sandeep; Singh, Onkar

    2017-01-01

    Background: Carpal tunnel syndrome (CTS) is the most common peripheral entrapment neuropathy. Limited incision techniques for carpal tunnel release are gaining popularity. The main advantages of these techniques are less scar load, less pillar pain, shorter recovery, and return-to-work time. However, the completeness of release, and risk of neurovascular injury are always a concern. We devised a method of limited incision release with two mini-incisions and use of nasal speculum and a probe. We aimed to evaluate the clinical and neurological outcome of this technique. Materials and Methods: Twenty seven cases (9 male and 18 female, age 28–56 years) of isolated CTS cases were enrolled in the study. A total of 33 hands (six bilateral) underwent limited incision carpal tunnel release. In this study, two mini-incisions were used and release was done with the help of nasal speculum. Evaluation preoperatively and in 6 months and at 1-year postoperatively was done, namely, (a) clinical status examination, (b) motor testing using grip and pinch dynamometer, and (c) neurological outcome measure using nerve conduction study. Results: All the patients had good clinical and neurological outcome with no recurrence during followup. The first symptom to get relieved was night pains, with a mean of 4.5 days (range 2–14 days). Compared to pain, improvement of sensory symptoms was delayed; the mean duration was 42.8 days (range 30–90 days). Scar tenderness was present only for a mean duration of 9 days (range 7–21 days). The mean duration for patients to resume their daily activities was12 days (range 7–28 days) and to work was 32 days (range 21–90 days). The hand grip showed mean values of 45.12 ± 16.16 g/mm2 preoperatively, 62.45 ± 18.86 g/mm2 at 6 months postoperatively, and 74.87 ± 20.35 g/mm2 at 1-year postoperatively. The key pinch showed mean values of 11.27 ± 3.51 g/mm2 preoperatively, 20.181 ± 3.94 g/mm2 at 6 months postoperatively, and 27.96 ± 94.42 g/mm2

  17. Zegerid--immediate-release omeprazole.

    PubMed

    2005-04-11

    The FDA has approved marketing of Zegerid powder for oral suspension (Santarus), an immediate-release formulation of the proton-pump inhibitor (PPI) omeprazole (Prilosec, and others). All other oral PPIs are delayed-release, enteric-coated formulations designed to prevent degradation of the drug by gastric acid. Each 20- or 40-mg packet of Zegerid contains 1680 mg sodium bicarbonate, which protects the drug from gastric acid degradation. A dose of Zegerid contains 460 mg of sodium, which may be excessive for some patients. Zegerid is the first oral PPI to be approved by the FDA for reduction of risk of upper GI bleeding in critically ill patients. The drug may be useful for patients who are unable to swallow and have nasogastric (NG) tubes in place. Zegerid cost $70.00 for 14 days' treatment, compared to less than $10 for 14 tablets of Prilosec OTC.

  18. Neutron-absorber release device

    DOEpatents

    VAN Erp, Jan B.; Kimont, Edward L.

    1976-01-01

    A resettable device is provided for supporting an object, sensing when an environment reaches a critical temperature and releasing the object when the critical temperature is reached. It includes a flexible container having a material inside with a melting point at the critical temperature. The object's weight is supported by the solid material which gives rigidity to the container until the critical temperature is reached at which point the material in the container melts. The flexible container with the now fluid material inside has insufficient strength to support the object which is thereby released. Biasing means forces the container back to its original shape so that when the temperature falls below the melting temperature the material again solidifies, and the object may again be supported by the device.

  19. 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.

  20. 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.

  1. 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.

  2. A high-throughput radiometric kinase assay

    PubMed Central

    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 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

  3. 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.

  4. EDTA interference in electrochemiluminescence ACTH assay.

    PubMed

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer

    2016-11-01

    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  5. Bacillus Spore Inactivation Methods Affect Detection Assays

    PubMed Central

    Dang, Jessica L.; Heroux, Karen; Kearney, John; Arasteh, Ameneh; Gostomski, Mark; Emanuel, Peter A.

