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Sample records for double antibody method

  1. Occurrence and significance of antibody to liver-specific membrane lipoprotein by double-antibody immunoprecipitation method in sera of patients with acute and chronic liver diseases.

    PubMed

    Kakumu, S; Arakawa, Y; Goji, H; Kashio, T; Yata, K

    1979-04-01

    A double-antibody immunoprecipitation method was developed for detecting antibody to liver-specific membrane lipoprotein (anti-LSP) in sera of patients with various liver diseases and primary nonhepatic autoimmune diseases. Liver-specific membrane lipoprotein prepared from normal rat livers was labeled with 125I (chloramine-T) and monospecific antibody raised in rabbits. Cross-reactivity and absorption studies demonstrated that the assay used was highly specific. The frequency and titer of anti-LSP were similar for HBsAg-positive and -negative patients with both acute and chronic liver diseases. Patients with chronic active hepatitis had the highest frequenzy (25 of 44 cases, 57%) when compared with those with chronic persistent hepatitis (5 of 23 cases, 22%) and nonalcoholic cirrhosis (8 of 21 cases, 38%). Of the anti-LSP positive cases, the mean titer in patients with chronic active hepatitis tended to be the highest. In patients recovered from acute viral hepatitis, anti-LSP was transiently positive (7 of 20 cases, 35%) in the acute phase. In those who progressed to chronic hepatitis, a late rise as well as an early rise occurred in 6 of 10 patients before the diagnosis was made. Two of 6 patients with primary biliary cirrhosis had anti-LSP, but none of 41 patients with other nonviral liver diseases and none of 60 patients with primary nonhepatic autoimmune diseases. These data indicate that an autoimmune reaction directed against LSP can be initiated during the acute phase of viral hepatitis and it may persist in chronic hepatitis in both HBsAg-positive and -negative cases.

  2. Chromosome doubling method

    DOEpatents

    Kato, Akio

    2006-11-14

    The invention provides methods for chromosome doubling in plants. The technique overcomes the low yields of doubled progeny associated with the use of prior techniques for doubling chromosomes in plants such as grasses. The technique can be used in large scale applications and has been demonstrated to be highly effective in maize. Following treatment in accordance with the invention, plants remain amenable to self fertilization, thereby allowing the efficient isolation of doubled progeny plants.

  3. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    DOEpatents

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  4. Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay.

    PubMed

    Aggerbeck, H; Nørgaard-Pedersen, B; Heron, I

    1996-04-19

    A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.

  5. Base specific binding of deoxyguanylate and deoxycytidylate antibodies to double stranded DNA.

    PubMed Central

    Jacob, A; Jacob, T M

    1982-01-01

    Antibodies raised in rabbits against deoxyguanylate and deoxycytidylate bind to 3H-lambda double stranded DNA and the binding is base specific. The concentrations of antibody populations that bind to double stranded DNA are much less than those binding to denatured DNA. Due to their low concentrations, these antibodies were not detected in earlier studies. These antibodies are expected to be useful to probe the conformational flexibilities of double stranded DNAs. PMID:6217448

  6. Methods of identification employing antibody profiles

    DOEpatents

    Francoeur, Ann-Michele

    1993-12-14

    An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of the set of individual-specific antibodies that are part of the unique antibody repertoire present in animals, by reacting an effective amount of such antibodies with a particular panel, of n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly be used to distinguish genetically, or otherwise similar individuals, or their body parts containing individual-specific antibodies.

  7. Individual-specific antibody identification methods

    DOEpatents

    Francoeur, Ann -Michele

    1989-11-14

    An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of a hithertofore unknown set of individual-specific, or IS antibodies, that are part of the unique antibody repertoire present in animals, by reacting an effective amount of IS antibodies with a particular panel, or n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly by used to distinguish genetically, or otherwise similar individuals, or their body parts containing IS antibodies. Identification of inanimate objects, particularly security documents, is similarly affected by associating with the documents, an effective amount of a particular individual's IS antibodies, or conversely, a particular panel of antigens, and forming antibody-antigen complexes with a particular panel of antigens, or a particular individual's IS antibodies, respectively. One embodiment of the instant identification method, termed the blocked fingerprint assay, has applications in the area of allergy testing, autoimmune diagnostics and therapeutics, and the detection of environmental antigens such as pathogens, chemicals, and toxins.

  8. Antibody humanization methods - a review and update.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh

    2013-01-01

    This article reviews recent advances achieved during recent years on various aspects of antibody humanization theories and techniques. Common methods for producing humanized antibodies including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting, as well as advantages and disadvantages of each of these methods and their applications are discussed.

  9. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, Françoise

    2009-01-01

    Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption but they may work in specific applications and be ineffective in other ones. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.

  10. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, Françoise

    2015-01-01

    Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption, but they may work in specific applications and be ineffective in others. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.

  11. Double-antibody based immunoassay for the detection of β-casein in bovine milk samples.

    PubMed

    Zhou, Y; Song, F; Li, Y S; Liu, J Q; Lu, S Y; Ren, H L; Liu, Z S; Zhang, Y Y; Yang, L; Li, Z H; Zhang, J H; Wang, X R

    2013-11-01

    The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1-10.0 μg mL(-1). The detection limit was 0.04 μg mL(-1). In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.

  12. Methods for Selecting Phage Display Antibody Libraries.

    PubMed

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  13. Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, F

    2001-07-01

    "Double-blotting" (DB) was developed to overcome the problem of non-specific binding of secondary antibodies in immunoblotting (IB). After it had been probed by the primary antibody, the membrane with the blotted proteins was assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules were thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remained bound to the first one. The second membrane could then be probed by the secondary antibodies without the risk of non-specific binding. This method was developed for the study of erythropoietin (EPO) in concentrated urine since a strong non-specific binding of biotinylated secondary antibodies to some urinary proteins had been observed using classical IB protocols.

  14. Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

    PubMed

    Lasne, Françoise

    2003-05-01

    "Double-blotting" (DB) has been developed to overcome the problem of nonspecific binding of secondary antibodies in immunoblotting (IB). After it has been probed by the primary antibody, the membrane with the blotted proteins is assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules are thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first one. The second membrane can then be probed by the secondary antibodies without the risk of nonspecific binding. This method has been developed for the study of erythropoietin (EPO) in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical IB protocols. However, its concept makes it usable in other applications that come up against this kind of problem.

  15. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food.

    PubMed

    Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-03-15

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 10(4)-2.8 × 10(6) cells/mL with a detection limit (LOD) of 0.9 × 10(3) cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens.

  16. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food

    PubMed Central

    Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 104–2.8 × 106 cells/mL with a detection limit (LOD) of 0.9 × 103 cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

  17. Method for preparation of single chain antibodies

    SciTech Connect

    Cheung, Nai-Kong V; Guo, Hong-fen

    2012-04-03

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  18. Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

    PubMed

    Isidro, Raymond A; Isidro, Angel A; Cruz, Myrella L; Hernandez, Siomara; Appleyard, Caroline B

    2015-12-01

    The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.

  19. Method for altering antibody light chain interactions

    DOEpatents

    Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

    2002-01-01

    A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

  20. Antibody humanization methods for development of therapeutic applications.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Feizi, Mohammad Ali Hosseinpour; Nejad, Ramezan Ali Khavari

    2014-04-01

    Recombinant antibody technologies are rapidly becoming available and showing considerable clinical success. However, the immunogenicity of murine-derived monoclonal antibodies is restrictive in cancer immunotherapy. Humanized antibodies can overcome these problems and are considered to be a promising alternative therapeutic agent. There are several approaches for antibody humanization. In this article we review various methods used in the antibody humanization process.

  1. Platelet antibody: review of detection methods

    SciTech Connect

    Schwartz, K.A.

    1988-10-01

    The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with /sup 125/I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated. 61 references.

  2. Double-antibody solid-phase radioimmunoassay: a simplified phase-separation procedure applied to various ligands

    SciTech Connect

    Tevaarwerk, G.J.M.; Boyle, D.A.; Hurst, C.J.; Anguish, I.; Uksik, P.

    1980-06-01

    The purpose was to develop a simplified and reliable method of separating free from antibody-bound ligand using a precipitating antibody linked to a cellulose derivative. Dose-response curves and control sera were set up in parallel for various pituitary and placental polypeptides, steroid hormones, insulin, glucagon, triiodothyronine, thyroxine, angiotensin I, calcitonin, gastrin, cyclic AMP, and digoxin. After first-antibody reactions had reached equilibrium, free and bound ligand were separated using a double-antibody solid-phase system in parallel with conventional methods, including dextran-coated charcoal, double-antibody precipitation, single-antibody solid phase, organic solvents, salt precipitation, and anion-exchange resins. The effect of variations in temperature, incubation time, protein content, pH, and amount of separating material added were studied. The results showed that separation was complete within 1 hr for small ligand molecules and within 2 hr for larger ones. Dose-response curves and control-sera results closely paralleled those obtained with conventional methods. The method was not affected by moderate variations in incubation variables. Nonspecific binding was less than 3% in all assays, while intra-assay and interassay coefficients of variation were similar to those obtained with conventional phase-separation methods. It is concluded that the method is a simple and rapid alternative phase-separation system. It has the advantage of being free from common nonspecific intersample variations, and can be applied to any assay system based on rabbit or guinea pig antibodies without preliminay time- or reagent-consuming titration or adjustments to establish optimum phase-separating conditions.

  3. [Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse].

    PubMed

    Liu, Chuanzhi; Niu, Yingying; Chen, Yuan'an; Wu, Cheng; Yu, Yuanhua

    2014-11-01

    We expressed 17-hydroxysteroid dehydrogenase10 (17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.

  4. Monoclonal antibodies and method for detecting dioxins and dibenzofurans

    DOEpatents

    Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

    1989-01-01

    Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

  5. A novel double antibody sandwich-lateral flow immunoassay for the rapid and simple detection of hepatitis C virus.

    PubMed

    Xiang, Tingxiu; Jiang, Zheng; Zheng, Jian; Lo, Chaoyu; Tsou, Harry; Ren, Guosheng; Zhang, Jun; Huang, Ailong; Lai, Guoqi

    2012-11-01

    The objective of this study was to screen for antigens of the hepatitis C virus (HCV) to establish a new double antibody sandwich-lateral flow immunoassay (DAS-LFIA) method for testing the presence of anti-HCV antibodies in human serum or plasma. A series of different recombinant HCV proteins in Escherichia coli cells were constructed, expressed, purified and the new DAS-LFIA strip was developed. The sensitivity and specificity of new the DAS-LFIA strip were evaluated by detecting 23 HCV-positive sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies, and 8 HCV-negative sera. A total of 300 clinical serum samples was examined by both the new DAS-LFIA strip and enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS 11.5 software. The sensitivity and specificity of the new DAS-LFIA strip were 100%. The lowest test line of the HCV DAS-LFIA strips was 2 NCU/ml. Additionally, the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%. In conclusion, our new testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV.

  6. On detecting interference from endogenous antibodies in immunoassays by doubling dilutions test.

    PubMed

    Ismail, Adel A A

    2007-01-01

    The clinical laboratory may perform additional test or tests to ascertain the integrity of immunoassay analyses when the results are in doubt. Doubling serial dilutions is one of these confirmatory tests. Doubling dilutions is a useful and informative test for detecting inaccuracy and potential interference from endogenous antibodies in immunoassays. Despite its technical simplicity, the science underpinning this test is highly complex. Appreciating such complexities could help in understanding the various outcomes of such tests from misleadingly inaccurate but virtually linear/parallel results, despite the presence of interfering antibodies in some immunoassays, to an array of non-linearity/non-parallelism synonymous with interference in others. A lack of parallelism/linearity could identify some 60% of samples with inaccurate and potentially misleading results caused by endogenous interfering antibodies. Assessing real-time binding kinetics using techniques such as non-linear regression analysis or linearised transformations such as the Scatchard plot could help in identifying more samples in which the doubling dilutions test remains linear and parallel.

  7. Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay.

    PubMed Central

    Blouin, C; Higgins, R; Gottschalk, M; Simard, J

    1994-01-01

    A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was standardized for the detection of specific antibodies following vaccination with Streptococcus suis capsular type 2 bacterins. No statistically significant increase of antibody titers was detected in vaccinated piglets compared to the nonvaccinated control group, even if a minority of piglets demonstrated an important postvaccinal response. Three of four vaccinated sows showed a low antibody response to vaccine and specific immunity was detected in piglets of only one litter of these three sows. Passive protection studies showed that none of the sera from vaccinated piglets were protective for mice whereas serum obtained from hyperimmunized pigs gave protection. PMID:8143253

  8. Composition and method for detecting cancer with technetium labeled antibody fragments

    SciTech Connect

    Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

    1984-10-23

    F(ab')/sub 2/ or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab')/sub 2/ or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab')/sub 2/ or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab')/sub 2/ or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab')/sub 2/ or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased.

  9. Methods for conjugating antibodies to nanocarriers.

    PubMed

    Wagh, Anil; Law, Benedict

    2013-01-01

    Antibodies are one of the most commonly used targeting ligands for nanocarriers, mainly because they are specific, have a strong binding affinity, and are available for a number of disease biomarkers. The bioconjugation chemistry can be a crucial factor in determining the targeting efficiency of drug delivery and should be chosen on a case-by-case basis. An antibody consists of a number of functional groups which offer many flexible options for bioconjugation. This chapter focuses on discussing some of the approaches including periodate oxidation, carbodiimide, maleimide, and heterofunctional linkers, for conjugating antibodies to different nanocarriers. The advantages and limitations are described herein. Specific examples are selected to demonstrate the experimental procedures and to illustrate the potential for applying to other nanocarrier system.

  10. Methods to Design and Synthesize Antibody-Drug Conjugates (ADCs)

    PubMed Central

    Yao, Houzong; Jiang, Feng; Lu, Aiping; Zhang, Ge

    2016-01-01

    Antibody-drug conjugates (ADCs) have become a promising targeted therapy strategy that combines the specificity, favorable pharmacokinetics and biodistributions of antibodies with the destructive potential of highly potent drugs. One of the biggest challenges in the development of ADCs is the application of suitable linkers for conjugating drugs to antibodies. Recently, the design and synthesis of linkers are making great progress. In this review, we present the methods that are currently used to synthesize antibody-drug conjugates by using thiols, amines, alcohols, aldehydes and azides. PMID:26848651

  11. Standardized Methods for Detection of Poliovirus Antibodies.

    PubMed

    Weldon, William C; Oberste, M Steven; Pallansch, Mark A

    2016-01-01

    Testing for neutralizing antibodies against polioviruses has been an established gold standard for assessing individual protection from disease, population immunity, vaccine efficacy studies, and other vaccine clinical trials. Detecting poliovirus specific IgM and IgA in sera and mucosal specimens has been proposed for evaluating the status of population mucosal immunity. More recently, there has been a renewed interest in using dried blood spot cards as a medium for sample collection to enhance surveillance of poliovirus immunity. Here, we describe the modified poliovirus microneutralization assay, poliovirus capture IgM and IgA ELISA assays, and dried blood spot polio serology procedures for the detection of antibodies against poliovirus serotypes 1, 2, and 3.

  12. Study report on a double isotope method of calcium absorption

    NASA Technical Reports Server (NTRS)

    1978-01-01

    Some of the pros and cons of three methods to study gastrointestinal calcium absorption are briefly discussed. The methods are: (1) a balance study; (2) a single isotope method; and (3) a double isotope method. A procedure for the double isotope method is also included.

  13. Method to directly radiolabel antibodies for diagnostic imaging and therapy

    DOEpatents

    Thakur, Mathew L.

    1994-01-01

    The invention is a novel method and kit for directly radiolabeling proteins such as antibodies or antibody fragments for diagnostic and therapeutic purposes. The method comprises incubating a protein-containing solution with a solution of sodium ascorbate; adding a required quantity of reduced radionuclide to the incubated protein. A kit is also provided wherein the protein and/or reducing agents may be in lyophilized form.

  14. Method to directly radiolabel antibodies for diagnostic imaging and therapy

    DOEpatents

    Thakur, Mathew L.

    1991-01-01

    The invention is a novel method and kit for directly radiolabeling proteins such as antibodies or antibody fragments for diagnostic and therapeutic purposes. The method comprises incubating a protein-containing solution with a solution of sodium ascorbate; adding a required quantity of reduced radionuclide to the incubated protein. A kit is also provided wherein the protein and/or reducing agents may be in lyophilized form.

  15. Method to directly radiolabel antibodies for diagnostic imaging and therapy

    SciTech Connect

    Thakur, M.L.

    1994-05-03

    The invention is a novel method and kit for directly radiolabeling proteins such as antibodies or antibody fragments for diagnostic and therapeutic purposes. The method comprises incubating a protein-containing solution with a solution of sodium ascorbate; adding a required quantity of reduced radionuclide to the incubated protein. A kit is also provided wherein the protein and/or reducing agents may be in lyophilized form. No Drawings

  16. Photoluminescence of double core/shell infrared (CdSeTe)/ZnS quantum dots conjugated to Pseudo rabies virus antibodies

    NASA Astrophysics Data System (ADS)

    Torchynska, T. V.; Casas Espinola, J. L.; Jaramillo Gómez, J. A.; Douda, J.; Gazarian, K.

    2013-06-01

    Double core CdSeTe/ZnS quantum dots (QDs) with emission at 800 nm (1.60 eV) have been studied by photoluminescence (PL) and Raman scattering methods in the non-conjugated state and after the conjugation to the Pseudo rabies virus (PRV) antibodies. The transformation of PL spectra, stimulated by the electric charge of antibodies, has been detected for the bioconjugated QDs. Raman scattering spectra are investigated with the aim to reveal the CdSeTe core compositions. The double core QD energy diagrams were designed that help to analyze the PL spectra and their transformation at the bioconjugation. It is revealed that the interface in double core QDs has the type II quantum well character that permits to explain the near IR optical transition (1.60 eV) in the double core QDs. It is shown that the essential transformation of PL spectra is useful for the study of QD bioconjugation with specific antibodies and can be a powerful technique in early medical diagnostics.

  17. Method for double-sided processing of thin film transistors

    DOEpatents

    Yuan, Hao-Chih; Wang, Guogong; Eriksson, Mark A.; Evans, Paul G.; Lagally, Max G.; Ma, Zhenqiang

    2008-04-08

    This invention provides methods for fabricating thin film electronic devices with both front- and backside processing capabilities. Using these methods, high temperature processing steps may be carried out during both frontside and backside processing. The methods are well-suited for fabricating back-gate and double-gate field effect transistors, double-sided bipolar transistors and 3D integrated circuits.

  18. Method for detection of antibodies for metallic elements

    DOEpatents

    Barrick, Charles W.; Clarke, Sara M.; Nordin, Carl W.

    1993-11-30

    An apparatus and method for detecting antibodies specific to non-protein antigens. The apparatus is an immunological plate containing a plurality of plastic projections coated with a non-protein material. Assays utilizing the plate are capable of stabilizing the non-protein antigens with detection levels for antibodies specific to the antigens on a nanogram level. A screening assay with the apparatus allows for early detection of exposure to non-protein materials. Specifically metallic elements are detected.

  19. Method for detection of antibodies for metallic elements

    DOEpatents

    Barrick, C.W.; Clarke, S.M.; Nordin, C.W.

    1993-11-30

    An apparatus and method for detecting antibodies specific to non-protein antigens. The apparatus is an immunological plate containing a plurality of plastic projections coated with a non-protein material. Assays utilizing the plate are capable of stabilizing the non-protein antigens with detection levels for antibodies specific to the antigens on a nanogram level. A screening assay with the apparatus allows for early detection of exposure to non-protein materials. Specifically metallic elements are detected. 10 figures.

  20. Endomysium antibodies in coeliac disease: an improved method.

    PubMed Central

    Ladinser, B; Rossipal, E; Pittschieler, K

    1994-01-01

    The ultra structural binding sites of endomysium antibodies have been studied on human umbilical cord tissue. The sensitivity and specificity of IgA endomysium antibodies were compared with recently described methods using basement membrane of smooth muscle of monkey oesophagus. Thirty adults affected by coeliac disease (10 in remission) and 75 healthy adult controls with normal intestinal mucosa (35 false antigliadin positive) were investigated. Sensitivity and correlation of endomysium antibodies with total villous atrophy in untreated coeliac disease patients were 100% on the human umbilical cord smooth muscles, and only 90% on the muscular layer of primate oesophagus. Indirect immunofluorescence was superior to peroxidase staining in detecting these IgA antibodies. The easy availability and enhanced testing sensitivity of the umbilical cord is an advance towards a better diagnostic tool for coeliac disease. Images p777-a PMID:8020804

  1. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods.

    PubMed

    Vlasak, J; Ionescu, R

    2008-12-01

    The expanding field of monoclonal antibody-based pharmaceuticals has triggered increased interest in analytical characterization of these large proteins and in understanding of their heterogeneity and degradation pathways. As a result, a large number of enzymatic modifications as well as chemical and physical degradations have been reported in monoclonal antibodies in recent years. Most heterogeneity is related to changes in the surface charge of the antibody, either directly, as a change in the number of charged residues, or indirectly as a chemical or physical alteration that changes surface-charge distribution. This review presents an overview of the sources of charge-related heterogeneity in monoclonal antibodies and the methods used for their detection. A detailed section is dedicated to deamidation of asparagine and isomerization of aspartic acid residues, two ubiquitous degradation pathways detected in antibodies and other proteins as well. Finally, kinetic modeling of the accumulation of antibody variants is presented as a tool to determine the expected fraction of molecules that have undergone one or more degradation reactions.

  2. A Generic Method for Design of Oligomer-Specific Antibodies

    PubMed Central

    Brännström, Kristoffer; Lindhagen-Persson, Malin; Gharibyan, Anna L.; Iakovleva, Irina; Vestling, Monika; Sellin, Mikael E.; Brännström, Thomas; Morozova-Roche, Ludmilla; Forsgren, Lars; Olofsson, Anders

    2014-01-01

    Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the Aβ peptide and α-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies. PMID:24618582

  3. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  4. Advanced Doubling Adding Method for Radiative Transfer in Planetary Atmospheres

    NASA Astrophysics Data System (ADS)

    Liu, Quanhua; Weng, Fuzhong

    2006-12-01

    The doubling adding method (DA) is one of the most accurate tools for detailed multiple-scattering calculations. The principle of the method goes back to the nineteenth century in a problem dealing with reflection and transmission by glass plates. Since then the doubling adding method has been widely used as a reference tool for other radiative transfer models. The method has never been used in operational applications owing to tremendous demand on computational resources from the model. This study derives an analytical expression replacing the most complicated thermal source terms in the doubling adding method. The new development is called the advanced doubling adding (ADA) method. Thanks also to the efficiency of matrix and vector manipulations in FORTRAN 90/95, the advanced doubling adding method is about 60 times faster than the doubling adding method. The radiance (i.e., forward) computation code of ADA is easily translated into tangent linear and adjoint codes for radiance gradient calculations. The simplicity in forward and Jacobian computation codes is very useful for operational applications and for the consistency between the forward and adjoint calculations in satellite data assimilation.

  5. A new method for the formulation of double nanoemulsions.

    PubMed

    Ding, Shukai; Anton, Nicolas; Akram, Salman; Er-Rafik, Meriem; Anton, Halina; Klymchenko, Andrey; Yu, Wei; Vandamme, Thierry F; Serra, Christophe A

    2017-02-22

    Double emulsions are very attractive systems for many reasons; the most important of these are their capacity to encapsulate hydrophilic and lipophilic molecules simultaneously in a single particle and their potentiality to protect fragile hydrophilic molecules from the continuous phase. Double emulsions represent a technology that is widely present down to the micrometer scale; however, double nanoemulsions, with their new potential applications as nanomedicines or diagnosis agents, currently present a significant challenge. In this study, we propose an original two-step approach for the fabrication of double nanoemulsions with a final size below 200 nm. The process consists of the formulation of a primary water-in-oil (w1/O) nanoemulsion by high-pressure homogenization, followed by the re-emulsification of this primary emulsion by a low-energy method to preserve the double nanostructure. Various characterization techniques were undertaken to confirm the double structure and to evaluate the encapsulation efficiency of a small hydrophilic probe in the inner aqueous droplets. Complementary fluorescence confocal and cryo-TEM microscopy experiments were conducted to characterize and confirm the double structure of the double nanoemulsion.

  6. A double-sandwich ELISA for identification of monoclonal antibodies suitable for sandwich immunoassays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sandwich immunoassay (sIA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated...

  7. A new method for the evaluation of antibody affinity based on serial dilutions of studied antibody-antigen mixture.

    PubMed

    Bobrovnik, S A

    2002-01-01

    A new method for the evaluation of the affinity of bivalent antibodies were suggested. This method is based on the previously published by the author the idea of using so-called coordinates of dilutions. It was shown that the suggested method allows to evaluate the affinity of antibodies with high accuracy using this simple approach. It is supposed that at some conditions the suggested method could have substantial advantages in comparison to the traditional methods. This method allows to analyze situations when antibodies are already in a mixture with antigen, for example in the bloodstream in the case of infections or autoimmune diseases. The method provides useful approach for the evaluation not only antibody affinity, but also the concentration of circulated antigen.

  8. Comparison of ABO Antibody Titers on the Basis of the Antibody Detection Method Used

    PubMed Central

    Kang, Seon Joo; Baik, Sae Yun

    2014-01-01

    Background Detection methods for ABO antibody (Ab) titers vary across laboratories, and the results are different depending on the method used. We aimed to compare titer values using different detection methods for the measurement of ABO Ab titers. Methods For ABO Ab detection, pooled group A or B red blood cells (RBCs) were reacted with each of 20 sera from blood groups A, B, or O without dithiothreitol treatment. The room-temperature (RT) incubation technique and the indirect antiglobulin test (IAT) were used in the tube test and gel card test. Flow cytometry (FCM) was performed by using anti-IgM and anti-IgG Abs. Results Regardless of the blood groups tested, the FCM assay with anti-IgM showed the highest titer compared to the tube test and gel card test with RT incubation in both. The tube test with IAT showed a higher titer than the gel card test with IAT (Gel-IAT) or FCM with anti-IgG in blood group A and B, while Gel-IAT showed the highest titer relative to the other tests, only for the anti-A Ab in blood group O. Conclusions There were significant differences in the titers depending on the detection method used, and each method showed a different detection capacity for each ABO Ab depending on the ABO blood group tested. Therefore, caution should be exercised in interpreting ABO Ab titer results, taking into consideration the detection method used and the blood group. PMID:24982835

  9. An ultrasensitive squamous cell carcinoma antigen biosensing platform utilizing double-antibody single-channel amplification strategy.

    PubMed

    Ren, Xiang; Wu, Dan; Wang, Yuhuan; Zhang, Yunhui; Fan, Dawei; Pang, Xuehui; Li, Yueyun; Du, Bin; Wei, Qin

    2015-10-15

    A novel electrochemical immunosensor was developed for ultrasensitive detection of squamous cell carcinoma antigen (SCCA), which was based on the double-antibody single-channel amplification strategy. For the first time, human immunoglobulin antibody (anti-HIgG) was used as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA). In this strategy, SCCA can be detected without using mesoporous nanometers to amplify the signal. In addition, Pd icosahedrons were first used as the connecter to immobilize the antibodies and strengthen the sensitivity. Only one touch point exists under the limited condition between a sphere and another shape in geometry, thus the Pd icosahedron is an excellent candidate as the role of connecter. Gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were synthesized as the transducing materials. The fabricated immunosensor exhibited an excellent detection limit of 2.8 pg/mL and wide linear range of 0.01-5 ng/mL. This kind of immunosensor would provide a potential application in clinical diagnosis.

  10. Double wall vacuum tubing and method of manufacture

    DOEpatents

    Stahl, Charles R.; Gibson, Michael A.; Knudsen, Christian W.

    1989-01-01

    An evacuated double wall tubing is shown together with a method for the manufacture of such tubing which includes providing a first pipe of predetermined larger diameter and a second pipe having an O.D. substantially smaller than the I.D. of the first pipe. An evacuation opening is then in the first pipe. The second pipe is inserted inside the first pipe with an annular space therebetween. The pipes are welded together at one end. A stretching tool is secured to the other end of the second pipe after welding. The second pipe is then prestressed mechanically with the stretching tool an amount sufficient to prevent substantial buckling of the second pipe under normal operating conditions of the double wall pipe. The other ends of the first pipe and the prestressed second pipe are welded together, preferably by explosion welding, without the introduction of mechanical spacers between the pipes. The annulus between the pipes is evacuated through the evacuation opening, and the evacuation opening is finally sealed. The first pipe is preferably of steel and the second pipe is preferably of titanium. The pipes may be of a size and wall thickness sufficient for the double wall pipe to be structurally load bearing or may be of a size and wall thickness insufficient for the double wall pipe to be structurally load bearing, and the double wall pipe positioned with a sliding fit inside a third pipe of a load-bearing size.

  11. Method for removal of human antibodies to native DNA from serum

    SciTech Connect

    Diamond, B.A.

    1987-09-01

    A method is described for removing human anti-native DNA antibody from a liquid sample comprising coupling monoclonal, antiidiotypic antibodies capable of binding to a shared idiotype on human anti-native DNA antibody to a medium. The idiotype shares between genetically nonidentical individuals, contacting a liquid sample to the medium to permit binding of human anti-native DNA antibody in the sample to the anti-idiotypic antibodies and separating the sample from the medium to remove the human anti-native DNA antibodies therefrom.

  12. Double sided circuit board and a method for its manufacture

    DOEpatents

    Lindenmeyer, Carl W.

    1989-07-04

    Conductance between the sides of a large double sided printed circuit board is provided using a method which eliminates the need for chemical immersion or photographic exposure of the entire large board. A plurality of through-holes are drilled or punched in a substratum according to the desired pattern, conductive laminae are made to adhere to both sides of the substratum covering the holes and the laminae are pressed together and permanently joined within the holes, providing conductive paths.

  13. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo

    PubMed Central

    Dai, Hua; Xu, Zheng-zhong; Wang, Meiling; Chen, Jun-hua; Chen, Xiang; Pan, Zhi-ming; Jiao, Xin-an

    2016-01-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ. PMID:27127340

  14. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo.

    PubMed

    Dai, Hua; Xu, Zheng-Zhong; Wang, Meiling; Chen, Jun-Hua; Chen, Xiang; Pan, Zhi-Ming; Jiao, Xin-An

    2016-04-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ.

  15. Preexisting Antibodies to an F(ab')2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment.

    PubMed

    Ruppel, Jane; Brady, Ann; Elliott, Rebecca; Leddy, Cecilia; Palencia, Marco; Coleman, Daniel; Couch, Jessica A; Wakshull, Eric

    2016-01-01

    Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab')2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab')2 CDR using anti-DLL4 F(ab')2 and a control F(ab')2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.

  16. Preexisting Antibodies to an F(ab′)2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment

    PubMed Central

    Brady, Ann; Leddy, Cecilia; Coleman, Daniel; Couch, Jessica A.

    2016-01-01

    Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab′)2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab′)2 CDR using anti-DLL4 F(ab′)2 and a control F(ab′)2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials. PMID:27413757

  17. Method for detecting pathogens attached to specific antibodies

    DOEpatents

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2005-01-25

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  18. Methods for the homology modeling of antibody variable regions.

    PubMed

    Sircar, Aroop

    2012-01-01

    Antibodies are one of the critical molecules of our immune system and are unique in their enormous diversity required for recognizing various antigens. Antibodies are protein molecules and their antigen interacting region, the fragment variable (F (V)), is typically composed of a light (V (L)) and heavy (V (H)) chain. In particular, three loops each at the tip of the V (L) and the V (H), known as the complementarity determining region (CDR) loops, are responsible for binding to the antigen. While the framework regions of the V (L) and V (H) are relatively constant across the entire repertoire of antibodies, the conformation of the CDR loops varies extensively to enable the antibody to recognize different antigens. Three-dimensional structures of antibodies illustrating the V (L)-V (H) relative orientation and the CDR conformations are needed to gain insight into antibody stability, immunogenicity, and antibody-antigen interactions. Computational modeling provides a fast and inexpensive route for generating antibody structural models. This chapter highlights the various features crucial for creating a successful antibody homology model.

  19. Completed double layer boundary element method for periodic suspensions

    NASA Astrophysics Data System (ADS)

    Fan, X.-J.; Phan-Thien, N.; Zheng, R.

    In this paper, a traction-based boundary element method is formulated and implemented for periodic suspensions. Hydrodynamic interaction of particles at infinity is handled by O'Brien's method (1979), which is suitably modified for the adjoint double layer using the mean field values of the traction and the background flow. After a deflation of the extreme eigenvalue -1 of the adjoint double layer operator, an iterative solution strategy is implemented, which solves for the traction field on the surfaces of a group of near-by particles sequentially. Ewald's summation technique is employed, by expressing the adjoint double layer kernel in two sums, one converges rapidly in real space, and the other, in the reciprocal Fourier space. The implementation is tested on a periodic suspension of spheres and spheroids in simple and elongated face-centred cubic arrays, and proved to be very accurate when compared to established results. New results for the intrinsic viscosities of periodic suspensions of cubes and spheroids from moderate to high volume fractions are reported. Based on the numerical data for suspensions of spheroids, a simple modification of the constitutive equation of Hinch and Leal (1972), which was derived for dilute suspension of spheroids, is reported, allowing the constitutive equation to reasonably fit the numerical data at moderate to high concentrations.

  20. A method for generating double-ring-shaped vector beams

    NASA Astrophysics Data System (ADS)

    Huan, Chen; Xiao-Hui, Ling; Zhi-Hong, Chen; Qian-Guang, Li; Hao, Lv; Hua-Qing, Yu; Xu-Nong, Yi

    2016-07-01

    We propose a method for generating double-ring-shaped vector beams. A step phase introduced by a spatial light modulator (SLM) first makes the incident laser beam have a nodal cycle. This phase is dynamic in nature because it depends on the optical length. Then a Pancharatnam-Berry phase (PBP) optical element is used to manipulate the local polarization of the optical field by modulating the geometric phase. The experimental results show that this scheme can effectively create double-ring-shaped vector beams. It provides much greater flexibility to manipulate the phase and polarization by simultaneously modulating the dynamic and the geometric phases. Project supported by the National Natural Science Foundation of China (Grant No. 11547017), the Hubei Engineering University Research Foundation, China (Grant No. z2014001), and the Natural Science Foundation of Hubei Province, China (Grant No. 2014CFB578).

  1. Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.

    PubMed

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.

  2. Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Alves, Nathan J; Bilgicer, Basar

    2013-05-21

    This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

  3. New Methods for Improved Double Circular-Arc Helical Gears

    NASA Technical Reports Server (NTRS)

    Litvin, Faydor L.; Lu, Jian

    1997-01-01

    The authors have extended the application of double circular-arc helical gears for internal gear drives. The geometry of the pinion and gear tooth surfaces has been determined. The influence of errors of alignment on the transmission errors and the shift of the bearing contact have been investigated. Application of a predesigned parabolic function for the reduction of transmission errors was proposed. Methods of grinding of the pinion-gear tooth surfaces by a disk-shaped tool and a grinding worm were proposed.

  4. Analysis of Double Ring Resonators using Method of Equating Fields

    NASA Astrophysics Data System (ADS)

    Althaf, Shahana

    Optical ring resonators have the potential to be integral parts of large scale photonic circuits. My thesis theoretically analyzes parallel coupled double ring resonators (DRRs) in detail. The analysis is performed using the method of equating fields (MEF) which provides an in depth understanding about the transmitted and reflected light paths in the structure. Equations for the transmitted and reflected fields are derived; these equations allow for unequal ring lengths and coupling coefficients. Sanity checks including comparison with previously studied structures are performed in the final chapter in order to prove the correctness of the obtained results.

  5. A double-correlation tremor-location method

    NASA Astrophysics Data System (ADS)

    Li, Ka Lok; Sgattoni, Giulia; Sadeghisorkhani, Hamzeh; Roberts, Roland; Gudmundsson, Olafur

    2016-12-01

    A double-correlation method is introduced to locate tremor sources based on stacks of complex, doubly-correlated tremor records of multiple triplets of seismographs back projected to hypothetical source locations in a geographic grid. Peaks in the resulting stack of moduli are inferred source locations. The stack of the moduli is a robust measure of energy radiated from a point source or point sources even when the velocity information is imprecise. Application to real data shows how double correlation focuses the source mapping compared to the common single correlation approach. Synthetic tests demonstrate the robustness of the method and its resolution limitations which are controlled by the station geometry, the finite frequency of the signal, the quality of the used velocity information and noise level. Both random noise and signal or noise correlated at time shifts that are inconsistent with the assumed velocity structure can be effectively suppressed. Assuming a surface-wave velocity, we can constrain the source location even if the surface-wave component does not dominate. The method can also in principle be used with body waves in three dimensions, although this requires more data and seismographs placed near the source for depth resolution.

  6. A double-correlation tremor-location method

    NASA Astrophysics Data System (ADS)

    Li, Ka Lok; Sgattoni, Giulia; Sadeghisorkhani, Hamzeh; Roberts, Roland; Gudmundsson, Olafur

    2017-02-01

    A double-correlation method is introduced to locate tremor sources based on stacks of complex, doubly-correlated tremor records of multiple triplets of seismographs back projected to hypothetical source locations in a geographic grid. Peaks in the resulting stack of moduli are inferred source locations. The stack of the moduli is a robust measure of energy radiated from a point source or point sources even when the velocity information is imprecise. Application to real data shows how double correlation focuses the source mapping compared to the common single correlation approach. Synthetic tests demonstrate the robustness of the method and its resolution limitations which are controlled by the station geometry, the finite frequency of the signal, the quality of the used velocity information and noise level. Both random noise and signal or noise correlated at time shifts that are inconsistent with the assumed velocity structure can be effectively suppressed. Assuming a surface wave velocity, we can constrain the source location even if the surface wave component does not dominate. The method can also in principle be used with body waves in 3-D, although this requires more data and seismographs placed near the source for depth resolution.

  7. Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

    PubMed

    Menzel, Ulrike; Greiff, Victor; Khan, Tarik A; Haessler, Ulrike; Hellmann, Ina; Friedensohn, Simon; Cook, Skylar C; Pogson, Mark; Reddy, Sai T

    2014-01-01

    High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

  8. Gold Nanorods, DNA Origami, and Porous Silicon Nanoparticle-functionalized Biocompatible Double Emulsion for Versatile Targeted Therapeutics and Antibody Combination Therapy.

    PubMed

    Kong, Feng; Zhang, Hongbo; Qu, Xiangmeng; Zhang, Xu; Chen, Dong; Ding, Ruihua; Mäkilä, Ermei; Salonen, Jarno; Santos, Hélder A; Hai, Mingtan

    2016-12-01

    Gold nanorods, DNA origami, and porous silicon nanoparticle-functionalized biocompatible double emulsion are developed for versatile molecular targeted therapeutics and antibody combination therapy. This advanced photothermal responsive all-in-one biocompatible platform can be easily formed with great therapeutics loading capacity for different cancer treatments with synergism and multidrug resistance inhibition, which has great potential in advancing biomedical applications.

  9. Immunodetection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: potential for high-throughput diagnostic methods.

    PubMed

    Tatineni, Satyanarayana; Sarath, Gautam; Seifers, Dallas; French, Roy

    2013-04-01

    Triticum mosaic virus (TriMV), an economically important virus infecting wheat in the Great Plains region of the USA, is the type species of the Poacevirus genus in the family Potyviridae. Sensitive and high-throughput serology-based detection methods are crucial for the management of TriMV and germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was expressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form recombinant CP (rCP) in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western immuno-blot and enzyme-linked immunosorbent assays (ELISA). In direct antigen coating (DAC)-ELISA, antibodies reacted specifically, beyond 1:20,000 dilution with TriMV in crude sap, but not with healthy extracts, and antiserum at a 1:10,000 dilution detected TriMV in crude sap up to 1:4860 dilution. Notably, rabbit anti-TriMV IgG and anti-TriMV IgG-alkaline phosphatase conjugate reacted positively with native virions in crude sap in a double antibody sandwich-ELISA, suggesting that these antibodies can be used as coating antibodies which is crucial for any 'sandwich' type of assays. Finally, the recombinant antibodies reacted positively in ELISA with representative TriMV isolates collected from fields, suggesting that antibodies generated against rCP can be used for sensitive, large-scale, and broad-spectrum detection of TriMV.

  10. Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

    PubMed

    Marcon, Edyta; Jain, Harshika; Bhattacharya, Anandi; Guo, Hongbo; Phanse, Sadhna; Pu, Shuye; Byram, Gregory; Collins, Ben C; Dowdell, Evan; Fenner, Maria; Guo, Xinghua; Hutchinson, Ashley; Kennedy, Jacob J; Krastins, Bryan; Larsen, Brett; Lin, Zhen-Yuan; Lopez, Mary F; Loppnau, Peter; Miersch, Shane; Nguyen, Tin; Olsen, Jonathan B; Paduch, Marcin; Ravichandran, Mani; Seitova, Alma; Vadali, Gouri; Vogelsang, Maryann S; Whiteaker, Jeffrey R; Zhong, Guoqing; Zhong, Nan; Zhao, Lei; Aebersold, Ruedi; Arrowsmith, Cheryl H; Emili, Andrew; Frappier, Lori; Gingras, Anne-Claude; Gstaiger, Matthias; Paulovich, Amanda G; Koide, Shohei; Kossiakoff, Anthony A; Sidhu, Sachdev S; Wodak, Shoshana J; Gräslund, Susanne; Greenblatt, Jack F; Edwards, Aled M

    2015-08-01

    Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

  11. Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

    NASA Astrophysics Data System (ADS)

    Huy, Tran Quang; Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi; Tuan, Mai Anh

    2011-06-01

    In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

  12. AN APPRAISAL OF THE FLUORESCENT ANTIBODY METHOD IN GONORRHOEA.

    PubMed

    OVCINNIKOV, N M

    1963-01-01

    Fluorescent antibody procedures have in a short time become valued techniques for the detection of many pathogenic micro-organisms, and are used in syphilis, for instance. A fluorescent antibody test has also been proposed for use in gonorrhoea, but the author of this paper suggests that there are still many questions to be answered before that test can be recommended for widespread practical application. In particular, large-scale further research is necessary on the antigenic structure of Neisseria gonorrhoeae, and other organisms of the Neisseria group, before reliance can be placed on the strict specificity of this test.The author also describes the fluorescent antibody technique for gonorrhoea used in the USSR, discussing its advantages and disadvantages.

  13. Machine learning methods enable predictive modeling of antibody feature:function relationships in RV144 vaccinees.

    PubMed

    Choi, Ickwon; Chung, Amy W; Suscovich, Todd J; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Kaewkungwal, Jaranit; O'Connell, Robert J; Francis, Donald; Robb, Merlin L; Michael, Nelson L; Kim, Jerome H; Alter, Galit; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-04-01

    The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.

  14. Field evaluation of mercury vapor analytical methods: comparison of the "double amalgam method" and ISO 17733.

    PubMed

    Takaya, Mitsutoshi; Joeng, Jee Yeon; Ishihara, Nobuo; Serita, Fumio; Kohyama, Norihiko

    2006-04-01

    In this study, a gold amalgam method called the "Double amalgam method" was compared with the ISO 17733 method for mercury vapor analysis method. In terms of sensitivity and ease of operation, the amalgamation method is superior to the oxidation method. Two parallel samplings were carried out in this research at a button battery factory, where the mercury vapor level in the air was about 0.001 mg/m3 and at a fluorescent lamp factory, where the mercury vapor level was about 0.015 mg/m3. In the both cases, the measured values of the two showed good agreement with each other. As these two workplaces represent typical mercury levels in industries today, the double amalgam method is applicable to working environment measurement.

  15. Antibody-directed double suicide gene therapy targeting of MUC1- positive leukemia cells in vitro and in vivo.

    PubMed

    Dong, Xiao-Ya; Wang, Wen-Qian; Zhao, Yu; Li, Xu-Dong; Fang, Zhi-Gang; Lin, Dong-Jun; Xiao, Ruo-Zhi; Huang, Ren-Wei; Pan, Guang-Jin; Liu, Jia-Jun

    2013-10-01

    Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

  16. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

    1994-01-01

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

  17. Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

    1994-08-02

    Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

  18. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    DOEpatents

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  19. [Laboratory-based evaluation of "INOVA/QUANTA Lite" to determine antinuclear antibodies (ANA) and autoantibodies to double-stranded DNA, SS-A and SS-B].

    PubMed

    Yamauchi, Megumi S; Shingaki, Naohiko; Yamane, Nobuhisa

    2012-05-01

    We evaluated QUANTA Lite reagent series (INOVA Diagnostics, CA, USA) to determine antinuclear antibodies (ANA) and autoantibodies to double-stranded (ds) DNA, SS-A and SS-B, in parallel with MESACUP (Medical & Biological Laboratories, Nagoya). Overall agreements between two reagents for qualitative interpretation ranged from 77.5% (ANA) to 99.0%(anti-SS-B antibodies). When we compared to the results by indirect fluorescent antibody (IFA) test on HEp-2 cells, QUANTA Lite ANA demonstrated better sensitivity and specificity; 92.2% versus 76.5% in sensitivity and 92.1% versus 86.8% in specificity. Also, determining anti-chromatin antibodies and IFA test onto Chrithidia luciliae demonstrated greater interpretive correlation to detect anti-ds DNA by QUANTA Lite than by MESACUP. All the discrepant sera to which QUANTA Lite SS-A gave positive interpretations were confirmed to contain the antibodies specific to SS-A 52kDa antigen, which is supplemented to QUANTA Lite capture-probes. With these results, we can conclude that QUANTA Lite has superiorities over MESACUP; (1) to detect a variety of autoantibodies consisting of ANA, (2) to have a better correlation with confirmatory tests to detect anti-ds DNA antibodies, (3)to detect additional autoantibodies specific to SS-A 52kDa antigen, and (4) to have an enough compatibility in determining anti-SS-B antibodies.

  20. Hg stable isotope analysis by the double-spike method.

    PubMed

    Mead, Chris; Johnson, Thomas M

    2010-06-01

    Recent publications suggest great potential for analysis of Hg stable isotope abundances to elucidate sources and/or chemical processes that control the environmental impact of mercury. We have developed a new MC-ICP-MS method for analysis of mercury isotope ratios using the double-spike approach, in which a solution containing enriched (196)Hg and (204)Hg is mixed with samples and provides a means to correct for instrumental mass bias and most isotopic fractionation that may occur during sample preparation and introduction into the instrument. Large amounts of isotopic fractionation induced by sample preparation and introduction into the instrument (e.g., by batch reactors) are corrected for. This may greatly enhance various Hg pre-concentration methods by correcting for minor fractionation that may occur during preparation and removing the need to demonstrate 100% recovery. Current precision, when ratios are normalized to the daily average, is 0.06 per thousand, 0.06 per thousand, 0.05 per thousand, and 0.05 per thousand (2sigma) for (202)Hg/(198)Hg, (201)Hg/(198)Hg, (200)Hg/(198)Hg, and (199)Hg/(198)Hg, respectively. This is slightly better than previously published methods. Additionally, this precision was attained despite the presence of large amounts of other Hg isotopes (e.g., 5.0% atom percent (198)Hg) in the spike solution; substantially better precision could be achieved if purer (196)Hg were used.

  1. Subgroup conflicts? Try the psychodramatic "double triad method".

    PubMed

    Verhofstadt-Denève, Leni M F

    2012-04-01

    The present article suggests the application of a psychodramatic action method for tackling subgroup conflicts in which the direct dialogue between representatives of two opposing subgroups is prepared step by step through an indirect dialogue strategy within two triads, a strategy known as the Double Triad Method (DTM). In order to achieve integration in the group as a whole, it is important that all the members of both subgroups participate actively during the entire process. The first part of the article briefly explores the theoretical background, with a special emphasis on the Phenomenological-Dialectical Personality Model (Phe-Di PModel). In the second part, the DTM procedure is systematically described through its five action stages, each accompanied with 1) a spatial representation of the consecutive actions, 2) some illustrative statements for each stage, and 3) a theoretical interpretation of the dialectically involved personality dimensions in both protagonists. The article concludes with a discussion and suggestions for more extensive applications of the DTM method, including the question of its relationships to Agazarian's functional subgrouping, psychodrama, and sociodrama.

  2. A novel antibody humanization method based on epitopes scanning and molecular dynamics simulation.

    PubMed

    Zhang, Ding; Chen, Cai-Feng; Zhao, Bin-Bin; Gong, Lu-Lu; Jin, Wen-Jing; Liu, Jing-Jun; Wang, Jing-Fei; Wang, Tian-Tian; Yuan, Xiao-Hui; He, You-Wen

    2013-01-01

    1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization.

  3. A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

    PubMed Central

    Suzuki, Takuo; Miyazaki, Chihiro; Ishii-Watabe, Akiko; Tada, Minoru; Sakai-Kato, Kumiko; Kawanishi, Toru; Kawasaki, Nana

    2015-01-01

    Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies. PMID:25891896

  4. Method for Extracting Viral Hemagglutination-Inhibiting Antibodies from the Nonspecific Inhibitors of Serum

    PubMed Central

    Altemeier, William A.; Mundon, Francis K.; Top, Franklin H.; Russell, Philip K.