    2001-01-01

    Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment. PMID:11472945

  6. Development of an objective comedogenicity assay.

    PubMed

    Tucker, S B; Flannigan, S A; Dunbar, M; Drotman, R B

    1986-06-01

    The rabbit ear comedogenicity assay is useful as a screening procedure for evaluating agents that come in contact with human skin. Controversy exists regarding the reliability of this assay because of differences in results from various laboratories. The subjective nature of the standard method of grading may also contribute to this variation. We use a more objective comedogenicity assay that utilizes increasing follicular orifice size on the rabbit ear as a measure of comedogenic activity. A generally linear increase in the degree of follicular orifice area was noted with several agents evaluated over a four-week application period. Further, a noninvasive Silastic elastomer mold was used to allow measurement of the same follicular orifice areas over time.

  7. Enzyme immunometric assay for leukotriene C4.

    PubMed

    Volland, H; Vulliez Le Normand, B; Mamas, S; Grassi, J; Créminon, C; Ezan, E; Pradelles, P

    1994-09-30

    An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

  8. Intracellular accumulation of indium ions released from nanoparticles induces oxidative stress, proinflammatory response and DNA damage.

    PubMed

    Tabei, Yosuke; Sonoda, Akinari; Nakajima, Yoshihiro; Biju, Vasudevanpillai; Makita, Yoji; Yoshida, Yasukazu; Horie, Masanori

    2016-02-01

    Due to the widespread use of indium tin oxide (ITO), it is important to investigate its effect on human health. In this study, we evaluated the cellular effects of ITO nanoparticles (NPs), indium chloride (InCl3) and tin chloride (SnCl3) using human lung epithelial A549 cells. Transmission electron microscopy and inductively coupled plasma mass spectrometry were employed to study cellular ITO NP uptake. Interestingly, greater uptake of ITO NPs was observed, as compared with soluble salts. ITO NP species released could be divided into two types: 'indium release ITO' or 'tin release ITO'. We incubated A549 cells with indium release ITO, tin release ITO, InCl3 or SnCl2 and investigated oxidative stress, proinflammatory response, cytotoxicity and DNA damage. We found that intracellular reactive oxygen species were increased in cells incubated with indium release ITO, but not tin release ITO, InCl3 or SnCl2. Messenger RNA and protein levels of the inflammatory marker, interleukin-8, also increased following exposure to indium release ITO. Furthermore, the alkaline comet assay revealed that intracellular accumulation of indium ions induced DNA damage. Our results demonstrate that the accumulation of ionic indium, but not ionic tin, from ITO NPs in the intracellular matrix has extensive cellular effects.

  9. Intracellular accumulation of indium ions released from nanoparticles induces oxidative stress, proinflammatory response and DNA damage

    PubMed Central

    Tabei, Yosuke; Sonoda, Akinari; Nakajima, Yoshihiro; Biju, Vasudevanpillai; Makita, Yoji; Yoshida, Yasukazu; Horie, Masanori

    2016-01-01

    Due to the widespread use of indium tin oxide (ITO), it is important to investigate its effect on human health. In this study, we evaluated the cellular effects of ITO nanoparticles (NPs), indium chloride (InCl3) and tin chloride (SnCl3) using human lung epithelial A549 cells. Transmission electron microscopy and inductively coupled plasma mass spectrometry were employed to study cellular ITO NP uptake. Interestingly, greater uptake of ITO NPs was observed, as compared with soluble salts. ITO NP species released could be divided into two types: ‘indium release ITO’ or ‘tin release ITO’. We incubated A549 cells with indium release ITO, tin release ITO, InCl3 or SnCl2 and investigated oxidative stress, proinflammatory response, cytotoxicity and DNA damage. We found that intracellular reactive oxygen species were increased in cells incubated with indium release ITO, but not tin release ITO, InCl3 or SnCl2. Messenger RNA and protein levels of the inflammatory marker, interleukin-8, also increased following exposure to indium release ITO. Furthermore, the alkaline comet assay revealed that intracellular accumulation of indium ions induced DNA damage. Our results demonstrate that the accumulation of ionic indium, but not ionic tin, from ITO NPs in the intracellular matrix has extensive cellular effects. PMID:26378248