    1970-01-01

    Various methods are used to remove nonspecific inhibitors from sera before titering viral hemagglutination-inhibiting antibodies. These methods have several undesirable features; some are tedious and time-consuming, some remove antibody along with nonspecific inhibitors, and different techniques are usually required to remove the nonspecific inhibitors for different viruses. This communication describes a single method that uses diethylaminoethyl-Sephadex to extract the immunoglobulin G antibodies for several viruses from nonspecific inhibitors. The procedure is fast, simple to perform, and removed the nonspecific inhibitors for influenza, Western equine encephalitis, dengue-2, and rubella viruses. Images PMID:5463576

  5. Development of a Double Antibody Sandwich ELISA for West Nile Virus Detection Using Monoclonal Antibodies against Non-Structural Protein 1

    PubMed Central

    Ding, Xi-Xia; Li, Xiao-Feng; Deng, Yong-Qiang; Guo, Yong-Hui; Hao, Wei; Qin, Cheng-Feng; Fu, Ning

    2014-01-01

    The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans. PMID:25303282

  6. Evaluation of the BioPlex 2200 ANA screen for the detection of antinuclear antibodies and comparison with conventional methods.

    PubMed

    Desplat-Jego, Sophie; Bardin, Nathalie; Larida, Bruno; Sanmarco, Marielle

    2007-08-01

    BioPlex 2200 multiplexed assays system is an automatic method allowing detection of antinuclear antibodies (ANA). The aim of our study was to evaluate the determination of 13 autoantibodies against chromatinic and nonchromatinic nuclear antigens by the BioPlex 2200 system and to compare the results achieved by this method to those obtained with our routinely used immunoassays. One thousand and four serum samples consecutively sent for ANA detection were routinely tested by indirect immunofluorescence (IIF) on HEp2 cells. Among them, 321 were also analyzed by dsDNA enzyme immunoassay (EliA) test and 657 by double immunodiffusion (DID) for extractable nuclear antigen (ENA) antibodies. All the sera were evaluated by the BioPlex 2200 ANA screen kit allowing simultaneous detection of antibodies against the following antigens: dsDNA, chromatin, SSA-52 kDa, SSA-60 kDa, SSB, Sm, Sm/RNP, RNP-A, RNP-68 kDa, Scl70, centromere B, Jo-1, and P ribosomal proteins. The kappa coefficient between BioPlex 2200 and routine tests for detection of ANA on HEp2 cells, anti-dsDNA, and anti-ENA antibodies was, respectively, 0.31, 0.66, and 0.61. The comparison with our routine tests showed numerous discrepancies between IIF ANA screening and BioPlex but a good concordance for detection of anti-dsDNA and anti-ENA specificities. BioPlex 2200 system is a rapid and sensitive method for simultaneous quantitative detection of several autoantibodies. It is perfectly well adapted to determine ANA antigenic specificities of samples found positive using initial IIF screening. The capability of this multiplexed technology to analyze simultaneously 13 ANA autoantibodies leads to the rapid availability of an "autoimmune connective tissue disease serologic profile."

  7. Comparison of conventional staining methods and monoclonal antibody-based methods for Cryptosporidium oocyst detection.

    PubMed Central

    Arrowood, M J; Sterling, C R

    1989-01-01

    The sensitivity and specificity of seven microscopy-based Cryptosporidium oocyst detection methods were compared after application to unconcentrated fecal smears. The seven methods were as follows: (i) a commercial acid-fast (AF) stain (VOLU-SOL) method, (ii) Truant auramine-rhodamine (AR) stain method, (iii) fluorescein-conjugated C1B3 monoclonal antibody (MAb) direct fluorescence method, (iv) OW3 MAb indirect fluorescence method, (v) biotinylated OW3 indirect fluorescence method, (vi) biotinylated OW3-indirect diaminobenzidine (DAB) method, and (vii) biotinylated OW3-aminoethylcarbazole (AEC) method. A total of 281 randomly collected Formalin-fixed fecal samples (submitted to the Maricopa County Health Department, Phoenix, Ariz.) and 30 known positives (Formalin-fixed and K2Cr2O7-preserved stools from our laboratory) were examined in a blind test; 32 of 311 samples (10.3%) were confirmed positive. Of the confirmed positives, 40.6% were identified by the AF method, 93.8% were identified by the AR method, 93.8% were identified by the C1B3 method, 81.3% were identified by the OW3-DAB method, 71.9% were identified by the OW3-AEC method, 100% were identified by the OW3 indirect fluorescence method, and 100% were identified by the biotinylated OW3 indirect fluorescence method. False-positives were encountered by the AF and AR methods (52.0 and 85.7% specificity, respectively), while no false-positives encountered by the MAb-based methods. Oocysts in infected tissue sections were easily detected by the MAb-based methods. Images PMID:2475523

  8. Lubrication approximation in completed double layer boundary element method

    NASA Astrophysics Data System (ADS)

    Nasseri, S.; Phan-Thien, N.; Fan, X.-J.

    This paper reports on the results of the numerical simulation of the motion of solid spherical particles in shear Stokes flows. Using the completed double layer boundary element method (CDLBEM) via distributed computing under Parallel Virtual Machine (PVM), the effective viscosity of suspension has been calculated for a finite number of spheres in a cubic array, or in a random configuration. In the simulation presented here, the short range interactions via lubrication forces are also taken into account, via the range completer in the formulation, whenever the gap between two neighbouring particles is closer than a critical gap. The results for particles in a simple cubic array agree with the results of Nunan and Keller (1984) and Stoksian Dynamics of Brady etal. (1988). To evaluate the lubrication forces between particles in a random configuration, a critical gap of 0.2 of particle's radius is suggested and the results are tested against the experimental data of Thomas (1965) and empirical equation of Krieger-Dougherty (Krieger, 1972). Finally, the quasi-steady trajectories are obtained for time-varying configuration of 125 particles.

  9. Extended operator expansion method for neutrinoless double beta decay

    NASA Astrophysics Data System (ADS)

    Hirsch, M.; Kadowaki, O.; Klapdor-Kleingrothaus, H. V.; Muto, K.; Oda, T.

    1995-03-01

    Reliable calculations of nuclear matrix elements are a prerequisite for the determination of the effective neutrino mass and other particle physics parameters from neutrinoless double beta decay. Here, the operator expansion method is improved by including Coulomb, tensor and central interactions simultaneously. Furthermore, the formalism of the OEM is extended to those matrix elements necessary to extract the right-handed parameters < λ > and < η > from 0 νββ decay. OEM includes the dependence of the nuclear matrix elements on the intermediate states implicitly and can therefore be understood as a step beyond the closure approximation. Numerical studies are carried out for the isotope76Ge combining the OEM expressions with ground-state wave functions calculated within a proton-neutron quasiparticle Random Phase Approximation (pn-QRPA) model. The influence and relative importance of central, tensor and Coulomb interactions is investigated. Within the OEM, contributions from the Coulomb force are found to be negligible in 0 νββ decay, while the tensor force leads to a moderate change of the results, of the order of (10 30)%, giving a better agreement between sets of calculations which employ different NN-interactions. Generally, results of the OEM+QRPA calculation are similar to previous calculations of 0 νββ decay matrix elements, indicating that 0 νββ decay is not sensitive to model approximations and might therefore be more accurately calculated than the strongly suppressed 2 νββ decay matrix elements.

  10. Comparison of micro column technology with conventional tube methods for antibody detection

    PubMed Central

    Garg, Sachin; Saini, Nishant; Bedi, Ravneet Kaur; Basu, Sabita

    2017-01-01

    BACKGROUND AND OBJECTIVES: Conventional tube technique (CTT) has been the mainstay for antibody detection in pretransfusion testing. There have been rapid technological advances in blood banking and methodology of crossmatch has been modified to improve the sensitivity of these tests and to enable automation. This study was done to compare the efficacy of three crossmatch techniques: CTT, tube low-ionic-strength-saline indirect antiglobulin test (tube LISS-IAT), and micro column technology (MCT) used in the blood bank serology laboratory. MATERIALS AND METHODS: In this prospective study, 150 samples from patients who had received two or more transfusions on two different occasions (with at least 72 h between two transfusions) were subjected to cross match by three different techniques – CTT, LISS-IAT, and MCT. RESULTS: A total of 16 cases with antibodies were identified in 150 patients. Out of 16 cases, 14 were clinically significant (anti-c = 5, anti-K = 4, anti-E = 2, anti-S = 2, anti-Jka = 1) and 2 nonclinically significant antibody cases (anti-Lea). MCT detected all the 14 clinically significant antibody cases and no case of nonclinically significant antibody. Tube LISS-IAT detected 14 antibody cases including 2 cases of non-clinically significant antibody but failed to detect 1 case of anti-c and the only case of anti-Jka. CTT detected only 10 antibody cases including 2 cases of non-clinically significant antibody and but failed to detect 3 cases of anti-c, 1 case of anti-K, 1 case of anti-E, and the only case of anti-Jka. CONCLUSION: MCT was found to be most efficacious when compared to CTT and tube LISS-IAT in detecting clinically significant red cell antibodies; although MCT missed 2 cases of Lea antibody which were detected by CTT and LISS-IAT. PMID:28367023

  11. Development and evaluation of a double antibody sandwich ELISA for the detection of human sDC-SIGN.

    PubMed

    Chen, Shang-Liang; Li, Yan-Li; Tang, Yuan; Chen, Zhi-Cheng; Zhou, Jing; Zhou, Jia; Lu, Xiao; Zhao, Na; Chen, Zheng-Liang; Zuo, Daming

    2016-09-01

    sDC-SIGN is the soluble form of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, CD209), which is a molecule involved with pathogen recognition and immune regulation. However, there is no commercially available ELISA kit for detecting human sDC-SIGN, and the normal range of this molecule is unknown. Here, we describe an ELISA for detecting human sDC-SIGN with high specificity. First, sDC-SIGN protein was expressed and purified. Monoclonal and polyclonal antibodies were then raised against the purified protein and subsequently characterized. A sandwich ELISA was developed using polyclonal antibodies specific for sDC-SIGN for capture and a biotin-labeled monoclonal antibody specific for sDC-SIGN for detection of protein. This method has sensitivity up to 0.2 ng/ml. Using this ELISA, we found that the concentration of sDC-SIGN in sera of healthy volunteers ranges from 0-319 ng/ml with a mean concentration of 27.14 ng/ml. Interestingly, the concentration of sDC-SIGN in sera from patients with cancer or chronic hepatitis B virus (CHB) infection was lower than that of health controls. The mean concentrations of sDC-SIGN in cancer patients and chronic hepatitis B virus infection patients were 3.2 ng/ml and 3.8 ng/ml, respectively. We developed a sandwich ELISA for detecting human sDC-SIGN and demonstrated its use by assessing sera concentrations of sDC-SIGN in patients with cancer and chronic CHB infection compared to that of healthy controls.

  12. Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies.

    PubMed

    Ghosh, Raja

    2006-02-05

    This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.

  13. Improved method for the direct labeling of antibodies with Tc-99m

    SciTech Connect

    Rhodes, B.A.; Hawkins, E.; Budd, P. ); Deleide, G.; Seccamani, E.; Bonino, C. )

    1990-01-01

    Antibodies and antibody fragments have been treated with stannous chloride or organic reducing agents to reduce disulfide bonds, providing sulfhydryl groups for binding reduced Tc-99m. The reduced antibody, additives and stannous salts are lyophilized. To radiolabel, sodium pertechnetate solution is added, which dissolves the protein and other reagents. The pertechnetate is reduced by the stannous ions and becomes bound to the antibody. After radiolabeling the shelf-life of the product exceeds the half-life of the radionuclide. One of the more effective additives is human serum albumin, which serves as a carrier protein, agent to protect against autoradiolysis and possibly as a transfer ligand. Many different antibodies have been labeled using this method. The most widely studied antibody is an anti-melanoma fragment which has now been used clinically in more than 1000 cases and has been proven effective for the diagnostic localization of melanoma. In summary, a single vial, one step procedure for the direct labeling of antibodies in the presence of human serum albumin has been developed, extensively characterized, and clinically validated. The method is used with murine monoclonal IgG fragments, IgM and human gamma globulin. 4 refs., 10 figs.

  14. The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-double-stranded DNA antibodies from patients with systemic lupus erythematosus

    PubMed Central

    Tyrrell-Price, J; Lydyard, P M; Isenberg, D A

    2001-01-01

    IL-10 and IL-12 are cytokines which are important in regulating immune responses. Plasma levels of IL-10 and autoantibodies against double-stranded DNA (dsDNA) often mirror disease activity in patients with SLE. IL-12 secretion from SLE patients' blood mononuclear cells also correlates with disease activity, but has an inverse relationship. The aim of this study was to measure the effect of IL-10 and of IL-12 on the production of IgG autoantibodies from patients with SLE, both cross-sectionally and longitudinally. Peripheral blood mononuclear cells (PBMC) were cultured with IL-10 (at 20 ng/ml or 2 ng/ml) or IL-12 (at 2 ng/ml or 0·2 ng/ml) or without cytokine and the supernatanants tested for the production of double-stranded DNA antibodies (dsDNA abs), single-stranded DNA antibodies (ssDNA abs) and total IgG antibodies (IgG abs) by ELISA. The BILAG disease activity index was recorded at each patient visit (a global score of six or more is regarded as active disease). In general, treatment with IL-10 caused PBMCs from patients with inactive disease to increase their antissDNA and dsDNA ab production (by upto 354% and 186%, respectively) while patients with active disease decreased their antibody production (by upto 91% and 97%, respectively). Overall there was a correlation between disease activity and change in antissDNA and dsDNA ab production (r = − 0·51; P = 0·03 and r = − 0·48; P = 0·042, respectively). Treatment with IL-12 at 0·2 ng/ml inhibited antissDNA and dsDNA antibody production, having the greatest effect on patients with active disease (decreasing antissDNA and dsDNA antibody production by upto 75% and 73%, respectively). This resulted in a significant correlation between disease activity and change in antissDNA antibody production (r = − 0·76; P = 0·03), but significance was not reached with antidsDNA antibody production (P = 0·06). Together these data suggest that the effect of these cytokines on antibody production by SLE PBMCs

  15. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

    1991-01-01

    Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

  16. Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

    DOEpatents

    Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

    1991-10-01

    Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

  17. Double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor.

    PubMed

    Qi, Honglan; Li, Min; Zhang, Rui; Dong, Manman; Ling, Chen

    2013-08-20

    A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at -0.25V in 0.10M PBS (pH 7.0) containing 0.1mM hydroquinone and 2.0mM H2O2. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0×10(-10)g mL(-1) to 1.0×10(-8)g mL(-1) with a detection limit of 3×10(-11)g mL(-1). Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array.

  18. Antibody immobilization using pneumatic spray: comparison with the avidin-biotin bridge immobilization method.

    PubMed

    Figueroa, Jhon; Magaña, Sonia; Lim, Daniel V; Schlaf, Rudy

    2012-12-14

    The formation of a thin antibody film on a glass surface using pneumatic spray was investigated as a potential immobilization technique for capturing pathogenic targets. Goat-Escherichia coli O157:H7 IgG films were made by pneumatic spray and compared against the avidin-biotin bridge immobilized films by assaying with green fluorescent protein (GFP) transformed E. coli O157:H7 cells and fluorescent reporter antibodies. Functionality, stability, and immobilization of the films were tested. The pneumatic spray films had lower fluorescence intensity values than the avidin-biotin bridge films but resulted in similar detection for E. coli O157:H7 at 10(5)-10(7)cells/ml sample concentrations with no detection of non-E. coli O157:H7 strains. Both methods also resulted in similar percent capture efficiencies. The results demonstrated that immobilization of antibody via pneumatic spray did not render the antibody non-functional and produced stable antibody films. The amount of time necessary for immobilization of the antibody was reduced significantly from 24h for the avidin-biotin bridge to 7 min using the pneumatic spray technique, with additional benefits of greatly reduced use of materials and chemicals. The pneumatic spray technique promises to be an alternative for the immobilization of antibodies on glass slides for capturing pathogenic targets and use in biosensor type devices.

  19. Redoublement lexical, procede intensif (Lexical Doubling, Intensive Method).

    ERIC Educational Resources Information Center

    George, Kenneth E. M.

    1983-01-01

    An often-neglected aspect of daily language is syllable doubling or repetition, as in infant language ("nounou"), onomatopoeia ("ronron"), interjections or responses ("oui oui"), names ("Mimi"), or military slang ("coco" for "commandant"). The mechanisms and semantic functions of this phenomenon are outlined, drawing on examples from French…

  20. Methods of preparing and using single chain anti-tumor antibodies

    SciTech Connect

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  1. A novel screening method to assess developability of antibody-like molecules

    PubMed Central

    Kohli, Neeraj; Jain, Nidhi; Geddie, Melissa L; Razlog, Maja; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Monoclonal antibodies and antibody-like molecules represent a fast-growing class of bio-therapeutics that has rapidly transformed patient care in a variety of disease indications. The discovery of antibodies that bind to particular targets with high affinity is now a routine exercise and a variety of in vitro and in vivo techniques are available for this purpose. However, it is still challenging to identify antibodies that, in addition to having the desired biological effect, also express well, remain soluble at different pH levels, remain stable at high concentrations, can withstand high shear stress, and have minimal non-specific interactions. Many promising antibody programs have ultimately failed in development due to the problems associated with one of these factors. Here, we present a simple high-performance liquid chromatography (HPLC)-based screening method to assess these developability factors earlier in discovery process. This method is robust and requires only microgram quantities of proteins. Briefly, we show that for antibodies injected on a commercially available pre-packed Zenix HPLC column, the retention times are inversely related to their colloidal stability with antibodies prone to precipitation or aggregation retained longer on the column with broader peaks. By simply varying the salt content of running buffer, we were also able to estimate the nature of interactions between the antibodies and the column. We believe this approach should generally be applicable to assessment of the developability of other classes of bio-therapeutic molecules, and that the addition of this simple tool early in the discovery process will lead to selection of molecules with improved developability characteristics. PMID:25961854

  2. Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and αVβ3 Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells

    PubMed Central

    Li, Jiale; Zhou, Ping; Li, Lan; Zhang, Yan; Shao, Yang; Tang, Li; Tian, Shuangming

    2016-01-01

    Objective Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin αVβ3 over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-αVβ3 integrin monoclonal antibodies into cationic microbubbles (CMBsαvβ3), and evaluated its killing effect in HCC cells. Methods To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, αVβ3 integrin antibody was conjugated to CMBs, and CMBsαvβ3 was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBsαvβ3 was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBsαvβ3 were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBsαvβ3 and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBsαvβ3 and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBsαvβ3, HCC cells with CMBsαvβ3 were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL

  3. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  4. An impedance spectroscopy method for the detection and evaluation of Babesia bovis antibodies in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An immunosensor method for diagnosis of Babesia bovis in cattle based on impedance measurement is presented in this study. The method probes the interaction between serum antibodies against B. bovis infected cattle and recombinant protein, RAP-1, with C-terminal obtained from a Portuguese B. bovis s...

  5. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    PubMed Central

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  6. Methods for detection of West Nile virus antibodies in mosquito blood meals.

    PubMed

    Komar, Nicholas; Panella, Nicholas A; Young, Ginger R; Basile, Alison J

    2015-03-01

    We describe and compare 2 qualitative serologic techniques for detecting West Nile virus (WNV)-specific antibodies in mosquito blood meals. The techniques are the biotin microsphere immunoassay (b-MIA) and the inhibition platform of the VectorTest™ WNV antigen assay (VecTest-inhibition). To demonstrate the ability of these tests to detect WNV-neutralizing antibodies, we experimentally exposed feeding mosquitoes to blood containing 5 concentrations of 6B6C-1, a flavivirus-neutralizing monoclonal antibody. Antibody concentrations were quantified using the 90% plaque-reduction neutralization test (PRNT90). After 24 h of blood-meal digestion at 22.5°C, the threshold PRNT90 titer of detection was ≤18 for b-MIA and ≤50 for VecTest-inhibition. Both tests reliably detected antibodies in 3 of 3 blood meals that had been digested for up to 30 h, or were about 25% digested. The b-MIA was also applied to mosquitoes that had engorged on avian blood in Arizona following a WNV epidemic in 2010. There was no significant difference in the WNV antibody prevalence determined by b-MIA (52% of 71 avian blood meals) compared to the WNV-neutralizing antibody prevalence in birds determined by direct sampling (49% of 234 birds). VecTest-inhibition requires fewer resources and may be used in the field without a laboratory, but consumes the entire blood meal and relies on subjective interpretation of results. The b-MIA requires a laboratory and sophisticated equipment and reagents. Results for b-MIA are analyzed objectively and can be applied to mosquito blood meals with greater confidence than the VecTest-inhibition method and thus can contribute substantially to research and surveillance programs that would benefit from the detection of specific WNV antibodies in mosquito blood meals.

  7. Augmentation of influenza antibody response in elderly men by thymosin alpha one. A double-blind placebo-controlled clinical study.

    PubMed

    Gravenstein, S; Duthie, E H; Miller, B A; Roecker, E; Drinka, P; Prathipati, K; Ershler, W B

    1989-01-01

    Influenza remains a major cause of illness and death in elderly people despite current vaccination programs. One factor is an immunization failure rate in the elderly that may be as high as 50%. To test whether administration of thymosin alpha 1 would result in greater antibody production, we administered it (900 micrograms/m2 subcutaneously twice weekly for eight doses) in conjunction with the 1986 trivalent influenza vaccine. Ninety men (65-99 years old, mean age 77.3 years) were randomized double-blind to receive thymosin alpha 1 or placebo by the same schedule; the sera from 85 of these men were acceptable for analysis. The two groups were similar with respect to underlying disease, medications, and age. No toxicity was observed in either group. Antibody response rate was defined as a four-fold rise in antibody titer over 3-6 weeks following vaccination and was measured by an enzyme-linked immunosorbent assay (ELISA). Analysis was performed on treatment groups and subgroups divided by the mean age: the older group consisted of subjects aged 77 years and older, and the younger group those aged from 65-76 years. Baseline and change in absolute antibody levels were compared by t test and using age as a continuous variable by multiple regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Interferometric Methods of Measuring Refractive Indices and Double-Refraction of Fibres.

    ERIC Educational Resources Information Center

    Hamza, A. A.; El-Kader, H. I. Abd

    1986-01-01

    Presents two methods used to measure the refractive indices and double-refraction of fibers. Experiments are described, with one involving the use of Pluta microscope in the double-beam interference technique, the other employing the multiple-beam technique. Immersion liquids are discussed that can be used in the experiments. (TW)

  9. Double Cross-Validation in Multiple Regression: A Method of Estimating the Stability of Results.

    ERIC Educational Resources Information Center

    Rowell, R. Kevin

    In multiple regression analysis, where resulting predictive equation effectiveness is subject to shrinkage, it is especially important to evaluate result replicability. Double cross-validation is an empirical method by which an estimate of invariance or stability can be obtained from research data. A procedure for double cross-validation is…

  10. Glycation of antibodies: Modification, methods and potential effects on biological functions.

    PubMed

    Wei, Bingchuan; Berning, Kelsey; Quan, Cynthia; Zhang, Yonghua Taylor

    2017-03-08

    Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

  11. Method to directly radiolabel antibodies for diagnostic imaging and therapy

    SciTech Connect

    Thakur, M.L.

    1991-04-30

    This patent describes a method for directly labeling proteins with radionuclides for use in diagnostic imaging and therapy. It comprises: the steps of incubating a protein-containing solution with a solution of sodium ascorbate; adding a required quantity of reduced radionuclide to the incubated protein-containing solution and incubating.

  12. Inverse scattering method and soliton double solution family for the general symplectic gravity model

    SciTech Connect

    Gao Yajun

    2008-08-15

    A previously established Hauser-Ernst-type extended double-complex linear system is slightly modified and used to develop an inverse scattering method for the stationary axisymmetric general symplectic gravity model. The reduction procedures in this inverse scattering method are found to be fairly simple, which makes the inverse scattering method applied fine and effective. As an application, a concrete family of soliton double solutions for the considered theory is obtained.

  13. High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation.

    PubMed

    Li, Xiaojuan; Xu, Wei; Wang, Yi; Zhao, Jia; Liu, Yan-Hui; Richardson, Daisy; Li, Huijuan; Shameem, Mohammed; Yang, Xiaoyu

    2016-08-19

    Oxidation of therapeutic monoclonal antibodies (mAbs) often occurs on surface exposed methionine and tryptophan residues during their production in cell culture, purification, and storage, and can potentially impact the binding to their targets. Characterization of site specific oxidation is critical for antibody quality control. Antibody oxidation is commonly determined by peptide mapping/LC-MS methods, which normally require a long (up to 24h) digestion step. The prolonged sample preparation procedure could result in oxidation artifacts of susceptible methionine and tryptophan residues. In this paper, we developed a rapid and simple UV based peptide mapping method that incorporates an 8-min trypsin in-solution digestion protocol for analysis of oxidation. This method is able to determine oxidation levels at specific residues of a mAb based on the peptide UV traces within <1h, from either TBHP treated or UV light stressed samples. This is the simplest and fastest method reported thus far for site specific oxidation analysis, and can be applied for routine or high throughput analysis of mAb oxidation during various stability and degradation studies. By using the UV trace, the method allows more accurate measurement than mass spectrometry and can be potentially implemented as a release assay. It has been successfully used to monitor antibody oxidation in real time stability studies.