  10. Thermo-responsive hydrogels for intravitreal injection and biomolecule release

    NASA Astrophysics Data System (ADS)

    Drapala, Pawel

    In this dissertation, we develop an injectable polymer system to enable localized and prolonged release of therapeutic biomolecules for improved treatment of Age-Related Macular Degeneration (AMD). Thermo-responsive hydrogels derived from N-isopropylacrylamide (NIPAAm) and cross-linked with poly(ethylene glycol) (PEG) poly(L-Lactic acid) (PLLA) copolymer were synthesized via free-radical polymerization. These materials were investigated for (a) phase change behavior, (b) in-vitro degradation, (c) capacity for controlled drug delivery, and (d) biocompatibility. The volume-phase transition temperature (VPTT) of the PNIPAAm- co-PEG-b-PLLA hydrogels was adjusted using hydrophilic and hydrophobic moieties so that it is ca. 33°C. These hydrogels did not initially show evidence of degradation at 37°C due to physical cross-links of collapsed PNIPAAm. Only after addition of glutathione chain transfer agents (CTA)s to the precursor did the collapsed hydrogels become fully soluble at 37°C. CTAs significantly affected the release kinetics of biomolecules; addition of 1.0 mg/mL glutathione to 3 mM cross-linker accelerated hydrogel degradation, resulting in 100% release in less than 2 days. This work also explored the effect of PEGylation in order to tether biomolecules to the polymer matrix. It was demonstrated that non-site-specific PEGylation can postpone the burst release of solutes (up to 10 days in hydrogels with 0.5 mg/mL glutathione). Cell viability assays showed that at least two 20-minute buffer extraction steps were needed to remove cytotoxic elements from the hydrogels. Clinically-used therapeutic biomolecules LucentisRTM and AvastinRTM were demonstrated to be both stable and bioactive after release form PNIPAAm-co-PEG-b-PLLA hydrogels. The thermo-responsive hydrogels presented here offer a promising platform for the localized delivery of proteins such as recombinant antibodies.

  11. Cortisol Release in Response to UVB Exposure in Xiphophorus Fish

    PubMed Central

    Contreras, Adam J.; Boswell, Mikki; Downs, Kevin P.; Pasquali, Amanda; Walter, Ronald B.

    2014-01-01

    Xiphophorus fishes are comprised of 26 known species. Interspecies hybridization between select species has been utilized to produce experimental models to study melanoma development. Xiphophorus melanoma induction protocols utilize ultraviolet light (UVB) to induce DNA damage and associated downstream tumorigenesis. However, the impact of induced stress caused by the UVB treatment of the experimental animals undergoing tumor induction protocols has not been assessed. Stress is an adaptive physiological response to excessive or unpredictable environmental stimuli. The stress response in fishes may be measured by assay of cortisol released into the water. Here, we present results from investigations of stress response during experimental treatment and UVB exposure in X. maculatus Jp 163 B, X. couchianus, and F1 interspecies hybrids produced from the mating X. maculatus Jp 163 B x X. couchianus. Overall, cortisol release rates for males and females after UVB exposure showed no statistical differences. At lower UVB doses (8 and 16 kJ/m2), X. couchianus exhibited 2 fold higher levels of DNA damage then either X. maculatus or the F1 hybrid. However, based on cortisol release rates, none of the fish types tested induced a primary stress response at the UVB lower doses (8 and 16 kJ/m2). In contrast, at a very high UVB dose (32 kJ/m2) both X. maculatus and the F1 hybrid showed a 5 fold increase in cortisol release rate. To determine the effect of pigmentation on UVB induced stress, wild type and albino X. hellerii were exposed to UVB (32 kJ/m2). Albino X. hellerii exhibited 3.7 fold increase in cortisol release while wild type X. hellerii did not exhibit a significant cortisol response to UVB. Overall, the data suggest the rather low UVB doses often employed in tumour induction protocols do not induce a primary stress response in Xiphophorus fishes. PMID:24625568