  14. A method for double-labeling sputum cells for p53 and cytokeratin

    SciTech Connect

    Neft, R.E.; Tierney, L.A.; Belinsky, S.A.

    1995-12-01

    Molecular and immunological techniques may enhance the usefulness of sputum cytology as a screening tool for lung cancer. These techniques may also be useful in detecting and following the early progression of disease from metaplasia to dysplasia, carcinoma in situ, and finally to invasive carcinoma. Longitudinal information on the evolution of these malignant changes in the respiratory epithelium can be gained by prospective study of populations at high risk for lung cancer. This work is significant because double-labeling of cells in sputum with p53 and cytokeratin antibodies facilitates rapid screening of p53 positive neoplastic and preneoplastic lung cells by brightfield and fluorescence microscopy.

  15. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

    1992-01-01

    Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

  16. Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

    DOEpatents

    Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

    1992-04-28

    Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

  17. Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae.

    PubMed

    Okada, Munenori; Asai, Tetsuo; Futo, Satoshi; Mori, Yasuyuki; Mukai, Tetsuya; Yazawa, Shigeto; Uto, Takehiko; Shibata, Isao; Sato, Shizuo

    2005-02-25

    To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.

  18. Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

    PubMed Central

    Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

    2016-01-01

    Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. PMID:25180293

  19. Double Seronegative Myasthenia Gravis with Anti-LRP4 Antibodies Presenting with Dropped Head and Acute Respiratory Insufficiency

    PubMed Central

    Beck, Goichi; Yabumoto, Taiki; Baba, Kousuke; Sasaki, Tsutomu; Higuchi, Osamu; Matsuo, Hidenori; Mochizuki, Hideki

    2016-01-01

    We herein report the case of a 72-year-old man demonstrating myasthenia gravis (MG) with a dropped head and acute respiratory insufficiency. There was no ocular, bulbar, or limb involvement. The patient was seronegative for anti-acetylcholine receptor (AChR) antibodies and anti-muscle-specific tyrosine kinase (MuSK) antibodies. Subsequent tests showed seropositivity for anti-low-density lipoprotein receptor-related protein 4 (LRP4) antibodies. The addition of steroid pulse therapy resulted in a full remission of his respiratory symptoms. This presentation suggests that LRP4-positive MG should be considered in the differential diagnosis of patients presenting with acute respiratory insufficiency without either cranial or limb involvement. PMID:27853084

  20. Immunoglobulin (Ig) M antibody against myelin associated glycoprotein (MAG): A comparison of methods.

    PubMed

    Jaskowski, T D; Martins, T B; Litwin, C M; Hill, H R

    2004-01-01

    The presence of immunoglobulin (Ig)M antibody against myelin associated glycoprotein (MAG) has been associated with autoimmune demyelinating, sensorimotor neuropathies. Approximately 50% of patients with IgM paraproteinemia and associated peripheral neuropathy possess antibodies against MAG. These autoantibodies are thought to interfere with the process of myelination, myelin maintenance, or axon-Schwann cell interaction. The detection of these autoantibodies is useful to the clinician and is suggestive of active demyelination in a peripheral neuropathy. Our objective in this study was to compare the results obtained using three different methods (dual enzyme immunoassay [EIA], immunofluorescent antibody [IFA] and Western blot [WB]) for detecting IgM antibody against MAG in patients suspected of having autoimmune demyelinating neuropathies. Since the dual EIA utilized two different antigens, results from this assay were separated into two groups: MAG and sulfate-3-glucuronyl paragloboside (SGPG). When compared to WB (gold standard), percent agreement, sensitivity, and specificity for EIA and IFA are as follows: MAG EIA (68.3, 100.0, and 60.6); SGPG EIA (95.1, 100.0, and 93.9); and myelin IFA (97.6, 100.0, and 97.0). The authors conclude that the SGPG EIA and myelin IFA compared well with the standard WB method when detecting IgM antibody against MAG (100 kD). Many sera demonstrated reactivity on the MAG EIA that were negative by WB (100 kD glycoprotein). The authors recommend screening for MAG IgM in suspected patient sera by SGPG EIA or myelin IFA and utilizing these same methods to titer sera confirmed positive by WB.

  1. Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis.

    PubMed

    Barrs, V R; Ujvari, B; Dhand, N K; Peters, I R; Talbot, J; Johnson, L R; Billen, F; Martin, P; Beatty, J A; Belov, K

    2015-03-01

    Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1). Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.

  2. [Study of nucleic acid structure by immunochemical methods. I. Antibodies specific for 6-sulfo-5,6-dihydro-4-methoxyaminopyrimidinone-2].

    PubMed

    Poverennyĭ, A M; Podgorodnichenko, V K; Monastyrskaia, G S; Bryskina, L E; Sverdlov, E D

    1978-01-01

    Immunization of animals with DNA modified by a mixture of bisulphite and O-methylhydroxylamine and methylated bovine serum albumin results in production of antibodies mainly reacting with modified DNA. Antibodies that react with denatured DNA were produced in minute quantity. It was shown that elicited antibodies possess a high specificity and have the ability to recognize only nucleotides with a double modification. The immune sera were fractionated by Sephadex G-200 column chromatography and the antibody activity was demonstrable in the 19S and 7S fractions. The attempts to induce synthesis of antibodies by injection of DNA modified by O-methylhydroxylamine failed.

  3. Printability beyond the limits: Alternative double printing method for inkjet

    NASA Astrophysics Data System (ADS)

    Parraman, Carinna; Wang, Yu

    2009-01-01

    For artists wishing to print onto heavy weight coated and uncoated papers, the opportunity to improve colour density and saturation is always desirable. The paper presents research into methods for mixing and printing colours using the latest multi-primary inkjet printing system. The objective is to investigate the colour printability of the system printing on a fine art paper. The cellular Yule-Nielsen modified spectral Neugebauer model is employed to characterise the printing process. And the preliminary experiment result shows the effectiveness of the proposed method.

  4. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    PubMed

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  5. The double-assignment method for the exponential chaotic tabu search in quadratic assignment problems

    NASA Astrophysics Data System (ADS)

    Shibata, Kazuaki; Horio, Yoshihiko; Aihara, Kazuyuki

    The quadratic assignment problem (QAP) is one of the NP-hard combinatorial optimization problems. An exponential chaotic tabu search using a 2-opt algorithm driven by chaotic neuro-dynamics has been proposed as one heuristic method for solving QAPs. In this paper we first propose a new local search, the double-assignment method, suitable for the exponential chaotic tabu search, which adopts features of the Lin-Kernighan algorithm. We then introduce chaotic neuro-dynamics into the double-assignment method to propose a novel exponential chaotic tabu search. We further improve the proposed exponential chaotic tabu search with the double-assignment method by enhancing the effect of chaotic neuro-dynamics.

  6. Direct-space methods in phase extension and phase refinement. IV. The double-histogram method.

    PubMed

    Refaat, L S; Tate, C; Woolfson, M M

    1996-03-01

    In the conventional histogram-matching technique for phase extension and refinement for proteins a simple one-to-one transformation is made in the protein region to modify calculated density so that it will have some target histogram in addition to solvent flattening. This work describes an investigation where the density modification takes into account not only the current calculated density at a grid point but also some characteristic of the environment of the grid point within some distance R. This characteristic can be one of the local maximum density, the local minimum density or the local variance of density. The grid points are divided into ten groups, each containing the same number of grid points, for ten different ranges of value of the local characteristic. The ten groups are modified to give different histograms, each corresponding to that obtained under the same circumstances from a structure similar to the one under investigation. This process is referred to as the double-histogram matching method. Other processes which have been investigated are the weighting of structure factors when calculating maps with estimated phases and also the use of a factor to dampen the change of density and so control the refinement process. Two protein structures were used in numerical trials, RNApl [Bezborodova, Ermekbaeva, Shlyapnikov, Polyakov & Bezborodov (1988). Biokhimiya, 53, 965-973] and 2-Zn insulin [Baker, Blundell, Cutfield, Cutfield, Dodson, Dodson, Hodgkin, Hubbard, lsaacs, Reynolds, Sakabe, Sakabe & Vijayan (1988). Philos. Trans. R. Soc. London Ser. B, 319, 456--469]. Comparison of the proposed procedures with the normal histogram-matching technique without structure-factor weighting or damping gives mean phase errors reduced by up to 10 degrees with map correlation coefficients improved by as much as 0.14. Compared to the normal histogram used with weighting of structure factors and damping, the improvement due to the use of the double-histogram method is

  7. Fabrication of double layer optical tissue phantom by spin coating method: mimicking epidermal and dermal layer

    NASA Astrophysics Data System (ADS)

    Park, Jihoon; Bae, Yunjin; Bae, Youngwoo; Kang, Heesung; Lee, Kyoung-Joung; Jung, Byungjo

    2013-02-01

    Methodologies to fabricate a solid optical tissue phantom (OTP) mimicking epidermal thin-layer have been developed for in vitro human skin experiment. However, there are cumbersome and time-consuming efforts in fabrication process such as a custom-made casting and calculation of solvent volume before curing process. In a previous study, we introduced a new methodology based on spin coating method (SCM) which is utilized to fabricate a thin-layer OTP analogous to epidermal thickness. In this study, a double layer solid OTP which has epidermal and dermal layers was fabricated to mimic the morphological and optical similarity of human tissue. The structural characteristic and optical properties of fabricated double layer OTP were measured using optical coherence tomography and inverse adding doubling algorithms, respectively. It is expected that the new methodology based on the SCM may be usefully used in the fabrication of double layer OTP.

  8. [An experience of treatment of double positive myeloperoxidase-antineutrophil cytoplasmic antibodies (MPO-ANCA) and anti-glomerular basement membrane antibodies in Goodpasture's syndrome onset of crescentic glomerulonephritis].

    PubMed

    Takeda, T; Takeda, T; Naiki, Y; Yonekawa, S; Sakaguchi, M; Iwamoto, I; Tanaka, H; Hasegawa, H; Imada, A; Kanamaru, A; Hiruma, S; Maekura, S; Hashimoto, S; Yamazumi, T

    1998-11-01

    A 68-year-old woman was admitted to Kinki University Hospital because of progressive renal failure. She had been well until two months before admission. Laboratory data were as follows: serum creatinine 4.1 mg/dl, BUN 69 mg/dl, MPO-ANCA 33 EU, anti-glomerular basement membrane antibodies (AGBMA) 118 U. Histological findings showed cellular and fibrocellular crescents in many glomeruli. Therefore, we diagnosed rapidly progressive glomerulonephritis (RPGN) due to MPO-ANCA and anti-GBM associated renal disease. The patient was started on prednisolone and double filtration plasmapheresis (DFPP) therapy. Subsequently, the values of MPO-ANCA and AGBMA decreased. However, the patient's condition suddenly worsened and she died of interstitial pneumonia. Autopsy examination revealed crescentic glomerulonephritis and alveolar hemorrhage with linear deposition of IgG along the glomerular and alveolar capillary walls by immunofluorescence studies. We considered this to be a rare case of Goodpasture's syndrome associated with not only anti-GBM antibodies, but also MPO-ANCA.

  9. A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

    PubMed

    Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

    2015-08-01

    Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity.

  10. Caprylic acid precipitation method for impurity reduction: an alternative to conventional chromatography for monoclonal antibody purification.

    PubMed

    Brodsky, Yan; Zhang, Cheng; Yigzaw, Yinges; Vedantham, Ganesh

    2012-10-01

    We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes.

  11. [The comparative evaluation of the diagnostic value of methods of detection of antibodies to citrullinized proteins under rheumatoid arthritis].

    PubMed

    Novikov, A A; Cherkasova, M V; Aleksandrova, E N; Popkova, T V; Luchikhina, E L; Rytikova, N S; Nasonov, E L

    2012-10-01

    The hyper production of large specter of autoantibodies, primarily rheumatoid factors and antibodies to citrullinized proteins, is a characteristic sign of rheumatoid arthritis. The detection of these antibodies plays an important role in diagnosing the disease, especially on its early stages. The study compared the diagnostic accuracy of different methods of detection of antibodies to citrullinized proteins under rheumatoid arthritis. The examined sample included 144 patients aged 33-58 years with reliable diagnosis of rheumatoid arthritis. The patients with systemic lupus erythematous, osteoarthritis, psoriatic arthritis, OVERLAP syndrome, ankylosing spondylitis and conditionally healthy donors consisted the comparative group. To detect antibodies to citrullinized proteins the methods of enzyme immunoassay, electrochemiluminescence, immunochromatography were applied. The study demonstrated that all the methods of detection of antibodies to citrullinized proteins have adequate diagnostic value to be implemented both in a routine clinical diagnostic practice and on the stage of screening of patients.

  12. A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75(NTR.).

    PubMed

    Tani, Hiroaki; Osbourn, Jane K; Walker, Edward H; Rush, Robert A; Ferguson, Ian A

    2013-01-01

    The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.

  13. Epidermal growth factor receptor double targeting by a tyrosine kinase inhibitor (Iressa) and a monoclonal antibody (Cetuximab). Impact on cell growth and molecular factors

    PubMed Central

    Fischel, J-L; Formento, P; Milano, G

    2005-01-01

    Among the recent advances in the molecular targeted therapy of cancer, the applications focused on epidermal growth factor receptor (EGFR) are currently the most promising and the most advanced at clinical level. In view of the different modes of action of monoclonal antibodies and tyrosine kinase inhibitors (TKI), it is tempting to examine the effect of a combination between these two EGFR targeting approaches. It was the purpose of the present study to test this combination at experimental level by using two epidermoid human cell lines CAL 33 and CAL 39. As C225 (Cetuximab®) and ZD1839 (Iressa®) are, respectively, the most clinically advanced drugs in the category of anti-EGFR drugs, the experiments were performed using these two representative compounds. The combination of C225 and ZD1839 was antagonistic whatever the cell line considered. These antagonistic effects were corroborated by molecular changes in apoptosis (PARP) and EGFR signalling (phospho-p42–44). Drugs alone led to a diminution in EGFR levels, while their combination increased the cellular expression in EGFR. These data suggest that new and tempting treatment strategies on the EGFR target consisting in a double hit with a monoclonal antibody and a TKI must be considered with caution. PMID:15756277

  14. An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

    PubMed

    Ghosh, Inca; Sun, Luo; Evans, Thomas C; Xu, Ming-Qun

    2004-10-01

    Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

  15. Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial

    PubMed Central

    Ackerman, Margaret; Saunders, Kevin O.; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Michael, Nelson L.; O’Connell, Robert J.; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Phogat, Sanjay; Alam, S. Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S.; Montefiori, David C.; Harrison, Stephen C.; Haynes, Barton F.

    2017-01-01

    The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. Trial Registration: ClinicalTrials.gov NCT01435135 PMID:28235027

  16. Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial.

    PubMed

    Easterhoff, David; Moody, M Anthony; Fera, Daniela; Cheng, Hao; Ackerman, Margaret; Wiehe, Kevin; Saunders, Kevin O; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Kim, Jerome; Michael, Nelson L; O'Connell, Robert J; Excler, Jean-Louis; Robb, Merlin L; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Tartaglia, James; Phogat, Sanjay; Kepler, Thomas B; Alam, S Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S; Montefiori, David C; Tomaras, Georgia D; Harrison, Stephen C; Haynes, Barton F

    2017-02-01

    The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains.

  17. POLYTENE CHROMOSOME SQUASH METHODS FOR STUDYING TRANSCRIPTION AND EPIGENETIC CHROMATIN MODIFICATION IN DROSOPHILA USING ANTIBODIES

    PubMed Central

    Johansen, Kristen M.; Cai, Weili; Deng, Huai; Bao, Xiaomin; Zhang, Weiguo; Girton, Jack; Johansen, Jørgen

    2009-01-01

    The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure. PMID:19272452

  18. Microscopic quantification of bacterial invasion by a novel antibody-independent staining method.

    PubMed

    Agerer, Franziska; Waeckerle, Stephanie; Hauck, Christof R

    2004-10-01

    Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Gram-positive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms.

  19. Method based on the double sideband technique for the dynamic tracking of micrometric particles

    NASA Astrophysics Data System (ADS)

    Ramirez, Claudio; Lizana, Angel; Iemmi, Claudio; Campos, Juan

    2016-06-01

    Digital holography (DH) methods are of interest in a large number of applications. Recently, the double sideband (DSB) technique was proposed, which is a DH based method that, by using double filtering, provides reconstructed images without distortions and is free of twin images by using an in-line configuration. In this work, we implement a method for the investigation of the mobility of particles based on the DSB technique. Particle holographic images obtained using the DSB method are processed with digital picture recognition methods, allowing us to accurately track the spatial position of particles. The dynamic nature of the method is achieved experimentally by using a spatial light modulator. The suitability of the proposed tracking method is validated by determining the trajectory and velocity described by glass microspheres in movement.

  20. Diagnostic advantage of double immunohistochemistry using two mutation-specific anti-IDH antibodies (HMab-1 and MsMab-1) in gliomas.

    PubMed

    Takano, Shingo; Kato, Yukinari; Yamamoto, Tetsuya; Liu, Xing; Ishikawa, Eiichi; Kaneko, Mika K; Ogasawara, Satoshi; Matsuda, Masahide; Noguchi, Masayuki; Matsumura, Akira

    2015-07-01

    Isocitrate dehydrogenase (IDH) mutation is a valuable prognostic marker and a tool for decision-making for glioma treatment. An algorithm for IDH mutation screening was recently proposed--it consists of a two-step process of an initial search for the most common IDH1-R132H mutation using immunohistochemistry (IHC)-based assay, followed by DNA-based analysis of IHC-negative or -equivocal cases. Here, we report that immunohistochemistry using two mutation-specific anti-IDH monoclonal antibodies (mAbs)--an IDH1-R132H-specific mAb (clone HMab-1) and a multi-specific mAb (clone MsMab-1)--is easy and reliable for IDH mutation screening. We investigated the IDH status of 54 grade III gliomas. For the first screening, we used HMab-1 IHC and for the second, (of HMab-1-negative cases) we used MsMab-1 IHC. The double IHC screening results were confirmed using sequence analysis (100% specificity and 100% sensitivity). Thirty of 54 cases (55.6%) had IDH mutations and the remaining 24 were of the IDH wild type; moreover, the screening results predicted grade III glioma prognosis. IDH sequencing procedures are popular but inconsistent across laboratories. By contrast, double IHC screening using HMab-1 and MsMab-1 is very reliable for detecting IDH1/2 mutations and can predict survival in grade III glioma patients.

  1. A new hybrid double divisor ratio spectra method for the analysis of ternary mixtures

    NASA Astrophysics Data System (ADS)

    Youssef, Rasha M.; Maher, Hadir M.

    2008-10-01

    A new spectrophotometric method was developed for the simultaneous determination of ternary mixtures, without prior separation steps. This method is based on convolution of the double divisor ratio spectra, obtained by dividing the absorption spectrum of the ternary mixture by a standard spectrum of two of the three compounds in the mixture, using combined trigonometric Fourier functions. The magnitude of the Fourier function coefficients, at either maximum or minimum points, is related to the concentration of each drug in the mixture. The mathematical explanation of the procedure is illustrated. The method was applied for the assay of a model mixture consisting of isoniazid (ISN), rifampicin (RIF) and pyrazinamide (PYZ) in synthetic mixtures, commercial tablets and human urine samples. The developed method was compared with the double divisor ratio spectra derivative method (DDRD) and derivative ratio spectra-zero-crossing method (DRSZ). Linearity, validation, accuracy, precision, limits of detection, limits of quantitation, and other aspects of analytical validation are included in the text.

  2. A new hybrid double divisor ratio spectra method for the analysis of ternary mixtures.

    PubMed

    Youssef, Rasha M; Maher, Hadir M

    2008-10-01

    A new spectrophotometric method was developed for the simultaneous determination of ternary mixtures, without prior separation steps. This method is based on convolution of the double divisor ratio spectra, obtained by dividing the absorption spectrum of the ternary mixture by a standard spectrum of two of the three compounds in the mixture, using combined trigonometric Fourier functions. The magnitude of the Fourier function coefficients, at either maximum or minimum points, is related to the concentration of each drug in the mixture. The mathematical explanation of the procedure is illustrated. The method was applied for the assay of a model mixture consisting of isoniazid (ISN), rifampicin (RIF) and pyrazinamide (PYZ) in synthetic mixtures, commercial tablets and human urine samples. The developed method was compared with the double divisor ratio spectra derivative method (DDRD) and derivative ratio spectra-zero-crossing method (DRSZ). Linearity, validation, accuracy, precision, limits of detection, limits of quantitation, and other aspects of analytical validation are included in the text.

  3. Monoclonal antibody-based serological methods for maize chlorotic mottle virus detection in China*

    PubMed Central

    Wu, Jian-xiang; Wang, Qiang; Liu, Huan; Qian, Ya-juan; Xie, Yan; Zhou, Xue-ping

    2013-01-01

    Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64 000, and 1:3 276 800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China. PMID:23825140

  4. Qualification of a microfluidics-based electrophoretic method for impurity testing of monoclonal antibodies.

    PubMed

    Antes, Bernhard; Oberkleiner, Philipp; Nechansky, Andreas; Szolar, Oliver H J

    2010-02-05

    In this work, we present a comprehensive evaluation of the Agilent Bioanalyzer, a microfluidics-based electrophoretic device that was used for impurity testing of a monoclonal antibody (mAb). We compared the system to SDS-PAGE, both operated under non-reducing conditions and found a significant improvement of accuracy for the Bioanalyzer. In addition, the latter exhibited a larger assay range and lower limit of quantitation (LOQ) based on a predefined total error limit of +/-30%. However, during method qualification applying a three-factor nested design with two operators performing duplicate measurements per day, each on 4 different days, we observed unpredictable recurring quantitative outliers using the chip-based system. In-depth analysis on multiple runs with various chip lots confirmed the above finding and indicated that most likely on-chip dye labeling and/or post-column background fluorescence elimination are not compatible with the large size of the intact antibody as similar findings were observed for myosin used as upper marker for time correction. Interestingly, after reducing the intact antibody into light and heavy chain, we resolved the outlier issue. Eventually, requalification of the micro-fabricated analytical device under reducing conditions revealed only 1 out of 32 quality control samples (QCs) exceeding the +/-30% total error limits.

  5. Antibody gene transfer with adeno-associated viral vectors as a method for HIV prevention.

    PubMed

    Brady, Jacqueline M; Baltimore, David; Balazs, Alejandro B

    2017-01-01

    Broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus (HIV) show great promise in HIV prevention as they are capable of potently neutralizing a considerable breadth of genetically diverse strains. Passive transfer of monoclonal bNAb proteins can confer protection in animal models of HIV infection at modest concentrations, inspiring efforts to develop an HIV vaccine capable of eliciting bNAb responses. However, these antibodies demonstrate high degrees of somatic mutation and other unique characteristics that may hinder the ability of conventional approaches to consistently and effectively produce bNAb analogs. As an alternative strategy, we and others have proposed vector-mediated gene transfer to generate long-term, systemic production of bNAbs in the absence of immunization. Herein, we review the use of adeno-associated virus (AAV) vectors for delivery of HIV bNAbs and antibody-like proteins and summarize both the advantages and disadvantages of this strategy as a method for HIV prevention.

  6. A double expansion method for the frequency response of finite-length beams with periodic parameters

    NASA Astrophysics Data System (ADS)

    Ying, Z. G.; Ni, Y. Q.

    2017-03-01

    A double expansion method for the frequency response of finite-length beams with periodic distribution parameters is proposed. The vibration response of the beam with spatial periodic parameters under harmonic excitations is studied. The frequency response of the periodic beam is the function of parametric period and then can be expressed by the series with the product of periodic and non-periodic functions. The procedure of the double expansion method includes the following two main steps: first, the frequency response function and periodic parameters are expanded by using identical periodic functions based on the extension of the Floquet-Bloch theorem, and the period-parametric differential equation for the frequency response is converted into a series of linear differential equations with constant coefficients; second, the solutions to the linear differential equations are expanded by using modal functions which satisfy the boundary conditions, and the linear differential equations are converted into algebraic equations according to the Galerkin method. The expansion coefficients are obtained by solving the algebraic equations and then the frequency response function is finally determined. The proposed double expansion method can uncouple the effects of the periodic expansion and modal expansion so that the expansion terms are determined respectively. The modal number considered in the second expansion can be reduced remarkably in comparison with the direct expansion method. The proposed double expansion method can be extended and applied to the other structures with periodic distribution parameters for dynamics analysis. Numerical results on the frequency response of the finite-length periodic beam with various parametric wave numbers and wave amplitude ratios are given to illustrate the effective application of the proposed method and the new frequency response characteristics, including the parameter-excited modal resonance, doubling-peak frequency response

  7. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

    PubMed

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

  8. Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods

    PubMed Central

    2013-01-01

    Background Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method. Results Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes. Conclusions It was found that RT-IHC can be used

  9. Association of circulating antibodies against double-stranded and single-stranded DNA with thyroid autoantibodies in Graves' disease and Hashimoto's thyroiditis patients.