  12. Real-time imaging of renin release in vitro.

    PubMed

    Peti-Peterdi, János; Fintha, Attila; Fuson, Amanda L; Tousson, Albert; Chow, Robert H

    2004-08-01

    Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin, we isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold increase in the number of both individual granules and renin-positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 microM isoproterenol. There appeared to be a quantal release of the granular contents; i.e., an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further classic signs of granule exocytosis, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS)-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool for the further study of the mechanism, regulation, and dynamics of renin release.

  13. Magnesium Modulates Actin Binding and ADP Release in Myosin Motors*

    PubMed Central

    Swenson, Anja M.; Trivedi, Darshan V.; Rauscher, Anna A.; Wang, Yuan; Takagi, Yasuharu; Palmer, Bradley M.; Málnási-Csizmadia, András; Debold, Edward P.; Yengo, Christopher M.

    2014-01-01

    We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg2+-dependent manner (0.3–9.0 mm free Mg2+) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg2+ in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg2+ in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg2+ coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg2+ concentrations, demonstrating that the ADP release rate constant is slowed by Mg2+ in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg2+ reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg2+ inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg2+-dependent alterations in actin binding. Overall, our results suggest that Mg2+ reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins. PMID:25006251

  14. Matters of life and death. How neutrophils die or survive along NET release and is "NETosis" = necroptosis?

    PubMed

    Desai, Jyaysi; Mulay, Shrikant R; Nakazawa, Daigo; Anders, Hans-Joachim

    2016-06-01

    Neutrophil extracellular trap (NET) formation is a hallmark of many disorders that involve neutrophil recruitment, tissue damage, and inflammation. As NET formation is often associated with neutrophil death, the term "NETosis" has become popular. Upon discovery that neutrophils may survive NET release, apparent misnomers, such as "vital NETosis," have been proposed. Meanwhile, it has become obvious that certain stimuli can trigger neutrophil necroptosis, a process associated with NET-like chromatin release. Here, we discuss the relationship between NET release and neutrophil death in view highlighting that many assays used in the field do not properly distinguish between the two. An updated nomenclature is needed replacing the term "NETosis" to meet the growing variety of settings leading to chromatin release with and without neutrophil death. Dissecting which triggers of NET release involve which signaling pathway will help to define drugable molecular targets that inhibit NET release and/or neutrophil necrosis in specific disorders.

  15. Sensitive radioenzymatic assay for catechol drugs

    SciTech Connect

    Durrett, L.R.; Ziegler, M.G.

    1980-01-01

    This assay measures picogram quantities of catechol drugs and endogenous catecholamines in body tissues and fluids. The catechols are converted to their 3H-O-methyl metabolites during incubation with 3H-S-adenosylmethionine then separated by solvent extraction and thin-layer chromatography. Most drugs containing the catechol structure can be radiolabeled and separated from norepinephrine and epinephrine by this technique to provide simultaneous measurement of endogenous and exogenously administered catechols. The disposition of isoproterenol in tissues and fluids of man and experimental animals is measured to illustrate the utility of this assay. The reactivity of several commonly administered catechol drugs with COMT is described and the possible implications discussed.

  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. Continuous Fluorescence Assay for Peptidoglycan Glycosyltransferases.

    PubMed

    Egan, Alexander J F; Vollmer, Waldemar

    2016-01-01

    Bacterial cell wall peptidoglycan is synthesized from its precursor lipid II by two enzymatic reactions. First, glycosyltransferases polymerize the glycan strands and second, DD-transpeptidases form cross-links between peptides of neighboring strands. Most bacteria possess bifunctional peptidoglycan synthesis enzymes capable of catalyzing both reactions. Here, we describe a continuous fluorescence glycosyltransferase assay using Dansyl-labeled lipid II as substrate. Progression of the reaction is monitored by the reduction in fluorescence over time. The assay is suitable to investigate the effect of protein interaction partners on the glycan strand synthesis activity of peptidoglycan polymerases.

  18. 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.

  19. Miniaturized detection system for handheld PCR assays

    NASA Astrophysics Data System (ADS)

    Richards, James B.; Benett, William J.; Stratton, Paul; Hadley, Dean R.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.

    2000-12-01

    We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

  20. Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis.

    PubMed

    Shipovskov, Stepan; Saunders, Aaron M; Nielsen, Jesper S; Hansen, Majken H; Gothelf, Kurt V; Ferapontova, Elena E

    2012-01-01

    Attomole (10(-18)mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16 amole detection of 1563 nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the "alarm signal" range (from 1 to 100 cells per 100mL).

  1. A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

    PubMed

    Kage, Karen L; Richardson, Paul L; Traphagen, Linda; Severin, Jean; Pereda-Lopez, Ana; Lubben, Thomas; Davis-Taber, Rachel; Vos, Melissa H; Bartley, Diane; Walter, Karl; Harlan, John; Solomon, Larry; Warrior, Usha; Holzman, Thomas F; Faltynek, Connie; Surowy, Carol S; Scott, Victoria E

    2007-03-30

    Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

  2. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

    PubMed Central

    Ackerman, Margaret E.; Moldt, Brian; Wyatt, Richard T; Dugast, Anne-Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stephanie; Berger, Christoph T.; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J; Burton, Dennis R.; Alter, Galit

    2011-01-01

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors—allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function. PMID:21192942

  3. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    PubMed

    Dozier, Jonathan K; Distefano, Mark D

    2012-02-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  4. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

    PubMed Central

    Dozier, Jonathan K; Distefano, Mark D

    2012-01-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays use either radiolabeled substrates and are discontinuous, or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format and that it can reproduce IC50 values for several previously reported FDPS inhibitors. This new method offers a simple, safe and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target. PMID:22085443

  5. 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/

  6. Fluoride release from restorative materials.

    PubMed

    Basso, Gabriela Romanini; Della Bona, Alvaro; Gobbi, Delton Luiz; Cecchetti, Dileta

    2011-01-01

    The aim of this study was to evaluate in vitro fluoride (F) release from 4 restorative materials (3M ESPE): Ketak Molar Easymix [KME - conventional glass ionomer cement (GIC)]; Rely-X luting 2 [RL2 - resin-modified GIC (RMGIC)]; Vitremer (VIT- RMGIC); and Filtek Z250 [Z250 - negative control]. Disc-shaped specimens were fabricated according to the manufacturer's instructions and placed into 10 mL of reverse osmosis water at 37°C until the analyses were done using a liquid membrane for selective F ion electrode (Orion 710). F release was evaluated every 6 h in the first day and thereafter daily during 28 days (d). The results were analyzed statistically by two-way ANOVA and Tukey's test (α=0.05). Mean F release and standard deviation values (in ppm) were: KME: 6 h- 0.34 ± 0.04; 24 h- 1.22 ± 0.30; 7 d- 0.29 ± 0.09; 14 d- 0.20 ± 0.04; 28 d- 0.16 ± 0.01; RL2: 6 h- 2.46 ± 0.48; 24 h-12.33 ± 2.93; 7 d- 1.37 ± 0.38; 14 d- 0.80 ± 0.13; 28 d- 0.80 ± 0.21; VIT: 6 h- 0.98 ± 0.35; 24 h- 4.35 ± 1.22; 7 d- 0.66 ± 0.23; 14 d- 0.40 ± 0.07; 28 d- 0.39 ± 0.08; Z250: 6 h- 0.029 ± 0.001; 24 h- 0.024 ± 0.009; 7 d- 0.023 ± 0.004; 14 d- 0.025 ± 0.001; 28 d- 0.028 ± 0.001. RL2 RMGIC released more F than the other materials in all periods. The greatest release of F occurred in the first 24 h.