    PubMed

    Pedro, A B P; Romaldini, J H; Americo, C; Takei, K

    2006-01-01

    The occurrence of antinuclear antibody (ANA), rheumatoid factor (RF), antibodies to double-stranded DNA (anti-dsDNA) and to single-stranded DNA (anti-ssDNA) was investigated in 51 patients with autoimmune thyroid diseases (AITD), and in 25 matched control subjects. In comparison with controls, the prevalence of anti-dsDNA was 74.5% in AITD patients (p=0.0001), 82.0% in 39 hyperthyroid Graves' disease (GD) (p=0.0001), and 50.0% in 12 euthyroid Hashimoto's thyroiditis (HT) patients (p=0.0001). The prevalence of anti-ssDNA was 90.1% in AITD (94.8% in GD and 75% in HT; p=0.001). The concentration of both anti-dsDNA and anti-ssDNA were higher (p=0.002) in AITD, in GD (p=0.001), and in HT (p=0.01) patients than in controls. Two patients with AITD were identified as positive for ANA. RF was detected in 4 AITD patients. Positive correlation was noted between anti-dsDNA with T4 (p=0.001), T3 (p=0.002), thyroid peroxidase antibody (anti-TPO) (p=0.0001), and TSH (p=0.001) values but not with thyroglobulin antibody (anti-Tg). Serum anti-ssDNA values were also correlated with T3 (p=0.0001), TSH (p=0.003), and anti-TPO (p=0.0001). However, by using a multiple regression analysis only anti-TPO remained associated with anti-dsDNA and both anti-Tg and anti-TPO with anti-ssDNA values. The predisposition to develop systemic autoimmune disorders is not influenced by thyroid hormones. The elevated prevalence of serum anti-dsDNA and anti-ssDNA in AITD patients points out that we must be aware of the risk for predisposition for the development of other systemic autoimmune diseases.

  10. Comparison of four serological methods for the detection of diphtheria anti-toxin antibody.

    PubMed

    Walory, J; Grzesiowski, P; Hryniewicz, W

    2000-11-01

    The aim of this study was to compare four serological methods for the detection of Corynebacterium diphtheriae IgG anti-toxin antibodies (IgG-DTAb) in human serum. One hundred serum samples were evaluated for C. diphtheriae IgG-DTAb by four different methods: passive haemagglutination (PHA), latex agglutination test (LA), toxoid enzyme-linked immunosorbent assay (Toxoid-ELISA), and toxin-binding inhibition enzyme-linked immunosorbent assay (ToBI-ELISA). As the external standardisation the neutralisation test for C. diphtheriae toxin in Vero cells (TN Vero) was used. For internal standardisation of IgG-DTAb titres, the WHO standard serum of human diphtheria antitoxin was used. The study revealed a poor correlation between the reference test and the PHA (r=0.34 Pearson's correlation coefficient), an acceptable correlation for the LA (r=0.74), a good correlation for the Toxoid-ELISA (r=0.81) and a very good correlation for ToBI-ELISA (r=0.93). The sensitivity measurements of PHA, LA, Toxoid-ELISA and ToBI-ELISA tests, were 14, 100, 94, 96% respectively and the corresponding specificity characteristics were 86, 76, 94, 90 respectively. Of the four evaluated methods, the ToBI-ELISA could be recommended for scientific and precise laboratory assays of diphtheria antibody levels in humans. For screening purposes the Toxoid-ELISA could be used, but the accuracy of antibody titres below 0.1 IU/ml, considered as the limits of protection, is questionable. Both tests offer very useful alternatives to the in vitro diphtheria toxin neutralisation test in Vero cells. Because of their unsatisfactory correlation and sensitivity as compared to the reference method, PHA and LA should be avoided and replaced by one of the two enzyme immunoassays.

  11. Detection of vibrio cholerae O1 by using cerium oxide nanowires - based immunosensor with different antibody immobilization methods

    NASA Astrophysics Data System (ADS)

    Tam, Phuong Dinh; Hoang, Nguyen Luong; Lan, Hoang; Vuong, Pham Hung; Anh, Ta Thi Nhat; Huy, Tran Quang; Thuy, Nguyen Thanh

    2016-05-01

    In this work, we evaluated the effects of different antibody immobilization strategies on the response of a CeO2-nanowires (NWs)-based immunosensor for Vibrio cholerae O1 detection. Accordingly, the changes in the electron-transfer resistance ( R et ) from before to after cells bind to an antibody-modified electrode prepared by using three different methods of antibody immobilization were determined. The values were 16.2%, 8.3%, and 6.65% for the method that utilized protein A, antibodies activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS), and absorption, respectively. Cyclic voltammetry confirmed that the change in the current was highest for the immunosensors prepared using protein A (11%), followed by those prepared with EDC/NHS-activated antibodies (9%), and finally, those prepared through absorption (7.5%). The order of the antibody immobilization strategies in terms of resulting immunosensor detection limit and sensitivity was as follows order: absorption (3.2 × 103 CFU/mL; 45.1 Ω/CFU·mL-1) < EDC/NHS-activated antibody (1.0 × 103 CFU/mL; 50.6 Ω/CFU·mL-1) < protein A (1.0 × 102 CFU/mL; 65.8 Ω/CFU·mL-1). Thus, we confirmed that the protein A - mediated method showed significantly high cell binding efficiencies compared to the random immobilization method.

  12. The Effect of Three Methods of Supporting the Double Bass on Muscle Tension.

    ERIC Educational Resources Information Center

    Dennis, Allan

    1984-01-01

    Using different methods of holding the double bass, college students performed Beethoven's Symphony No. 9. Audio recordings of performance were rated. Muscle tension readings from the left arm, right arm, upper back, and lower back were taken, using electromyography. Results suggest nonsignificant differences in both performance quality and muscle…

  13. Error analysis of mixed finite element methods for wave propagation in double negative metamaterials

    NASA Astrophysics Data System (ADS)

    Li, Jichun

    2007-12-01

    In this paper, we develop both semi-discrete and fully discrete mixed finite element methods for modeling wave propagation in three-dimensional double negative metamaterials. Optimal error estimates are proved for Nedelec spaces under the assumption of smooth solutions. To our best knowledge, this is the first error analysis obtained for Maxwell's equations when metamaterials are involved.

  14. Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses.

    PubMed

    Kim, Kyoung-Ho; Bae, Jin-Woo

    2011-11-01

    Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

  15. Clinical association of mixed connective tissue disease and granulomatosis with polyangiitis: a case report and systematic screening of anti-U1RNP and anti-PR3 auto-antibody double positivity in ten European hospitals.

    PubMed

    Tubery, Amandine; Fortenfant, Françoise; Combe, Bernard; Abreu, Isabelle; Bossuyt, Xavier; Chretien, Pascale; Desplat-Jégo, Sophie; Fabien, Nicole; Hue, Sophie; Johanet, Catherine; Lakomy, Daniela; Vincent, Thierry; Daïen, Claire I

    2016-12-01

    We report here the case of a 50-years-old man treated for mixed connective tissue disease (MCTD) positive for anti-U1 ribonucleoprotein (U1RNP) antibodies who secondarily developed a granulomatosis with polyangiitis (GPA) associated with anti-proteinase 3 anti-neutrophil cytoplasmic antibodies (PR3-ANCA). We then evaluated the frequency of the association between anti-U1RNP and anti-PR3-ANCA antibodies by a systematic retrospective study in ten European hospitals. Overall, out of 11,921 samples analyzed for both auto-antibodies, 18 cases of anti-U1RNP and anti-PR3-ANCA double positivity were found and only one patient presented with both MCTD and GPA symptoms. Our retrospective analysis indicates that anti-U1RNP and anti-PR3-ANCA antibodies double positivity is infrequent and very rarely associated with both MTCD and GPA. Our observation describes for the first time the coexistence of MTCD and severe GPA in a Caucasian patient. Association of anti-U1RNP and ANCA antibodies was rarely reported in the literature. Eleven cases of MCTD and ANCA vasculitis have been reported to date, with only two cases with anti-PR3-ANCA association, and only one vasculitis. The seven other cases reported in the literature presented with an association of MCTD and microscopic polyangiitis which appears to be a more frequent presentation than MTCD associated with GPA.

  16. Separation of antibody drug conjugate species by RPLC: A generic method development approach.

    PubMed

    Fekete, Szabolcs; Molnár, Imre; Guillarme, Davy

    2017-04-15

    This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

  17. A new method for multilayered, site-directed immobilization of antibody on polystyrene surface.

    PubMed

    Feng, Bo; Wang, Caiyun; Xie, Xiaomei; Feng, Xi; Li, Yuqin; Cao, Zhijian

    2014-07-18

    Polystyrene is a common substrate material for protein adsorption in biosensors and bioassays. Here, we present a new method for multilayered, site-directed immobilization of antibody on polystyrene surface through the linkage of a genetically engineered ligand and the assembly of staphylococcal protein A (SPA) with immunoglobulin G (IgG). In this method, antibodies were stacked on polystyrene surface layer by layer in a potential three-dimensional way and exposed the analyte-binding sites well. Enzyme-linked immunosorbent assay (ELISA) revealed that the new method showed a 32-fold higher detection sensitivity compared with the conventional one. Pull-down assay and Western blot analysis further confirmed that it is different from the ones of monolayer adsorption according to the comparison of adsorption capacity. The differentiated introduction of functional ligands, which is the key of this method, might offer a unique idea as a way to interfere with the dynamic behavior of a protein complex during the process of adsorption.

  18. Mean centering of double divisor ratio spectra, a novel spectrophotometric method for analysis of ternary mixtures

    NASA Astrophysics Data System (ADS)

    Hassan, Said A.; Elzanfaly, Eman S.; Salem, Maissa Y.; El-Zeany, Badr A.

    2016-01-01

    A novel spectrophotometric method was developed for determination of ternary mixtures without previous separation, showing significant advantages over conventional methods. The new method is based on mean centering of double divisor ratio spectra. The mathematical explanation of the procedure is illustrated. The method was evaluated by determination of model ternary mixture and by the determination of Amlodipine (AML), Aliskiren (ALI) and Hydrochlorothiazide (HCT) in laboratory prepared mixtures and in a commercial pharmaceutical preparation. For proper presentation of the advantages and applicability of the new method, a comparative study was established between the new mean centering of double divisor ratio spectra (MCDD) and two similar methods used for analysis of ternary mixtures, namely mean centering (MC) and double divisor of ratio spectra-derivative spectrophotometry (DDRS-DS). The method was also compared with a reported one for analysis of the pharmaceutical preparation. The method was validated according to the ICH guidelines and accuracy, precision, repeatability and robustness were found to be within the acceptable limits.

  19. Mean centering of double divisor ratio spectra, a novel spectrophotometric method for analysis of ternary mixtures.

    PubMed

    Hassan, Said A; Elzanfaly, Eman S; Salem, Maissa Y; El-Zeany, Badr A

    2016-01-15

    A novel spectrophotometric method was developed for determination of ternary mixtures without previous separation, showing significant advantages over conventional methods. The new method is based on mean centering of double divisor ratio spectra. The mathematical explanation of the procedure is illustrated. The method was evaluated by determination of model ternary mixture and by the determination of Amlodipine (AML), Aliskiren (ALI) and Hydrochlorothiazide (HCT) in laboratory prepared mixtures and in a commercial pharmaceutical preparation. For proper presentation of the advantages and applicability of the new method, a comparative study was established between the new mean centering of double divisor ratio spectra (MCDD) and two similar methods used for analysis of ternary mixtures, namely mean centering (MC) and double divisor of ratio spectra-derivative spectrophotometry (DDRS-DS). The method was also compared with a reported one for analysis of the pharmaceutical preparation. The method was validated according to the ICH guidelines and accuracy, precision, repeatability and robustness were found to be within the acceptable limits.

  20. A Novel Method for Anti-HLA Antibody Detection Using Personalized Peptide Arrays

    PubMed Central

    Liu, Pan; Souma, Tomokazu; Wei, Andrew Zu-Sern; Xie, Xueying; Luo, Xunrong; Jin, Jing

    2016-01-01

    Background HLA mismatches are the primary cause of alloantibody-mediated rejection (AMR) in organ transplantation. To delineate antigenic and immunogenic potentials among individual HLA mismatches, information regarding antibody specificity at the epitope level, instead of the allelic level, is needed. Methods This study explores a direct screening method for HLA linear epitopes in kidney transplant patients. We custom synthesized a large panel of 15-residue HLA peptides in an array format and measured alloantibody reactivity to these peptides from the sera of post and/or pretransplant patients. Two design concepts for the arrays were followed: a standard array of a fixed panel of peptides or personalized arrays. The standard array contains 420 peptides derived from a predetermined set of HLA-DQ allelic antigens based on templates also used in the single-antigen beads assay. Results The array detected distinct antiserum patterns among transplant subjects and revealed epitope levels of specificity largely in accordance with the single-antigen results. Two personalized arrays that each included donor-derived peptides of HLA-A, -B, -C, -DQ, and -DR sequences were separately designed for 2 transplant subjects. The personalized arrays detected de novo antibodies following transplantation. The new method also showed superior sensitivity to a single-antigen assay in one of the cases whose pathological diagnosis of AMR occurred before single-antigen assay could detect antibodies. Conclusions This pilot study proved the feasibility of using personalized peptide arrays to achieve detection of alloantibodies for linear HLA epitopes associated with distinct donor-recipient mismatches. Single or multiple reactive epitopes may occur on an individual HLA molecule, and donor-specific HLA-DQ-reactivity among 5 kidney transplant subjects revealed patterns of shared epitopes. PMID:27826602

  1. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

    PubMed

    Gong, Haibiao; Holcomb, Ilona; Ooi, Aik; Wang, Xiaohui; Majonis, Daniel; Unger, Marc A; Ramakrishnan, Ramesh

    2016-01-20

    The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and

  2. Double Fourier Harmonic Balance Method for Nonlinear Oscillators by Means of Bessel Series

    DTIC Science & Technology

    2014-10-16

    Double Fourier harmonic balance method for nonlinear oscillators by means of Bessel series T.C. Lipscombe∗1 and C.E. Mungan†2 1Catholic University of... harmonic balance method consists in expanding the displacement of an oscillator as a Fourier cosine series in time. A key modification is proposed here, in...than the exact value. Even better, the predicted frequency for the V-ramp case turns out to be exact. Keywords: Fourier expansion, harmonic balance

  3. A planar lens in LiNbO3 produced by the double proton exchange method

    NASA Astrophysics Data System (ADS)

    Volkov, V. A.; Gan'shin, V. A.; Kvasha, M. Iu.; Korkishko, Iu. N.; Fedotov, S. M.

    1989-08-01

    Experiments are reported in which a waveguide lens in lithium niobate was produced by the method of double proton-lithium exchange. In this process, the formation of a Ti:LiNbO3 light waveguide is replaced by the formation of an H:LiNbO3 waveguide of identical parameters. The performance characteristics of the waveguide lenses fabricated by the method proposed here are as good as those of TIPE lenses.

  4. Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody.

    PubMed

    Suba, Dávid; Urbányi, Zoltán; Salgó, András

    2015-10-10

    Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some "out-of-the-box" applications commercially available which provide easy and rapid isoelectric focusing solutions for investigating monoclonal antibody drug proteins. However use of these kits in routine testings requires high costs. A capillary isoelectric focusing method was developed and validated for identification testing of monoclonal antibody drug products with isoelectric point between 7.0 and 9.0. A method was developed providing good pH gradient for internal calibration (R(2)>0.99) and good resolution between all of the isoform peaks (R=2), minimizing the time and complexity of sample preparation (no urea or salt used). The method is highly reproducible and it is suitable for validation and method transfer to any QC laboratories. Another advantage of the method is that it operates with commercially available chemicals which can be purchased from any suppliers. The interaction with capillary walls (avoid precipitation and adsorption as far as possible) was minimized and synthetic isoelectric small molecular markers were used instead of peptide or protein based markers. The developed method was validated according to the recent ICH guideline (Q2(R1)). Relative standard deviation results were below 0.2% for isoelectric points and below 4% according to the normalized migration times. The method is robust to buffer components with different lot numbers and neutral capillaries with different type of inner coatings. The fluoro-carbon coated column was chosen because of costs-effectivity aspects.

  5. Double-Blind Study To Evaluate Flow Cytometry Analysis of Anti-Live Trypomastigote Antibodies for Monitoring Treatment Efficacy in Cases of Human Chagas' Disease

    PubMed Central

    Martins-Filho, Olindo Assis; Eloi-Santos, Silvana Maria; Carvalho, Andréa Teixeira; Oliveira, Rodrigo Corrêa; Rassi, Anis; Luquetti, Alejandro Ostemayer; Rassi, Gustavo Gabriel; Brener, Zigman

    2002-01-01

    The validation of flow cytometry analysis of anti-live trypomastigote antibodies (FC-ALTA) to monitor cure after treatment of Chagas' disease was evaluated with serum samples from treated and nontreated chagasic patients. After optimization of the original technique, toward better sensitivity and applicability to field surveys, we design a double blind study of 94 coded samples classified into the following categories: patients not treated (NT) and patients treated but not cured (TNC), both presenting positive conventional serology and xenodiagnosis; patients treated and cured (TC), showing negative serology and xenodiagnosis; and patients treated under evaluation (TUE), who presented positive or oscillating conventional serology (CSA) but negative xenodiagnosis. Coded samples, diluted 1:256, were assayed by incubation with live cell culture trypomastigotes, which were subsequently stained with fluorescein isothiocyanate-conjugated anti-human immunoglobulin G, with prior fixation and analysis by flow cytometry. The results were expressed as the percentages of positive fluorescent parasites (PPFP) for each individual sample, establishing 20% PPFP as the cutoff between negative and positive results. Our data demonstrated that all NT and TNC presented positive results while all but one TC had a PPFP lower than 20%. Analysis of TUE demonstrated a wide degree of reactivity, with PPFP values that were negative (PPFP ≤ 20%), low positive (20% < PPFP ≤ 50%), and high positive (PPFP > 50%). As TUE with negative PPFP presented negative xenodiagnosis and positive or oscillating CSA, they were classified as dissociated according to the criteria of Krettli and Brener (J. Immunol. 128:2009-2012, 1982) and could indeed be considered cured after chemotherapy. This study demonstrates and validates the use of FC-ALTA to easily identify anti-live trypomastigote membrane-bound antibodies, offering another approach for investigating and monitoring the efficacy of specific

  6. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  7. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  8. Double freeform surfaces design for laser beam shaping with Monge-Ampère equation method

    NASA Astrophysics Data System (ADS)

    Zhang, Yaqin; Wu, Rengmao; Liu, Peng; Zheng, Zhenrong; Li, Haifeng; Liu, Xu

    2014-11-01

    This paper presents a method for designing double freeform surfaces to simultaneously control the intensity distribution and phase profile of the laser beam. Based on Snell’s law, the conservation law of energy and the constraint imposed on the optical path length between the input and output wavefronts, the double surfaces design is converted into an elliptic Monge-Ampère (MA) equation with a nonlinear boundary problem. A generalized approach is introduced to find the numerical solution of the design model. Two different layouts of the beam shaping system are introduced and detailed comparisons are also made between the two layouts. Design examples are given and the results indicate that good matching is achieved by the MA method with more than 98% of the energy efficiency. The MA method proposed in this paper provides a reasonably good means for laser beam shaping.

  9. Application of magnetic printing method to hard-disk media with double recording layers

    NASA Astrophysics Data System (ADS)

    Ono, Takuya; Kuboki, Yoshiyuki; Ajishi, Yoshifumi; Saito, Akira

    2003-05-01

    The magnetic printing method, which can duplicate soft magnetic patterns containing digital information such as servosignals formed on a master disk onto recording media, enables signals to be written to hard-disk media having high coercivities above 6000 Oe. We propose the application of the magnetic printing method to a hard-disk medium having double recording layers, one layer of which has high coercivity and is to be printed with digital information. This double recording layer medium is a hard-disk medium that has a magnetic read-only-memory (MROM) layer. In this study, we demonstrated a method for printing to this medium, which has MROM, and discussed the magnetic properties and recording performances of this medium.

  10. A joint sparse representation-based method for double-trial evoked potentials estimation.

    PubMed

    Yu, Nannan; Liu, Haikuan; Wang, Xiaoyan; Lu, Hanbing

    2013-12-01

    In this paper, we present a novel approach to solving an evoked potentials estimating problem. Generally, the evoked potentials in two consecutive trials obtained by repeated identical stimuli of the nerves are extremely similar. In order to trace evoked potentials, we propose a joint sparse representation-based double-trial evoked potentials estimation method, taking full advantage of this similarity. The estimation process is performed in three stages: first, according to the similarity of evoked potentials and the randomness of a spontaneous electroencephalogram, the two consecutive observations of evoked potentials are considered as superpositions of the common component and the unique components; second, making use of their characteristics, the two sparse dictionaries are constructed; and finally, we apply the joint sparse representation method in order to extract the common component of double-trial observations, instead of the evoked potential in each trial. A series of experiments carried out on simulated and human test responses confirmed the superior performance of our method.

  11. Two-Particle Coulomb Green Function Method with Projected Potential: Application to He Double Photoionization

    NASA Astrophysics Data System (ADS)

    Argenti, Luca; Colle, Renato

    2009-08-01

    A new method to compute fully differential double photoionization cross sections of atoms has been devised and fully developed for two-electron systems. The method exploits the Green function for two noninteracting electrons in the field of a nuclear charge to infer the effects of the residual potential projected on a set of L2-basis functions. Test calculations on helium at 100 eV excess energy indicate that, as long as the relevant part of the interaction potential is accounted for, the fully differential cross sections calculated in acceleration and velocity gauges converge in absolute value and reproduce measured angular distributions with a tunable accuracy. Generalization of the method to treat double photoionization of many-electron atoms is sketched.

  12. Two-particle coulomb Green function method with projected potential: application to He double photoionization.

    PubMed

    Argenti, Luca; Colle, Renato

    2009-12-31

    A new method to compute fully differential double photoionization cross sections of atoms has been devised and fully developed for two-electron systems. The method exploits the Green function for two noninteracting electrons in the field of a nuclear charge to infer the effects of the residual potential projected on a set of L(2)-basis functions. Test calculations on helium at 100 eV excess energy indicate that, as long as the relevant part of the interaction potential is accounted for, the fully differential cross sections calculated in acceleration and velocity gauges converge in absolute value and reproduce measured angular distributions with a tunable accuracy. Generalization of the method to treat double photoionization of many-electron atoms is sketched.

  13. [An efficient method for producing monoclonal antibodies against multi-pass membrane proteins].

    PubMed

    Yagi, Hideki; Masuko, Takashi

    2013-01-01

    Antibodies have greatly contributed to the development of medical science and pharmacology, because of their high specificity. The cell fusion method has developed monoclonal antibodies (mAb) technology, such that massive amounts of mAb with a uniform structure can be produced. Although mAb have been produced against many proteins so far, the production of mAb against multi-pass transmembrane proteins, such as G-protein coupled receptor (GPCR) and various transporter proteins has been extremely difficult. The complicated structures, poorly extracellular regions, and high hydrophobicity of multiple-transmembrane proteins make it difficult to produce mAb against them. Production of mAb that recognize the extracellular region of living cells is thought to be important in determining the ability of a protein. Based on these findings, we tried to produce mAb against a multi-pass transmembrane transporter using green fluorescent protein (GFP)-fused full-length target proteins as immunogens. Furthermore, the immunizing method has proved to be important in generating functional mAb. We succeeded in producing functional mAb that react against the extracellular region of a 12-pass transmembrane transporter in a living cell. Based on this success, we began to produce mAb against seven-transmembrane GPCR. In this symposium, we report on the results of producing mAb against S1P receptors, a type of GPCR.

  14. Indirect Fluorescent-Antibody Method for the Identification of Corynebacterium vaginale

    PubMed Central

    Vice, John L.; Smaron, Mary F.

    1973-01-01

    The indirect fluorescent-antibody technique was employed in an attempt to develop a rapid method of identification of Corynebacterium vaginale. Six reference strains and ten clinical isolates selected on the basis of morphology and conventional biochemical tests were compared. Antisera were prepared in rabbits against the six reference strains. The most satisfactory antiserum was that prepared using strain 14018 grown diphasically (14018 Di) as the antigen. Certain of the antisera did exhibit a cross-reacting titer when reacted against Corynebacterium diptheriae, Corynebacterium xerosis, or Lactobacillus acidophilus. However, antisera adsorbed with these bacteria did not exhibit a significant decrease in titer when reacted against the homologous strain. Various other species of Corynebacterium as well as species of Nocardia, Actinomyces, Hemophilus, and Streptococcus did not fluoresce with the antisera. A specific antiserum was prepared by adsorbing anti-14018 Di with L. acidophilus. The adsorption removed the cross-reacting antibody but did not affect the staining reaction with C. vaginale strains. All reference strains and clinical isolates characterized as C. vaginale gave a definite positive reaction with the adsorbed anti-14018 Di. The specificity of the reactions was assessed by adsorbing the antiserum with the homologous strain. The data suggest that the indirect staining method will be of value in the rapid presumptive identification of C. vaginale. Images PMID:4197767

  15. Applicability of predictive toxicology methods for monoclonal antibody therapeutics: status Quo and scope.

    PubMed

    Kizhedath, Arathi; Wilkinson, Simon; Glassey, Jarka

    2017-04-01

    Biopharmaceuticals, monoclonal antibody (mAb)-based therapeutics in particular, have positively impacted millions of lives. MAbs and related therapeutics are highly desirable from a biopharmaceutical perspective as they are highly target specific and well tolerated within the human system. Nevertheless, several mAbs have been discontinued or withdrawn based either on their inability to demonstrate efficacy and/or due to adverse effects. Approved monoclonal antibodies and derived therapeutics have been associated with adverse effects such as immunogenicity, cytokine release syndrome, progressive multifocal leukoencephalopathy, intravascular haemolysis, cardiac arrhythmias, abnormal liver function, gastrointestinal perforation, bronchospasm, intraocular inflammation, urticaria, nephritis, neuropathy, birth defects, fever and cough to name a few. The advances made in this field are also impeded by a lack of progress in bioprocess development strategies as well as increasing costs owing to attrition, wherein the lack of efficacy and safety accounts for nearly 60 % of all factors contributing to attrition. This reiterates the need for smarter preclinical development using quality by design-based approaches encompassing carefully designed predictive models during early stages of drug development. Different in vitro and in silico methods are extensively used for predicting biological activity as well as toxicity during small molecule drug development; however, their full potential has not been utilized for biological drug development. The scope of in vitro and in silico tools in early developmental stages of monoclonal antibody-based therapeutics production and how it contributes to lower attrition rates leading to faster development of potential drug candidates has been evaluated. The applicability of computational toxicology approaches in this context as well as the pitfalls and promises of extending such techniques to biopharmaceutical development has been highlighted.

  16. Quality assurance of monoclonal antibody pharmaceuticals based on their charge variants using microchip isoelectric focusing method.

    PubMed

    Kinoshita, Mitsuhiro; Nakatsuji, Yuki; Suzuki, Shigeo; Hayakawa, Takao; Kakehi, Kazuaki

    2013-09-27

    Monoclonal antibody (mAb) pharmaceuticals are much more complex than small-molecule drugs. Such complex characteristics raise challenging questions for regulatory evaluation. Although heterogeneity in mAbs based on their charge variants has been mainly evaluated using gel-based isoelectric focusing (IEF) method, recent development in capillary electrophoresis and microchip electrophoresis has made it possible to assure their heterogeneities in more easy and rapid manner. In the present paper, we customized the imaged microchip isoelectric focusing (mIEF) for the analysis of mAbs, and compared the customized version with the conventional capillary isoelectric focusing (cIEF) method, and found that mIEF has much higher performance in operations, and its resolving powers are comparable with those obtained by cIEF.

  17. Novel Microdilution Method to Assess Double and Triple Antibiotic Combination Therapy In Vitro.

    PubMed

    El-Azizi, Mohamed

    2016-01-01

    An in vitro microdilution method was developed to assess double and triple combinations of antibiotics. Five antibiotics including ciprofloxacin, amikacin, ceftazidime, piperacillin, and imipenem were tested against 10 clinical isolates of Pseudomonas aeruginosa. Each isolate was tested against ten double and nine triple combinations of the antibiotics. A 96-well plate was used to test three antibiotics, each one alone and in double and triple combinations against each isolate. The minimum bacteriostatic and bactericidal concentrations in combination were determined with respect to the most potent antibiotic. An Interaction Code (IC) was generated for each combination, where a numerical value was designated based on the 2-fold increase or decrease in the MICs with respect to the most potent antibiotic. The results of the combinations were verified by time-kill assay at constant concentrations of the antibiotics and in a chemostat. Only 13% of the double combinations were synergistic, whereas 5% showed antagonism. Forty-three percent of the triple combinations were synergistic with no antagonism observed, and 100% synergism was observed in combination of ciprofloxacin, amikacin, and ceftazidime. The presented protocol is simple and fast and can help the clinicians in the early selection of the effective antibiotic therapy for treatment of severe infections.

  18. Identification of antibody isotypes in biological fluids by means of micro-Raman spectroscopy and chemometric methods

    NASA Astrophysics Data System (ADS)

    Araujo-Andrade, C.; Pichardo-Molina, J. L.; Barbosa-Sabanero, G.; Frausto-Reyes, C.

    2008-02-01

    Clinical diagnosis of infections, generally are realized by serological methods, which identifies the antibodies presents in serum or tissue fluids of the patient. Antibodies are proteins present in our bodies that aid in the elimination of pathogens or antigens. Identification of antibodies isotypes is important because can help to predict when and whether patients will recover from infections and are commonly diagnosed by means of indirect methods such as serological test. In the other hand, the majority of these methods requires specific kits for the analysis, special sample preparation, chemical reagents, expensive equipment and require long time for getting results. In this work we show the feasibility to discriminate antibody isotypes in biological fluids like human colostrum by means of Raman spectroscopy and chemometrics. Spectra were obtained using an excitation wavelength of 514 nm over dried samples of human colostrum labeled previously as positives to specific IgG and IgM antibodies against Toxoplasma Gondii by means of ELISA test. Partial least square-discriminant analysis (PLS-DA) was used to discriminate among antibody isotypes by use second derivative of Raman spectra of colostrum samples.

  19. HIV Antibody Characterization as a Method to Quantify Reservoir Size During Curative Interventions

    PubMed Central

    Burbelo, Peter D.; Bayat, Ahmad; Rhodes, Craig S.; Hoh, Rebecca; Martin, Jeffrey N.; Fromentin, Rémi; Chomont, Nicolas; Hütter, Gero; Kovacs, Joseph A.; Deeks, Steven G.

    2014-01-01

    Quantitative humoral profiling of recent samples from a human immunodeficiency virus (HIV)–infected adult who was cured following a delta32/delta32 CCR5 stem cell transplant in 2007 revealed no antibodies against p24, matrix, nucleocapsid, integrase, protease, and gp120, but low levels of antibodies against reverse transcriptase, tat, and gp41. Antibody levels to these HIV proteins persisted at high and stable levels in most noncontrollers, elite controllers, and antiretroviral-treated subjects, but a rare subset of controllers had low levels of antibodies against matrix, reverse transcriptase, integrase, and/or protease. Comprehensive HIV antibody profiles may prove useful for monitoring curative interventions. PMID:24286982

  20. HIV antibody characterization as a method to quantify reservoir size during curative interventions.

    PubMed

    Burbelo, Peter D; Bayat, Ahmad; Rhodes, Craig S; Hoh, Rebecca; Martin, Jeffrey N; Fromentin, Rémi; Chomont, Nicolas; Hütter, Gero; Kovacs, Joseph A; Deeks, Steven G

    2014-05-15

    Quantitative humoral profiling of recent samples from a human immunodeficiency virus (HIV)-infected adult who was cured following a delta32/delta32 CCR5 stem cell transplant in 2007 revealed no antibodies against p24, matrix, nucleocapsid, integrase, protease, and gp120, but low levels of antibodies against reverse transcriptase, tat, and gp41. Antibody levels to these HIV proteins persisted at high and stable levels in most noncontrollers, elite controllers, and antiretroviral-treated subjects, but a rare subset of controllers had low levels of antibodies against matrix, reverse transcriptase, integrase, and/or protease. Comprehensive HIV antibody profiles may prove useful for monitoring curative interventions.

  1. Plasma-Derived C1 Esterase Inhibitor for Acute Antibody-Mediated Rejection Following Kidney Transplantation: Results of a Randomized Double-Blind Placebo-Controlled Pilot Study.

    PubMed

    Montgomery, R A; Orandi, B J; Racusen, L; Jackson, A M; Garonzik-Wang, J M; Shah, T; Woodle, E S; Sommerer, C; Fitts, D; Rockich, K; Zhang, P; Uknis, M E

    2016-05-16

    Antibody-mediated rejection (AMR) is typically treated with plasmapheresis (PP) and intravenous immunoglobulin (standard of care; SOC); however, there is an unmet need for more effective therapy. We report a phase 2b, multicenter double-blind randomized placebo-controlled pilot study to evaluate the use of human plasma-derived C1 esterase inhibitor (C1 INH) as add-on therapy to SOC for AMR. Eighteen patients received 20 000 units of C1 INH or placebo (C1 INH n = 9, placebo n = 9) in divided doses every other day for 2 weeks. No discontinuations, graft losses, deaths, or study drug-related serious adverse events occurred. While the study's primary end point, a difference between groups in day 20 pathology or graft survival, was not achieved, the C1 INH group demonstrated a trend toward sustained improvement in renal function. Six-month biopsies performed in 14 subjects (C1 INH = 7, placebo = 7) showed no transplant glomerulopathy (TG) (PTC+cg≥1b) in the C1 INH group, whereas 3 of 7 placebo subjects had TG. Endogenous C1 INH measured before and after PP demonstrated decreased functional C1 INH serum concentration by 43.3% (p < 0.05) for both cohorts (C1 INH and placebo) associated with PP, although exogenous C1 INH-treated patients achieved supraphysiological levels throughout. This new finding suggests that C1 INH replacement may be useful in the treatment of AMR.

  2. Double tracer autoradiographic method for sequential evaluation of regional cerebral perfusion

    SciTech Connect

    Matsuda, H.; Tsuji, S.; Oba, H.; Kinuya, K.; Terada, H.; Sumiya, H.; Shiba, K.; Mori, H.; Hisada, K.; Maeda, T. )

    1989-01-01

    A new double tracer autoradiographic method for the sequential evaluation of altered regional cerebral perfusion in the same animal is presented. This method is based on the sequential injection of two tracers, {sup 99m}Tc-hexamethylpropyleneamine oxime and N-isopropyl-({sup 125}I)p-iodoamphetamine. This method is validated in the assessment of brovincamine effects on regional cerebral perfusion in an experimental model of chronic brain ischemia in the rat. The drug enhanced perfusion recovery in low-flow areas, selectively in surrounding areas of infarction. The results suggest that this technique is of potential use in the study of neuropharmacological effects applied during the experiment.

  3. Simple, rapid /sup 125/I-labeled cyclosporine double antibody/polyethylene glycol radioimmunoassay used in a pediatric cardiac transplant program

    SciTech Connect

    Berk, L.S.; Webb, G.; Imperio, N.C.; Nehlsen-Cannarella, S.L.; Eby, W.C.

    1986-01-01

    We modified the Sandoz cyclosporine radioimmunoassay because of our need for frequent clinical monitoring of cyclosporine drug levels in allo- and xenograft pediatric cardiac transplant patients. With application of a commercially available (/sup 125/I)cyclosporine label in place of (/sup 3/H)cyclosporine and a second antibody/polyethylene glycol (PEG) method of separation in place of charcoal separation, we simplified and enhanced the speed and precision of assay performance. Studies of 140 whole blood samples comparing this new method to the (/sup 3/H)cyclosporine radioimmunoassay (RIA) method of Berk and colleagues yielded a coefficient of correlation of 0.96 (p less than 0.00001) with means of 626 and 667 ng/ml for (/sup 3/H)RIA and (/sup 125/I)RIA, respectively, and a regression equation of y = 28 + 1.02x. The major advantages are that total assay time is reduced to approximately 1 h; (/sup 125/I)cyclosporine label is used, avoiding the problems associated with liquid scintillation counting; and precision is enhanced by separating bound and free fractions with second antibody/PEG. These modifications should provide for greater ease of assay performance and improved clinical utility of cyclosporine monitoring not only in the pediatric but also in the adult transplant patient.

  4. A Double-difference Earthquake location algorithm: Method and application to the Northern Hayward Fault, California

    USGS Publications Warehouse

    Waldhauser, F.; Ellsworth, W.L.

    2000-01-01

    We have developed an efficient method to determine high-resolution hypocenter locations over large distances. The location method incorporates ordinary absolute travel-time measurements and/or cross-correlation P-and S-wave differential travel-time measurements. Residuals between observed and theoretical travel-time differences (or double-differences) are minimized for pairs of earthquakes at each station while linking together all observed event-station pairs. A least-squares solution is found by iteratively adjusting the vector difference between hypocentral pairs. The double-difference algorithm minimizes errors due to unmodeled velocity structure without the use of station corrections. Because catalog and cross-correlation data are combined into one system of equations, interevent distances within multiplets are determined to the accuracy of the cross-correlation data, while the relative locations between multiplets and uncorrelated events are simultaneously determined to the accuracy of the absolute travel-time data. Statistical resampling methods are used to estimate data accuracy and location errors. Uncertainties in double-difference locations are improved by more than an order of magnitude compared to catalog locations. The algorithm is tested, and its performance is demonstrated on two clusters of earthquakes located on the northern Hayward fault, California. There it colapses the diffuse catalog locations into sharp images of seismicity and reveals horizontal lineations of hypocenter that define the narrow regions on the fault where stress is released by brittle failure.

  5. High resolution image reconstruction method for a double-plane PET system with changeable spacing

    NASA Astrophysics Data System (ADS)

    Gu, Xiao-Yue; Zhou, Wei; Li, Lin; Wei, Long; Yin, Peng-Fei; Shang, Lei-Min; Yun, Ming-Kai; Lu, Zhen-Rui; Huang, Xian-Chao

    2016-05-01

    Breast-dedicated positron emission tomography (PET) imaging techniques have been developed in recent years. Their capacities to detect millimeter-sized breast tumors have been the subject of many studies. Some of them have been confirmed with good results in clinical applications. With regard to biopsy application, a double-plane detector arrangement is practicable, as it offers the convenience of breast immobilization. However, the serious blurring effect of the double-plane PET, with changeable spacing for different breast sizes, should be studied. We investigated a high resolution reconstruction method applicable for a double-plane PET. The distance between the detector planes is changeable. Geometric and blurring components were calculated in real-time for different detector distances, and accurate geometric sensitivity was obtained with a new tube area model. Resolution recovery was achieved by estimating blurring effects derived from simulated single gamma response information. The results showed that the new geometric modeling gave a more finite and smooth sensitivity weight in the double-plane PET. The blurring component yielded contrast recovery levels that could not be reached without blurring modeling, and improved visual recovery of the smallest spheres and better delineation of the structures in the reconstructed images were achieved with the blurring component. Statistical noise had lower variance at the voxel level with blurring modeling at matched resolution, compared to without blurring modeling. In distance-changeable double-plane PET, finite resolution modeling during reconstruction achieved resolution recovery, without noise amplification. Supported by Knowledge Innovation Project of The Chinese Academy of Sciences (KJCX2-EW-N06)

  6. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

    PubMed Central

    2011-01-01

    Background Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV. Results Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA. Conclusions The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome. PMID:21569615

  7. Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S-S Bonding Networks

    NASA Astrophysics Data System (ADS)

    Jones, Lisa M.; Zhang, Hao; Cui, Weidong; Kumar, Sandeep; Sperry, Justin B.; Carroll, James A.; Gross, Michael L.

    2013-06-01

    As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys → Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics.

  8. Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S–S Bonding Networks

    PubMed Central

    Jones, Lisa M.; Zhang, Hao; Cui, Weidong; Kumar, Sandeep; Sperry, Justin B.; Carroll, James A.; Gross, Michael L.

    2013-01-01

    As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys→Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics. PMID:23483515

  9. EDL configuration on a dissimilarly charged protrusion array via double Fourier series and perturbation method.

    PubMed

    Lin, Sung-Hwa; Hsu, Jyh-Ping; Tseng, Shiojenn; Kuo, Yung-Chih; Liu, Bo-Tau

    2011-11-01

    In this study, through the extension of an one-dimensional, dissimilarly charged protrusions surface model set up in our previous work, a novel dissimilarly charged protrusion array (DCPA) model immersed in an electrolyte solution, which could simulate realistically both the surface morphology and the surface charged condition profoundly concerned on a biological cell membrane, or on the surface of a micro-scale, modified particle used in biomedical engineering and water treatment, is proposed. Considering the condition of small protrusions, the electrical potential field due to the electrical double layer (EDL) on DCPA model is solved semi-analytically using both the double Fourier series and the perturbation method. The analysis from the numerical result reveals that, a small, dissimilarly charged protrusion can lead to a steep variation in the local EDL configuration, especially compared with that in the condition when the charged surface is taken roughly as a flat surface using a lumped, mean surface charge density.

  10. Double images encryption method with resistance against the specific attack based on an asymmetric algorithm.

    PubMed

    Wang, Xiaogang; Zhao, Daomu

    2012-05-21

    A double-image encryption technique that based on an asymmetric algorithm is proposed. In this method, the encryption process is different from the decryption and the encrypting keys are also different from the decrypting keys. In the nonlinear encryption process, the images are encoded into an amplitude cyphertext, and two phase-only masks (POMs) generated based on phase truncation are kept as keys for decryption. By using the classical double random phase encoding (DRPE) system, the primary images can be collected by an intensity detector that located at the output plane. Three random POMs that applied in the asymmetric encryption can be safely applied as public keys. Simulation results are presented to demonstrate the validity and security of the proposed protocol.

  11. Earthquake hypocenter relocation using double difference method in East Java and surrounding areas

    SciTech Connect

    C, Aprilia Puspita; Nugraha, Andri Dian; Puspito, Nanang T

    2015-04-24

    Determination of precise hypocenter location is very important in order to provide information about subsurface fault plane and for seismic hazard analysis. In this study, we have relocated hypocenter earthquakes in Eastern part of Java and surrounding areas from local earthquake data catalog compiled by Meteorological, Climatological, and Geophysical Agency of Indonesia (MCGA) in time period 2009-2012 by using the double-difference method. The results show that after relocation processes, there are significantly changes in position and orientation of earthquake hypocenter which is correlated with the geological setting in this region. We observed indication of double seismic zone at depths of 70-120 km within the subducting slab in south of eastern part of Java region. Our results will provide useful information for advance seismological studies and seismic hazard analysis in this study.

  12. Single-Mach and double-Mach reflection - Its representation in Ernst Mach's historical soot method

    NASA Astrophysics Data System (ADS)

    Krehl, P.

    In 1875 Ernst Mach discovered the effect of irregular interaction of shock waves, the so-called single Mach reflection (SMR), which for symmetric geometry is characterized by two triple points. He recorded their two trajectories on a soot-covered glass plate. Appearing as two mirror-symmetric V-branches, they form the well-known Mach soot funnel. Combining this soot method with the schlieren technique facilitates the interpretation of soot-recorded interaction phenomena as well as allows to resolve the soot removal mechanism in time. Increasing the dynamic recording range of the soot layer in terms of reflected shock pressures even renders visualization of double-Mach reflection (DMR) which, in the case of symmetric shock interaction, is characterized by a second concentric, external 'double-Mach funnel'. At transition of DMR to SMR it merges into the ordinary 'single-Mach funnel'.

  13. Earthquake hypocenter relocation using double difference method in East Java and surrounding areas

    NASA Astrophysics Data System (ADS)

    C, Aprilia Puspita; Nugraha, Andri Dian; Puspito, Nanang T.

    2015-04-01

    Determination of precise hypocenter location is very important in order to provide information about subsurface fault plane and for seismic hazard analysis. In this study, we have relocated hypocenter earthquakes in Eastern part of Java and surrounding areas from local earthquake data catalog compiled by Meteorological, Climatological, and Geophysical Agency of Indonesia (MCGA) in time period 2009-2012 by using the double-difference method. The results show that after relocation processes, there are significantly changes in position and orientation of earthquake hypocenter which is correlated with the geological setting in this region. We observed indication of double seismic zone at depths of 70-120 km within the subducting slab in south of eastern part of Java region. Our results will provide useful information for advance seismological studies and seismic hazard analysis in this study.

  14. Double labeling of human leukemic cells using /sup 3/H-cytarabine and monoclonal antibody against bromodeoxyuridine

    SciTech Connect

    Raza, A.; Preisler, H.D.

    1985-02-01

    A new technique using immunofluorescence and autoradiography is described, in which the DNA of cells in S phase are labeled with two different probes. This method makes it possible to study the relationship between DNA synthesis and the uptake and/or incorporation of chemotherapeutic agents into normal or neoplastic cells. An example is provided in which the incorporation of /sup 3/H-cytarabine into DNA is demonstrated to occur only in cells which were synthesizing DNA during exposure to /sup 3/H-cytarabine. Other radioactively labeled probes can be used as well.

  15. Production of Cytomegalovirus Dense Bodies by Scalable Bioprocess Methods Maintains Immunogenicity and Improves Neutralizing Antibody Titers.

    PubMed

    Schneider-Ohrum, Kirsten; Cayatte, Corinne; Liu, Yi; Wang, Zhaoti; Irrinki, Alivelu; Cataniag, Floro; Nguyen, Nga; Lambert, Stacie; Liu, Hui; Aslam, Shahin; Duke, Greg; McCarthy, Michael P; McCormick, Louise

    2016-11-15

    With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods and applied technically advanced assays to determine the relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding of the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate that (i) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, (ii) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, (iii) epithelial cell-tropic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between the UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH pentamer), (iv) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell-tropic DB or gH pentamer-deficient DB preparations, (v) UV-inactivated residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induced neutralizing antibody, preventing viral entry into epithelial cells, and (vi) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine.

  16. Anti-dsDNA antibodies in systemic lupus erythematosus: A combination of two quantitative methods and the ANA pattern is the most efficient strategy of detection.

    PubMed

    Almeida González, Delia; Roces Varela, Alfredo; Marcelino Rodríguez, Itahisa; González Vera, Alexander; Delgado Sánchez, Mónica; Aznar Esquivel, Antonio; Casañas Rodríguez, Carlos; Cabrera de León, Antonio

    2015-12-01

    Several methods have been used to measure anti-double-stranded DNA auto-antibody (anti-dsDNA). Our aim was to determine the most efficient strategy to test anti-dsDNA in systemic lupus erythematosus (SLE). In this study, anti-dsDNA and anti-nuclear antibody (ANA) tests were requested for 644 patients. Anti-dsDNA was tested by RIA, ELISA and CLIA in all patients. The results indicated that 78 patients had a positive anti-dsDNA test according to at least one of the methods. After a 3-year follow-up period only 26 patients were diagnosed with SLE. We evaluated each method and combination of methods. Specificity and positive predictive value (PPV) increased with the number of assay methods used (p=0.002 for trend), and PPV was 100% in patients whose results were positive by all three anti-dsDNA assay methods. The proportion of anti-dsDNA-positive patients who had SLE was highest (82%; p b 0.001) among those with a homogeneous pattern of ANA staining, followed by those with a speckled pattern. In ANA positive patients, when only RIA was considered, 59% of anti-dsDNA-positive patients had SLE, but when RIA and CLIA were both considered, all patients with positive results on both tests had SLE. The combination of RIA+CLIA in patients with homogeneous and speckled ANA staining showed a similar cost and higher sensitivity than RIA alone in ANA positive patients (p b 0.001). We conclude that the most efficient strategy was to combine simultaneously two quantitative and sensitive methods but only in patients with a homogeneous or speckled pattern of ANA staining. This approach maximized specificity and PPV, and reduced costs.

  17. Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Tran, Quang Huy; Hanh Nguyen, Thi Hong; Mai, Anh Tuan; Thuy Nguyen, Thi; Khue Vu, Quang; Nga Phan, Thi

    2012-03-01

    This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml-1 to 1 μg ml-1, and the limit of detection was about 10 ng ml-1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks.