  7. A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.

    PubMed

    Kraaij, Tineke; Tengström, Fredrik C; Kamerling, Sylvia W A; Pusey, Charles D; Scherer, H Ulrich; Toes, Rene E M; Rabelink, Ton J; van Kooten, Cees; Teng, Y K Onno

    2016-06-01

    A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.

  8. Validation and in vitro characterization of antibiotic-loaded bone cement release.

    PubMed

    Frutos Cabanillas, P; Díez Peña, E; Barrales-Rienda, J M; Frutos, G

    2000-11-19

    Antibiotic-loaded polymeric bone cement is used in orthopedic surgery to deliver local high concentrations of antibiotics to the tissues. The precise mechanism by which antibiotic is released from the polymeric matrix is still not very well known. This research was conducted to investigate the in vitro drug release behavior of antibiotic from acrylic bone cement. A spectrophotometric method for the quantitative analysis of gentamicin sulfate using o-phtaldialdehyde as a derivatizing reagent was thoroughly validated, in order to assure a minimum quality level of the measures. The method proved to be quick, reliable, less expensive than methods such as polarization fluorescence immunoassay and others, and therefore more convenient for the routine analysis of the numerous samples generated during in vitro liberation assays. The release of gentamicin from commercial CMW1 acrylic bone cement samples was investigated following proposed in vitro release experiments.

  9. Synapsins Differentially Control Dopamine and Serotonin Release

    PubMed Central

    Kile, Brian M.; Guillot, Thomas S.; Venton, B. Jill; Wetsel, William C.; Augustine, George J.; Wightman, R. Mark

    2010-01-01

    Synapsins are a family of synaptic vesicle proteins that are important for neurotransmitter release. Here we have used triple knockout (TKO) mice lacking all three synapsin genes to determine the roles of synapsins in the release of two monoamine neurotransmitters, dopamine and serotonin. Serotonin release evoked by electrical stimulation was identical in substantia nigra pars reticulata slices prepared from TKO and wild-type mice. In contrast, release of dopamine in response to electrical stimulation was approximately doubled in striatum of TKO mice, both in vivo and in striatal slices, in comparison to wild-type controls. This was due to loss of synapsin III, because deletion of synapsin III alone was sufficient to increase dopamine release. Deletion of synapsins also increased the sensitivity of dopamine release to extracellular calcium ions. Although cocaine did not affect the release of serotonin from nigral tissue, this drug did enhance dopamine release. Cocaine-induced facilitation of dopamine release was a function of external calcium, an effect that was reduced in TKO mice. We conclude that synapsins play different roles in the control of release of dopamine and serotonin, with release of dopamine being negatively regulated by synapsins, specifically synapsin III, while serotonin release appears to be relatively independent of synapsins. These results provide further support for the concept that synapsin function in presynaptic terminals varies according to the neurotransmitter being released. PMID:20660258

  10. Rapid Spectrophotometric Assay of Serum Iron

    DTIC Science & Technology

    1990-01-01

    conditions. Materials and methods MateriaLs Most of the chemicals used were purchased as iron -free reagents (G. Frederick Smith Co.. Columbus. OH). Water...Bedford. MA). The iron -releasing reagent was made to contain 0.5r Tween-20 (v/v), 0.55 thiourea (w/v) and 0.1% ascorbic acid (w/v). all dissolved in 0.1... reagent and 0.2 ml color-develop- ment reagent . A serum blank was prepared by adding to another test tube, 0.1 ml serum, 0.7 ml iron -releasing reagent and

  11. 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...

  12. 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...

  13. 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.

  14. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and anemia. (b) Classification. Class II. The special control for this device is FDA's “Document for...

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...