  18. Evaluation of the aggregation states of monoclonal antibodies by diverse and complementary methods.

    PubMed

    Yoshino, Tetsuya; Ishikawa, Tomoyoshi; Ishihara, Takashi; Takeuchi, Yoshimi; Yoshikawa, Hitomi; Yoshida, Hideaki; Yoshida, Hitoshi; Wakamatsu, Kaori

    2011-01-01

    Therapeutic monoclonal antibodies (MAbs) with high specificity and fewer adverse effects are becoming widely used for the treatment of various diseases. MAbs need to be stored and administered at high concentrations in solution, the conditions under which MAbs may aggregate. As aggregated MAbs compromise their safety and efficacy, aggregation should be prevented; thus, it is important to analyze the aggregation states of MAbs in detail. We obtained 2 MAbs against dinitrophenol (DNP) that exhibited different aggregation properties: anti-DNP1 exhibited a much higher aggregate content (dimer or trimer) than anti-DNP2 when analyzed by size-exclusion chromatography (SEC). As anti-DNP1 had a longer complementarity-determining region 3 (CDR3) light chain than anti-DNP2 by 2 amino acid residues, we hypothesized that the increased aggregation of DNP1 was due to these extra residues; therefore, we prepared mutant antibodies with shorter CDR3s to compare their aggregation properties. Anti-DNP1-ΔEI, with the same CDR3 length as anti-DNP2, exhibited no aggregates as expected. Anti-DNP1-ΔI, with 1 additional residue, exhibited a smaller peak than wild-type (WT) in SEC, whereas this mutant exhibited stronger thioflavin T fluorescence than WT, which is indicative of amyloid formation. In addition, the anti-DNP1-ΔI solution (but not others) became opalescent at 4°C and exhibited large visible particles, which are undetectable by SEC. The fragment antigen-binding region of this mutant was found to have lower thermal stability than the others by differential scanning calorimetry. These data suggest that diverse analytical methods should be applied to evaluate MAb aggregation, in addition to the commonly used SEC.

  19. Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves.

    PubMed

    Liu, Zhen; Chen, Zhe; Hong, Jian; Wang, Xuefeng; Zhou, Changyong; Zhou, Xueping; Wu, Jianxiang

    2016-08-01

    Citrus tristeza virus (CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein (CP) gene of CTV was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10(-6) to 10(-7) in indirect enzyme-linked immunosorbent assay (ELISA). Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich (TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and 1:10, 240 (w/v, g/mL), respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.

  20. Double hypernuclei experiment with hybrid emulsion method (J-PARC E07)

    NASA Astrophysics Data System (ADS)

    Ekawa, Hiroyuki; J-APRC E07 Collaboration

    2014-09-01

    Double hypernuclei are important probes to study the system with strangeness -2. In order to search for double hypernuclei, an upgrade experiment is planned at J-PARC K1.8 beam line. In the experiment, the KURAMA spectrometer system will detect Ξ- production in the (K- ,K+) reaction on a diamond target. SSDs located the upstream and the downstream of emulsion plates will record Ξ- tracks which flight toward emulsion plates precisely. Tracks in SSDs and emulsion will be automatically connected by a hybrid method. Discoveries of more than 10 new double hypernuclear species are expected, which enable us to discuss binding energy in terms of mass number dependence. On the other hand, we will also observe X rays from Ξ- atoms with a Germanium detector array installed close to the emulsion by tagging Ξ-stopped events. This will be the first measurement in the world and give information on the Ξ-potential shape at the nuclear surface region. Emulsion production has been completely done and a test experiment for some detectors of KURAMA spectrometer was carried out. In this talk, physics motivation and current status of the J-PARC E07 experiment will be reported.

  1. Second-order perturbation corrections to singles and doubles coupled-cluster methods: General theory and application to the valence optimized doubles model

    SciTech Connect

    Gwaltney, Steven R.; Sherrill, C. David; Head-Gordon, Martin; Krylov, Anna I.

    2000-09-01

    We present a general perturbative method for correcting a singles and doubles coupled-cluster energy. The coupled-cluster wave function is used to define a similarity-transformed Hamiltonian, which is partitioned into a zeroth-order part that the reference problem solves exactly plus a first-order perturbation. Standard perturbation theory through second-order provides the leading correction. Applied to the valence optimized doubles (VOD) approximation to the full-valence complete active space self-consistent field method, the second-order correction, which we call (2), captures dynamical correlation effects through external single, double, and semi-internal triple and quadruple substitutions. A factorization approximation reduces the cost of the quadruple substitutions to only sixth order in the size of the molecule. A series of numerical tests are presented showing that VOD(2) is stable and well-behaved provided that the VOD reference is also stable. The second-order correction is also general to standard unwindowed coupled-cluster energies such as the coupled-cluster singles and doubles (CCSD) method itself, and the equations presented here fully define the corresponding CCSD(2) energy. (c) 2000 American Institute of Physics.

  2. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  3. Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells.

    PubMed

    Yu, Changming; Pike, Gennett M; Rinkoski, Tommy A; Correia, Cristina; Kaufmann, Scott H; Federspiel, Mark J

    2015-08-11

    Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

  4. Development of a simple and quick immunochromatography method for detection of anti-HPV-16/-18 antibodies

    PubMed Central

    Endo, Fumiko; Tabata, Tsutomu; Sadato, Daichi; Kawamura, Machiko; Ando, Noriyuki; Ukaji, Masako; Kobayashi, Kaoru; Kobayashi, Yukuharu; Ikeda, Tomoaki; Shibasaki, Futoshi

    2017-01-01

    Immunochromatography (IC) is widely used to detect target molecules in biological fluids. Since this method can be performed without a special technique or device, IC is a convenient way to assess the existence of antibodies or pathogens such as viruses and bacteria, simply and quickly. In this study, we established an IC method to detect serum antibodies against oncogenic human papillomavirus (HPV)-16 and HPV-18 L1 proteins using recombinant L1 proteins produced by silkworms as antigens. Infection of oncogenic HPVs is a major risk factor of cervical cancer, which is one of the most common cancers in women worldwide. We first measured blood sera of two groups by magnetic beads enzyme-linked immunosorbent assay (MB-ELISA). For the first group, sera were collected prospectively from young women who planned to receive HPV vaccination. The second group consisted of children under 20 years of age, non-vaccinated healthy women, vaccinated healthy women, dysplasia, cervical intraepithelial neoplasia III, and cervical cancer patients. We confirmed that standard vaccination doses significantly increased serum HPV antibody concentrations, and the level was sustained at least more than 30 months after vaccination. In contrast, an increase in antibody concentration was not observed in patients with precancerous cervical changes and cervical cancer. We next measured the samples in both groups using the IC method we originally developed, and found that the measurement values of IC highly correlated with those of MB-ELISA. The simple and quick IC method would be a useful tool for rapid monitoring of L1 specific antibody levels in a non-laboratory environment. With less than one drop of serum, our IC can easily detect serum HPV-16/-18 antibodies within 15 minutes, without the need for electronic devices or techniques. PMID:28158224

  5. Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatography, part 1: optimization of the mobile phase.

    PubMed

    Rodriguez-Aller, Marta; Guillarme, Davy; Beck, Alain; Fekete, Szabolcs

    2016-01-25

    The goal of this work is to provide some recommendations for method development in HIC using monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as model drug candidates. The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC. Except the nature of the stationary phase, which was not discussed in this study, the most important parameter for modifying selectivity was the gradient steepness. The addition of organic solvent (up to 15% isopropanol) in the mobile phase was also found to be useful for mAbs analysis, since it could provide some changes in elution order, in some cases. On the contrary, isopropanol was not beneficial with ADCs, since the most hydrophobic DAR species (DAR6 and DAR8) cannot be eluted from the stationary phase under these conditions. This study also illustrates the possibility to perform HIC method development using optimization software, such as Drylab. The optimum conditions suggested by the software were tested using therapeutic mAbs and commercial cysteine linked ADC (brentuximab-vedotin) and the average retention time errors between predicted and experimental retention times were ∼ 1%.

  6. Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining.

    PubMed

    Cappella, Paolo; Gasparri, Fabio; Pulici, Maurizio; Moll, Jürgen

    2015-04-01

    Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry.

  7. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    NASA Astrophysics Data System (ADS)

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-11-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs.

  8. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    PubMed Central

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-01-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs. PMID:26585778

  9. Double-Ended Surface Walking Method for Pathway Building and Transition State Location of Complex Reactions.

    PubMed

    Zhang, Xiao-Jie; Shang, Cheng; Liu, Zhi-Pan

    2013-12-10

    Toward the activity prediction with large-scale computations, here a double-ended surface walking (DESW) method is developed for connecting two minima on a potential energy surface (PES) and locating the associated transition state (TS) using only the first derivatives. The method operates two images starting from the initial and the final states, respectively, to walk in a stepwise manner toward each other. The surface walking involves repeated bias potential addition and local relaxation with the constrained Broyden dimer method to correct the walking direction. We apply the method to a model PES, a large set of gas phase Baker reactions, and complex surface catalytic reactions, which demonstrates that the DESW method can establish a low energy pathway linking two minima even without iterative optimization of the pathway, from which the TS can be located readily. By comparing the efficiency of the new method with the existing methods, we show that the DESW method is much less computationally demanding and is applicable for reactions with complex PESs. We hope that the DESW method may be integrated with the PES sampling methods for automated reaction prediction.

  10. A novel method of preparing the monoform structure of catalytic antibody light chain.

    PubMed

    Hifumi, Emi; Matsumoto, Shingo; Nakashima, Hiroki; Itonaga, Shogo; Arakawa, Mitsue; Katayama, Yoshiki; Kato, Ryuichi; Uda, Taizo

    2016-02-01

    Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.

  11. A new virus discovered by immunocapture of double-stranded RNA, a rapid method for virus enrichment in metagenomic studies.

    PubMed

    Blouin, Arnaud G; Ross, Howard A; Hobson-Peters, Jody; O'Brien, Caitlin A; Warren, Ben; MacDiarmid, Robin

    2016-09-01

    Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Māori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples.

  12. A comparison of gel diffusion, fluorescent antibody and virus isolation methods in experimental and natural cases of infectious bursal disease.

    PubMed Central

    Ide, P R

    1975-01-01

    In studies with chicks inoculated with the Sk-1 strain of infectious bursal agent the bursa of Fabricius was found to be the tissue of choice for virus isolation as well as for use in the fluorescent antibody test and the agar gel diffusion test. In separate experiments positive results were obtained until postinoculation days 3 or 4 by the agar gel diffusion test, 5 or 6 by the fluorescent antibody test and 14 by the virus isolation method, respectively. Bursas from chickens involved in seven natural outbreaks of infectious bursal disease were then examined by these three methods. Virus was isolated from six outbreaks and infectious bursal agent antigen was demonstrated in three by the agar gel diffusion test method and seven (three by direct examination and four after one passage in chicks) by the fluorescent antibody test method. Passage in chicks was required when nonspecific fluorescence complicated the interpretation of fluorescent antibody test results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:164991

  13. A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies.

    PubMed

    Lee, Christina J; Seth, Gargi; Tsukuda, Joni; Hamilton, Robert W

    2009-03-01

    To meet increasing demands for efficient and streamlined production processes of therapeutic antibodies, improved methods of screening clones are required. In this article, we examined the potential of using antibody transcript levels as criteria for clone screening. We evaluated the QuantiGene Plex, a commercially available, high-throughput assay for simultaneously measuring multiple transcripts from cell lysate. Using the development of stable Chinese hamster ovary cell lines as examples, we investigated the relationship between transcript and antibody levels through several rounds of screening. First, we observed that measured heavy chain transcript levels are generally correlated with specific productivity, enabling the identification of high-producing clones from mRNA. Second, we observed that low ratios (< 1.5) of light to heavy chain transcript levels may be indicative of high antibody aggregation levels, allowing for the rapid identification and elimination of clones of questionable product quality. Therefore, an efficient process of identifying high-producing clones of desirable product quality is possible by using QuantiGene Plex assay to measure antibody transcript levels.

  14. Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

    PubMed Central

    Theil, K W; McCloskey, C M; Saif, L J; Redman, D R; Bohl, E H; Hancock, D D; Kohler, E M; Moorhead, P D

    1981-01-01

    A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays. Images PMID:6270190

  15. A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations.

    PubMed

    Zmolek, Wesley; Bañas, Stefanie; Barfield, Robyn M; Rabuka, David; Drake, Penelope M

    2016-10-01

    Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step.

  16. Could the novel ‘double-hole’ technique be an alternative for the inflow occlusion method?

    PubMed Central

    Bozok, Sahin; Gokhan, Ilhan; Izmir,, Kazdal; Berkan, Ozpak; Ismail, Yurekli; Mert, Kestelli; Serdar, Bayrak

    2016-01-01

    Summary Background Inflow occlusion on beating heart and cardiopulmonary bypass techniques have been proposed for the removal of foreign material, such as stents, catheters and mass lesions, from cardiac chambers. However, both techniques are not devoid of disadvantages and complications. In this article, we define an alternative, novel ‘double-hole’ technique, which is based on opening the right atrium without cardiopulmonary bypass . Methods Bovine hearts were obtained from a local supermarket. Two purse-string sutures were placed in the right atrium using 2-0 braided, non-absorbable polyester suture material, one close to the auricle, and the other close to the interatrial septum. The guidewire of a haemodialysis catheter was inserted through the superior vena cava into the right atrium and passed all the way through the right ventricle. Results We suggest that the double-hole technique may be useful, especially in revision cases with adhesions. Further research should be performed to document the efficacy and safety of this method. Conclusion We are aware that further extensive research is necessary to investigate the utility of this novel technique in contemporary cardiovascular surgery. We believe the doublehole technique has the potential to become a safe, practical and effective measure in the future. PMID:27078129

  17. Double hexagonal graphene ring synthesized using a growth-etching method

    NASA Astrophysics Data System (ADS)

    Liu, Jinyang; Xu, Yangyang; Cai, Hongbing; Zuo, Chuandong; Huang, Zhigao; Lin, Limei; Guo, Xiaomin; Chen, Zhendong; Lai, Fachun

    2016-07-01

    Precisely controlling the layer number, stacking order, edge configuration, shape and structure of graphene is extremely challenging but highly desirable in scientific research. In this report, a new concept named the growth-etching method has been explored to synthesize a graphene ring using the chemical vapor deposition process. The graphene ring is a hexagonal structure, which contains a hexagonal exterior edge and a hexagonal hole in the centre region. The most important concept introduced here is that the oxide nanoparticle derived from annealing is found to play a dual role. Firstly, it acts as a nucleation site to grow the hexagonal graphene domain and then it works as a defect for etching to form a hole. The evolution process of the graphene ring with the etching time was carefully studied. In addition, a double hexagonal graphene ring was successfully synthesized for the first time by repeating the growth-etching process, which not only confirms the validity and repeatability of the method developed here but may also be further extended to grow unique graphene nanostructures with three, four, or even tens of graphene rings. Finally, a schematic model was drawn to illustrate how the double hexagonal graphene ring is generated and propagated. The results shown here may provide valuable guidance for the design and growth of unique nanostructures of graphene and other two-dimensional materials.

  18. The incidence of antinuclear antibodies (ANA) detected by indirect immunofluorescence assay (IFA) method.

    PubMed

    Karamehic, Jasenko; Subasic, Djemo; Gavrankapetanovic, Faris; Zecevic, Lamija; Eminovic, Izet; Memic, Senaid; Seric, Natasa; Drace, Zahida

    2007-01-01

    Human anti-nuclear antibodies (ANA) in Systemic Lupus Erythematosus (SLE) react specificaly with DNA, RNA, several proteins and ribonucleoproteins. Systemic Lupus Erythematosus is the classic type of polysystemic autoimmune disease. The high frequency of ANA is determined in these patients. Actually, all SLE patients are ANA positive. ANA testing by IFA (Indirect Immunofluorescence Assay) is an excellent screening tool for SLE cases, but it is not so highly specific test. Patients with connective tissue diseases, such as rheumatoid arthritis, scleroderma and dermatomyositis are also frequently positive. Results of IFA ANA have relative low specific degree, and for this reason the titration of these specimens to the end point is usually recommended. Indirect immunoflourescence is reference method for ANA testing. Common substrates are thin sections of rodent organs or various types of cell lines. The cell line substrates are preferable to organ sections, because these rapidly dividing cells have higher level for detection of certain clinically relevant antigens such as (e.g., centromere, SSA (Ro) and Scl-70). In this paper we present the results evaluation of ANA incidence, detected by IFA in serum specimens of corresponding clinical patients, during 2005 and 2006.

  19. A double-observer method to estimate detection rate during aerial waterfowl surveys

    USGS Publications Warehouse

    Koneff, M.D.; Royle, J. Andrew; Otto, M.C.; Wortham, J.S.; Bidwell, J.K.

    2008-01-01

    We evaluated double-observer methods for aerial surveys as a means to adjust counts of waterfowl for incomplete detection. We conducted our study in eastern Canada and the northeast United States utilizing 3 aerial-survey crews flying 3 different types of fixed-wing aircraft. We reconciled counts of front- and rear-seat observers immediately following an observation by the rear-seat observer (i.e., on-the-fly reconciliation). We evaluated 6 a priori models containing a combination of several factors thought to influence detection probability including observer, seat position, aircraft type, and group size. We analyzed data for American black ducks (Anas rubripes) and mallards (A. platyrhynchos), which are among the most abundant duck species in this region. The best-supported model for both black ducks and mallards included observer effects. Sample sizes of black ducks were sufficient to estimate observer-specific detection rates for each crew. Estimated detection rates for black ducks were 0.62 (SE = 0.10), 0.63 (SE = 0.06), and 0.74 (SE = 0.07) for pilot-observers, 0.61 (SE = 0.08), 0.62 (SE = 0.06), and 0.81 (SE = 0.07) for other front-seat observers, and 0.43 (SE = 0.05), 0.58 (SE = 0.06), and 0.73 (SE = 0.04) for rear-seat observers. For mallards, sample sizes were adequate to generate stable maximum-likelihood estimates of observer-specific detection rates for only one aerial crew. Estimated observer-specific detection rates for that crew were 0.84 (SE = 0.04) for the pilot-observer, 0.74 (SE = 0.05) for the other front-seat observer, and 0.47 (SE = 0.03) for the rear-seat observer. Estimated observer detection rates were confounded by the position of the seat occupied by an observer, because observers did not switch seats, and by land-cover because vegetation and landform varied among crew areas. Double-observer methods with on-the-fly reconciliation, although not without challenges, offer one viable option to account for detection bias in aerial waterfowl

  20. A Rapid Detection Method of Brucella with Quantum Dots and Magnetic Beads Conjugated with Different Polyclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Song, Dandan; Qu, Xiaofeng; Liu, Yushen; Li, Li; Yin, Dehui; Li, Juan; Xu, Kun; Xie, Renguo; Zhai, Yue; Zhang, Huiwen; Bao, Hao; Zhao, Chao; Wang, Juan; Song, Xiuling; Song, Wenzhi

    2017-03-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Traditional methods for detection of Brucella spp. take 48-72 h that does not meet the need of rapid detection. Herein, a new rapid detection method of Brucella was developed based on polyclonal antibody-conjugating quantum dots and antibody-modified magnetic beads. First, polyclonal antibodies IgG and IgY were prepared and then the antibody conjugated with quantum dots (QDs) and immunomagnetic beads (IMB), respectively, which were activated by N-(3-dimethylaminopropyl)- N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form probes. We used the IMB probe to separate the Brucella and labeled by the QD probe, and then detected the fluorescence intensity with a fluorescence spectrometer. The detection method takes 105 min with a limit of detection of 103 CFU/mL and ranges from 10 to 105 CFU/mL ( R 2 = 0.9983), and it can be well used in real samples.

  1. [Determination of double bonds in olive and sunflower oils by ozonize method].

    PubMed

    Evteeva, N M

    2007-01-01

    Kinetics of spending double bonds of tocotherol and accumulation of peroxides during oxidation of olive and sunflower oils were investigated. Date on spending double bonds during oxidation of commercial oils were measured for the first time.

  2. DSMC calculations for the double ellipse. [direct simulation Monte Carlo method

    NASA Technical Reports Server (NTRS)

    Moss, James N.; Price, Joseph M.; Celenligil, M. Cevdet

    1990-01-01

    The direct simulation Monte Carlo (DSMC) method involves the simultaneous computation of the trajectories of thousands of simulated molecules in simulated physical space. Rarefied flow about the double ellipse for test case 6.4.1 has been calculated with the DSMC method of Bird. The gas is assumed to be nonreacting nitrogen flowing at a 30 degree incidence with respect to the body axis, and for the surface boundary conditions, the wall is assumed to be diffuse with full thermal accommodation and at a constant wall temperature of 620 K. A parametric study is presented that considers the effect of variations of computational domain, gas model, cell size, and freestream density on surface quantities.

  3. Simulation of electric double-layer capacitors: evaluation of constant potential method

    NASA Astrophysics Data System (ADS)

    Wang, Zhenxing; Laird, Brian; Yang, Yang; Olmsted, David; Asta, Mark

    2014-03-01

    Atomistic simulations can play an important role in understanding electric double-layer capacitors (EDLCs) at a molecular level. In such simulations, typically the electrode surface is modeled using fixed surface charges, which ignores the charge fluctuation induced by local fluctuations in the electrolyte solution. In this work we evaluate an explicit treatment of charges, namely constant potential method (CPM)[1], in which the electrode charges are dynamically updated to maintain constant electrode potential. We employ a model system with a graphite electrode and a LiClO4/acetonitrile electrolyte, examined as a function of electrode potential differences. Using various molecular and macroscopic properties as metrics, we compare CPM simulations on this system to results using fixed surface charges. Specifically, results for predicted capacity, electric potential gradient and solvent density profile are identical between the two methods; However, ion density profiles and solvation structure yield significantly different results.

  4. Simple method of monoclonal antibody production against mammalian cellular prion protein.

    PubMed

    Liu, Yong-sheng; Ding, Yao-zhong; Zhang, Jie; Chen, Hao-tai; Zhu, Xiao-ling; Cai, Xue-peng; Liu, Xiang-tao; Xie, Qing-ge

    2010-02-01

    Monoclonal antibodies (MAbs) against prion protein (PrP) are powerful tools for diagnosis and research in transmissible spongiform encephalopathies. Ten MAbs to recombinant/native cellular PrP (PrPc) in mammals were prepared with a simple method and identified in detail. Normal BALB/c mice were immunized with the recombinant bovine mature PrP (rbomPrP) and PrP27-30 (rboPrP27-30) expressed in Escherichia coli. The immunized splenocytes were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA). The characterizations of these MAbs, such as Ig, Ig subclass, titer, affinity index, specificity, epitopes recognized, and binding to recombinant/native PrPc of cattle, sheep, or human beings, were evaluated by Western blotting and indirect or sandwich ELISA. Ten MAbs could be divided into five groups depending on the results of indirect ELISA additivity test and their reaction to E. coli-expressed truncated-PrPs. Isotyping of the MAbs revealed that they belong to IgG1, IgG2a, and IgG2b subclass. Their indirect ELISA titers were between 10(3) and 10(6). Affinity constants were between 10(9) and 10(12) M(-1). Ten MAbs specifically reacted with the rbomPrP, without binding to prion-like protein Doppel and the lysates of E. coli. These MAbs could also respond to the recombinant mature PrP (rmPrP) of sheep and human beings. Also of interest was the ability of the MAbs to bind with dimer of rmPrP and PrP extracted from the brain tissue of cattle or sheep. We conclude that anti-PrP MAbs successfully prepared with a simple method could potentially be useful in mammalian prion research.

  5. Use of finite element analysis to optimize probe design for double sensor method-based thermometer.

    PubMed

    Sim, Soo Young; Joo, Kwang Min; Park, Kwang Suk

    2015-08-01

    Body temperature is an essential vital sign for assessing physiological functions. The double sensor method-based thermometer is a promising technology that may be applicable to body temperature monitoring in daily life. It continuously estimates deep tissue temperature from the intact skin surface. Despite its considerable potential for monitoring body temperature, its key design features have not been investigated. In this study, we considered four design factors: the cover material, insulator material, insulator radius, and insulator height. We also evaluated their effects on the performance of the double sensor thermometer in terms of accuracy, initial waiting time, and the ability to track changes in body temperature. The probe material and size influenced the accuracy and initial waiting time. Finite element analysis revealed that four thermometers of different sizes composed of an aluminum cover and foam insulator provided high accuracy (<0.1°C) under various ambient temperatures and blood perfusion rates: R=20mm, H=5mm; R=15mm, H=10mm; R=20mm, H=10mm; and R=15mm, H=15mm. The initial waiting time was approximately 10min with almost the same traceability of temperature change. Our findings may provide thermometer manufacturers with new insights into probe design and help them fabricate thermometers optimized for specific applications.

  6. Simple Method of Counterclockwise Isthmus Conduction Block by Comparing Double Potentials and Flutter Cycle Length

    PubMed Central

    Rhee, Kyoung-Suk; Kwon, Keun-Sang; Lee, Sun Hwa; Lee, Kang-Hyu; Lee, Sang Rok; Chae, Jei Keon; Kim, Won-Ho; Ko, Jae-Ki; Nam, Gi-Byoung; Kim, You-Ho

    2009-01-01

    Background and Objectives Local wide split double potentials are used as a parameter to determine complete conduction block during cavotricuspid isthmus ablation in patients with isthmus dependent atrial flutter. However, delayed slow conduction in that region can sometimes be very difficult to differentiate from complete block. Flutter cycle length (FCL) can be used to confirm isthmus conduction block, because FCL is a measure of conduction time around the tricuspid annulus (TA). This study was designed to determine which degree of splitting of the local electrograms is adequate to confirm complete isthmus block, using FCL as a reference. Subjects and Methods Cavotricuspid isthmus (CTI) ablation was performed in fifty consecutive patients. The interval between the pacing stimulus on the lateral side of the CTI and the first component of the double potentials on the block line (SD1) corresponded to the counterclockwise conduction time. The interval between the pacing stimulus and second component (SD2) represented the clockwise conduction time to the contralateral side of the ablation line. SD1 and SD2 were measured before and after complete isthmus block. Results An SD1+SD2 reaching 90% of the FCL identified the counterclockwise isthmus conduction block with 94% sensitivity and 100% specificity. Conclusion If the sum of SD1 and SD2 following isthmus ablation was close to the FCL, complete conduction block was predicted with high diagnostic accuracy and positive predictive value for at least counterclockwise conduction. PMID:20049138

  7. Comparing Regional Seismic Location Results Using Kriged Travel Time Correction Surfaces and Double-Difference Methods

    NASA Astrophysics Data System (ADS)

    Begnaud, M. L.; Velasco, A. A.; Steck, L. K.

    2001-12-01

    The use of kriged two-dimensional travel time correction surfaces improves the location of regional seismic events using a sparse network. For more closely-spaced regional events (approximately 150 km), using travel time correction surfaces does not always demonstrate a distinct improvement in relative locations. A new location program HYPODD [Waldhauser and Ellsworth, 2000] uses a double-difference location algorithm with events correlated based on travel times. While this algorithm has been tested on local events with a fairly dense seismic network, it has not been tested with regional networks that have large station-event distances. We will compare locations defined using two-dimensional travel time correction surfaces with those from the double-difference method [Flores et al., this issue]. We will utilize a data set of approximately 142 events for a region around the Mw=7.5 Tibet event of 08NOV1997, with manually-picked P and S arrivals as well as global catalog arrivals having a station-event distance ranging from 5.5 to over 30 degrees. The Tibet event provides a unique ground truth test due to the presence of a related surface rupture identified by Inferometric Synthetic Aperture Radar (InSAR) [Peltzer et al., 1999] and the determination of the Tibet event as associated with a vertical strike/slip fault [Velasco et al., 2000]. We will test which method produces relocations that better align with this surface rupture. In addition, a small (5 km) secondary rupture was identified with the main rupture, but with no associated seismic event. We will also test whether each method relocates any events near to this secondary rupture.

  8. The expression of acidic ribosomal phosphoproteins on the surface membrane of different tissues in autoimmune and normal mice which are the target molecules for anti-double-stranded DNA antibodies.

    PubMed Central

    Sun, K H; Liu, W T; Tang, S J; Tsai, C Y; Hsieh, S C; Wu, T H; Han, S H; Yu, C L

    1996-01-01

    Affinity-purified polyclonal anti-double-stranded DNA (anti-dsDNA) antibodies from patients with systemic lupus erythematosus (SLE) exert a cytostatic effect on cultured rat glomerular mesangial cells (MC). The cognate antigens expressed on the surface of MC have been proved to be acidic ribosomal phosphoproteins (P proteins) in our previous study. The mesangial cytostatic effect of anti-dsDNA antibodies is attributed to the cross-reactivity of the antibodies with membrane-expressed P proteins, but not to the effect of minute amounts of anti-ribosomal P proteins antibodies contained in the anti-dsDNA preparations. Immunofluorescence staining of the native cells demonstrated that anti-dsDNA antibodies bound to the surface of rat mesangial cells, rat brain astrocytes (RBA-1) and mouse fibroblasts (3T3). Anti-dsDNA antibodies also exert potent cytostatic effects on these cells in a dose-dependent manner. In addition, the plasma membranes of different cell lines and tissues from normal and autoimmune mice were isolated and probed by anti-dsDNA antibodies in Western blot analysis. We found the actively proliferating cells such as MC, RBA-1 and 3T3 may express both P0 (38,000 MW) and P1 (19,000 MW) on the surface membrane. In addition, the kidney, liver and spleen from either autoimmune MRL-lpr/lpr or BALB/c mice may constantly express P0 protein, but the expression of P1 is inconsistent. In contrast, brain and muscle from either mice failed to express P proteins on their surface. Unexpectedly, a high molecular weight substance (larger than 205,000 MW) with unknown nature appears in the membrane of brain and muscle tissues in both mice. Immunoprecipitation of the surface-biotinylated MC-lysate by anti-dsDNA antibodies further confirmed that P1 (19,000 MW) and P2 (17,000 MW) are really expressed on the cell surface. These results suggest that P proteins expressed on the surface of different tissues become the targets for anti-dsDNA antibodies mediating pleomorphic tissue

  9. Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses

    PubMed Central

    Pascale, Juan M.; Moreno, Brechla; Chackerian, Bryce; Peabody, David S.

    2017-01-01

    Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets. PMID:28152075

  10. Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses.

    PubMed

    Frietze, Kathryn M; Pascale, Juan M; Moreno, Brechla; Chackerian, Bryce; Peabody, David S

    2017-01-01

    Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets.

  11. Estimates of mumps seroprevalence may be influenced by antibody specificity and serologic method.

    PubMed

    Latner, Donald R; McGrew, Marcia; Williams, Nobia J; Sowers, Sun B; Bellini, William J; Hickman, Carole J

    2014-03-01

    Neutralizing antibodies are assumed to be essential for protection against mumps virus infection, but their measurement is labor- and time-intensive. For this reason, enzyme-linked immunosorbent assays (ELISAs) are typically used to measure mumps-specific IgG levels. However, since there is poor correlation between mumps neutralization titers and ELISAs that measure the presence of mumps-specific IgG levels, ELISAs that better correlate with neutralization are needed. To address this issue, we measured mumps antibody levels by plaque reduction neutralization, by a commercial ELISA (whole-virus antigen), and by ELISAs specific for the mumps nucleoprotein and hemagglutinin. The results indicate that differences in the antibody response to the individual mumps proteins could partially explain the lack of correlation among various serologic tests. Furthermore, the data indicate that some seropositive individuals have low levels of neutralizing antibody. If neutralizing antibody is important for protection, this suggests that previous estimates of immunity based on whole-virus ELISAs may be overstated.

  12. Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

    PubMed Central

    Meulenbroek, Elisabeth M.; Wouters, Diana; Zeerleder, Sacha

    2014-01-01

    Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation. PMID:24514151

  13. A method for triple fluorescence labeling with Vicia villosa agglutinin, an anti-parvalbumin antibody and an anti-G-protein-coupled receptor antibody.

    PubMed

    Bausch, S B

    1998-06-01

    The aim of the original study [S.B. Bausch, C. Chavkin, Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum, Dev. Brain Res., 97, 1996, 169-177] [3] was to develop a method for identifying a subset of mu opioid receptor-expressing interneurons in the rat subiculum for electrophysiological studies. Previous studies had shown that a subset of parvalbumin-positive neurons in the rat subiculum could be labeled with the lectin, Vicia villosa agglutinin (VVA) [C.T. Drake, K.A. Mulligan, T.L. Wimpey, A. Hendrickson, C. Chavkin, Characterization of Vicia villosa agglutinin-labeled GABAergic neurons in the hippocampal formation and in acutely dissociated hippocampus, Brain Res., 554, 1991, 176-185] [11], and that mu opioid receptor immunoreactivity (-IR) and parvalbumin-IR were colocalized in a subset of neurons in the hippocampus and dentate gyrus [S.B. Bausch, C. Chavkin, Colocalization of mu and delta opioid receptors with GABA, parvalbumin and a G-protein-coupled inwardly rectifying potassium channel in the rodent brain, Analgesia, 1, 1995, 282-285] [2]. We hypothesized that a subset of mu opioid receptor-expressing neurons in the subiculum also would express the calcium binding protein, parvalbumin, and could be labeled with VVA. Labeling of live neurons with VVA [11] then could be used to identify these neurons. This protocol was designed to triple-label neurons expressing the mu opioid receptor, parvalbumin and the carbohydrate group, N-acetylgalactosamine (which binds VVA [S.E. Tollefsen, R. Kornfeld, The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins, J. Biol. Chem., 258, 1983, 5172-5176][M.P. Woodward, W.W. Young, R.A. Bloodgood, Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation, J. Immunol. Methods, 78, 1985, 143-153] [25

  14. Efficacy and safety of tabalumab, an anti-BAFF monoclonal antibody, in patients with moderate-to-severe rheumatoid arthritis and inadequate response to TNF inhibitors: results of a randomised, double-blind, placebo-controlled, phase 3 study

    PubMed Central

    Schiff, Michael; Combe, Bernard; Dörner, Thomas; Kremer, Joel M; Huizinga, Thomas W; Veenhuizen, Melissa; Gill, Anne; Komocsar, Wendy; Berclaz, Pierre-Yves; Ortmann, Robert; Lee, Chin

    2015-01-01

    Background Tabalumab is a human monoclonal antibody that neutralises B-cell activating factor. Objectives To evaluate tabalumab efficacy and safety in patients with rheumatoid arthritis (RA). Methods This phase 3, randomised, double-blind, placebo-controlled study evaluated 456 patients with active RA after 24-week treatment with subcutaneous tabalumab (120 mg every 4 weeks (120/Q4W) or 90 mg every 2 weeks (90/Q2W)) versus placebo, with loading doses (240 or 180 mg) at week 0. Patients were allowed background disease-modifying antirheumatic drugs and previously discontinued ≥1 tumour necrosis factor α inhibitors for lack of efficacy/intolerance. Primary end point was American College of Rheumatology 20% (ACR20) response at 24 weeks. This study was terminated early due to futility. Results Most patients had moderate-to-high baseline disease activity. There was no significant difference in week 24 ACR20 responses between 120/Q4W, 90/Q2W, and placebo (17.6%, 24.3%, 20%) per non-responder imputation analysis. Mean percent changes in CD20+ B-cell count (−10.8%, −9.6%, +10.9%) demonstrated expected pharmacodynamic effects. Treatment-emergent adverse events (AEs) were similar (59.5%, 51.7%, 52.6%), as were AE discontinuations (2.6%, 2.7%, 2.6%), serious AEs (4.6%, 4.1%, 3.9%), serious infectious events (1.3%, 0, 0) and events of interest: infections (23.5%, 25.9%, 24%), injection site reactions (13.1%, 25.8%, 11%) and allergy/hypersensitivity (3.9%, 4.1%, 3.9%) reports. Incidence of treatment-emergent antidrug antibodies was similar to placebo (3.9%, 4.8%, 3.9%). No deaths or new/unexpected safety findings were reported. Conclusions Tabalumab did not demonstrate clinical efficacy in patients with RA in this phase 3 study, despite evidence of biological activity. There were no notable differences in safety parameters between tabalumab treatment groups and placebo. Trial registration number: NCT01202773. PMID:26535134

  15. Calculating the reflected radiation error between turbine blades and vanes based on double contour integral method

    NASA Astrophysics Data System (ADS)

    Feng, Chi; Li, Dong; Gao, Shan; Daniel, Ketui

    2016-11-01

    This paper presents a CFD (Computation Fluid Dynamic) simulation and experimental results for the reflected radiation error from turbine vanes when measuring turbine blade's temperature using a pyrometer. In the paper, an accurate reflection model based on discrete irregular surfaces is established. Double contour integral method is used to calculate view factor between the irregular surfaces. Calculated reflected radiation error was found to change with relative position between blades and vanes as temperature distribution of vanes and blades was simulated using CFD. Simulation results indicated that when the vanes suction surface temperature ranged from 860 K to 1060 K and the blades pressure surface average temperature is 805 K, pyrometer measurement error can reach up to 6.35%. Experimental results show that the maximum pyrometer absolute error of three different targets on the blade decreases from 6.52%, 4.15% and 1.35% to 0.89%, 0.82% and 0.69% respectively after error correction.

  16. Nonlinear free vibrations of curved double walled carbon nanotubes using differential quadrature method

    NASA Astrophysics Data System (ADS)

    Cigeroglu, Ender; Samandari, Hamed

    2014-11-01

    Nonlinear free vibration analysis of curved double-walled carbon nanotubes (DWNTs) embedded in an elastic medium is studied in this study. Nonlinearities considered are due to large deflection of carbon nanotubes (geometric nonlinearity) and nonlinear interlayer van der Waals forces between inner and outer tubes. The differential quadrature method (DQM) is utilized to discretize the partial differential equations of motion in spatial domain, which resulted in a nonlinear set of algebraic equations of motion. The effect of nonlinearities, different end conditions, initial curvature, and stiffness of the surrounding elastic medium, and vibrational modes on the nonlinear free vibration of DWCNTs is studied. Results show that it is possible to detect different vibration modes occurring at a single vibration frequency when CNTs vibrate in the out-of-phase vibration mode. Moreover, it is observed that boundary conditions have significant effect on the nonlinear natural frequencies of the DWCNT including multiple solutions.

  17. 1-D seismic velocity model and hypocenter relocation using double difference method around West Papua region

    SciTech Connect

    Sabtaji, Agung E-mail: agung.sabtaji@bmkg.go.id; Nugraha, Andri Dian

    2015-04-24

    West Papua region has fairly high of seismicity activities due to tectonic setting and many inland faults. In addition, the region has a unique and complex tectonic conditions and this situation lead to high potency of seismic hazard in the region. The precise earthquake hypocenter location is very important, which could provide high quality of earthquake parameter information and the subsurface structure in this region to the society. We conducted 1-D P-wave velocity using earthquake data catalog from BMKG for April, 2009 up to March, 2014 around West Papua region. The obtained 1-D seismic velocity then was used as input for improving hypocenter location using double-difference method. The relocated hypocenter location shows fairly clearly the pattern of intraslab earthquake beneath New Guinea Trench (NGT). The relocated hypocenters related to the inland fault are also observed more focus in location around the fault.

  18. Equation-of-motion coupled cluster method for high spin double electron attachment calculations

    SciTech Connect

    Musiał, Monika Lupa, Łukasz; Kucharski, Stanisław A.

    2014-03-21

    The new formulation of the equation-of-motion (EOM) coupled cluster (CC) approach applicable to the calculations of the double electron attachment (DEA) states for the high spin components is proposed. The new EOM equations are derived for the high spin triplet and quintet states. In both cases the new equations are easier to solve but the substantial simplification is observed in the case of quintets. Out of 21 diagrammatic terms contributing to the standard DEA-EOM-CCSDT equations for the R{sub 2} and R{sub 3} amplitudes only four terms survive contributing to the R{sub 3} part. The implemented method has been applied to the calculations of the excited states (singlets, triplets, and quintets) energies of the carbon and silicon atoms and potential energy curves for selected states of the Na{sub 2} (triplets) and B{sub 2} (quintets) molecules.

  19. Study of acoustic field modulation in the regenerator by double loudspeakers method.

    PubMed

    Zhou, Lihua; Xie, Xiujuan; Li, Qing

    2011-11-01

    A model to modulate acoustic field in a regenerator of a thermoacoustic system by the double loudspeakers method is presented in this paper. The equations are derived for acoustic field modulation. They represent the relations among acoustic field (complex pressure p(0), complex velocity u(0), and acoustic impedance Z(0)), driving parameters of loudspeakers (voltage amplitude and its phase difference), and operating parameters involved in a matrix H (frequency, temperature of regenerator). The range of acoustic field is adjustable and limited by the maximal driving voltages of loudspeakers according to driving parameters. The range is simulated and analyzed in the amplitude-phase and complex coordinate planes for a given or variable H. The simulated results indicate that the range has its intrinsic characteristics. The expected acoustic field in a regenerator can be obtained feasibly by the modulation.

  20. Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

    PubMed

    Cao, Mingyan; Mo, Wenjun David; Shannon, Anthony; Wei, Ziping; Washabaugh, Michael; Cash, Patricia

    Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing.

  1. Evaluation of antinuclear antibodies by indirect immunofluorescence and line immunoassay methods': four years' data from Turkey.

    PubMed

    Sener, Asli Gamze; Afsar, Ilhan; Demirci, Mustafa

    2014-12-01

    The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test. The objective of the study was to evaluate ANA patterns in a 4-year period retrospectively. All 19 996 serum samples that were sent to the Laboratory of Medical Microbiology of the tertiary Hospital by any hospital department between 1 January 2009 and 1 January 2013 with a request to test for ANA, anti-ENA or both were included in the study. Of these samples, 4375 (21.9%) were ANA-IIF-positive and 15621 (78.1%) were ANA-IIF-negative. The presented ANA-positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern. Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF-positive but LIA-negative, whereas the rest 31 samples were IIF-negative but LIA-positive. ANA patterns in 22 IIF-positive samples were homogenous (9), speckled (5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro-52, SSB/La and Scl-70 positivity were detected in 31 IIF-negative/LIA-positive samples by LIA. The present study comes forward with its overall scope, which covers 4-year data obtained in tertiary hospital located in the western part of Turkey.

  2. Standardized Method of Measuring Acanthamoeba Antibodies in Sera from Healthy Human Subjects

    PubMed Central

    Chappell, Cynthia L.; Wright, John A.; Coletta, Michael; Newsome, Anthony L.

    2001-01-01

    Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological comparisons. Previous serological studies have utilized a variety of antigen preparations and assay methods and reported disparate (3 to 100%) results. This study was designed to (i) optimize an enzyme-linked immunosorbent assay for detecting serum antibodies to each of the Acanthamoeba serogroups and (ii) test 55 healthy individuals for specific immunoglobulin G reactivity. The highest signal-to-background ratio was found when 3,000 fixed, intact trophozoites per well were used with a 1:10 serum dilution. Sera yielding optical densities of <0.25 against all three Acanthamoeba serogroups were used to define the cutoff for positive results. The highest background reactivity with these sera was seen with Acanthamoeba polyphaga (serogroup 2), followed by Acanthamoeba culbertsoni (serogroup 3) and Acanthamoeba astronyxis (serogroup 1). Of 55 subjects tested, the highest number of positive results was seen with A. polyphaga (81.8%), followed by A. astronyxis (52.8%) and A. culbertsoni (40%). Seven serum samples (12.7%) were negative for all three Acanthamoeba serogroups, 16 (29.1%) were positive for one serogroup only, 16 were positive for two serogroups, and 16 reacted to all three serogroups. Further analysis showed no significant associations between serogroup reactivity and age or gender. However, some ethnic differences were noted, especially with A. polyphaga antigens. In that case, serum samples from Hispanic subjects were 14.5 times less likely to be positive (P = 0.0025) and had lower mean absorbance values (P = 0.047) than those from Caucasian subjects. Overall, these data suggest that Acanthamoeba colonization or infection is more common than previously thought. Mild or asymptomatic infections may contribute to the observed serum reactivities. PMID:11427418

  3. A double-observer method for reducing bias in faecal pellet surveys of forest ungulates

    USGS Publications Warehouse

    Jenkins, K.J.; Manly, B.F.J.

    2008-01-01

    1. Faecal surveys are used widely to study variations in abundance and distribution of forest-dwelling mammals when direct enumeration is not feasible. The utility of faecal indices of abundance is limited, however, by observational bias and variation in faecal disappearance rates that obscure their relationship to population size. We developed methods to reduce variability in faecal surveys and improve reliability of faecal indices. 2. We used double-observer transect sampling to estimate observational bias of faecal surveys of Roosevelt elk Cervus elaphus roosevelti and Columbian black-tailed deer Odocoileus hemionus columbianus in Olympic National Park, Washington, USA. We also modelled differences in counts of faecal groups obtained from paired cleared and uncleared transect segments as a means to adjust standing crop faecal counts for a standard accumulation interval and to reduce bias resulting from variable decay rates. 3. Estimated detection probabilities of faecal groups ranged from < 0.2-1.0 depending upon the observer, whether the faecal group was from elk or deer, faecal group size, distance of the faecal group from the sampling transect, ground vegetation cover, and the interaction between faecal group size and distance from the transect. 4. Models of plot-clearing effects indicated that standing crop counts of deer faecal groups required 34% reduction on flat terrain and 53% reduction on sloping terrain to represent faeces accumulated over a standard 100-day interval, whereas counts of elk faecal groups required 0% and 46% reductions on flat and sloping terrain, respectively. 5. Synthesis and applications. Double-observer transect sampling provides a cost-effective means of reducing observational bias and variation in faecal decay rates that obscure the interpretation of faecal indices of large mammal abundance. Given the variation we observed in observational bias of faecal surveys and persistence of faeces, we emphasize the need for future

  4. ISS Double-Gimbaled CMG Subsystem Simulation Using the Agile Development Method

    NASA Technical Reports Server (NTRS)

    Inampudi, Ravi

    2016-01-01

    This paper presents an evolutionary approach in simulating a cluster of 4 Control Moment Gyros (CMG) on the International Space Station (ISS) using a common sense approach (the agile development method) for concurrent mathematical modeling and simulation of the CMG subsystem. This simulation is part of Training systems for the 21st Century simulator which will provide training for crew members, instructors, and flight controllers. The basic idea of how the CMGs on the space station are used for its non-propulsive attitude control is briefly explained to set up the context for simulating a CMG subsystem. Next different reference frames and the detailed equations of motion (EOM) for multiple double-gimbal variable-speed control moment gyroscopes (DGVs) are presented. Fixing some of the terms in the EOM becomes the special case EOM for ISS's double-gimbaled fixed speed CMGs. CMG simulation development using the agile development method is presented in which customer's requirements and solutions evolve through iterative analysis, design, coding, unit testing and acceptance testing. At the end of the iteration a set of features implemented in that iteration are demonstrated to the flight controllers thus creating a short feedback loop and helping in creating adaptive development cycles. The unified modeling language (UML) tool is used in illustrating the user stories, class designs and sequence diagrams. This incremental development approach of mathematical modeling and simulating the CMG subsystem involved the development team and the customer early on, thus improving the quality of the working CMG system in each iteration and helping the team to accurately predict the cost, schedule and delivery of the software.

  5. Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay.

    PubMed

    Jiao, Yongjun; Zeng, Xiaoyan; Guo, Xiling; Qi, Xian; Zhang, Xiao; Shi, Zhiyang; Zhou, Minghao; Bao, Changjun; Zhang, Wenshuai; Xu, Yan; Wang, Hua

    2012-02-01

    The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.

  6. Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes.

    PubMed

    Miyamoto, Shigehiko; Sano, Sotaro; Takahashi, Koji; Jikihara, Takaaki

    2015-03-15

    Because loop-mediated isothermal amplification (LAMP) can amplify substantial amounts of DNA under isothermal conditions, its applications for simple genetic testing have attracted considerable attention. A positive LAMP reaction is indicated by the turbidity caused by by-products or by the color change after adding a metallochromic indicator to the reaction solution, but these methods have certain limitations. Leuco crystal violet (LCV), a colorless dye obtained after sodium sulfite treatment of crystal violet (CV), was used as a new colorimetric method for detecting LAMP. LCV is reconverted into CV through contact with double-stranded DNA (dsDNA). Therefore, the positive reaction of LAMP is indicated by color change from colorless to violet. The assay is sensitive enough to detect LAMP products, with a detection limit of 7.1 ng/μl for dsDNA. It is also highly selective to dsDNA, and interference with single-stranded DNA and deoxynucleotide triphosphates (dNTPs) is not observed. LCV facilitates direct colorimetric detection of the main product rather than a by-product of the LAMP reaction; therefore, this method can be used under various reaction conditions such as those with added pyrophosphatase in solution. This colorimetric LAMP detection method using LCV is useful for point-of-care genetic testing given its simplicity.

  7. Gemcitabine Conjugated Chitosan and Double Antibodies (Abc-GC-Gemcitabine Nanoparticles) Enhanced Cytoplasmic Uptake of Gemcitabine and Inhibit Proliferation and Metastasis In Human SW1990 Pancreatic Cancer Cells

    PubMed Central

    Xiao, Jun; Yu, Haibo

    2017-01-01

    Background Pancreatic cancer is considered a chemoresistant neoplasm with extremely dismal prognosis and gemcitabine treatment is associated with many side effects and poor overall survival. The study aimed at developing a new nanobioconjugate, which specifically delivered gemcitabine and anti-EGFR antibody into pancreatic cancer cells. Material/Methods The novel nanodrug is based on chitosan platform, which is non-toxic, biocompatibility and biodegradable. We measured the effects of proliferation and metastasis on SW1990 by CCK-8 assay, colony formation assay, wound healing assay and Transwell assay. The expression of related proteins were evaluated by Western blot. Results We synthesized Abc-GC-gemcitabine nanoparticles successfully with the encapsulation rate of nanobioconjugates was 91.63% and the drug loadings was 9.97%. Both GC-gemcitabine microspheres solution (GC group) and Abc-GC-gemcitabine microspheres solution (Abc group) inhibited cells proliferation, colony formation, migration and invasion in SW1990 cells dramatically. Moreover, Abc-GC-gemcitabine microspheres expressed more significant inhibited action than GC-gemcitabine microspheres efficiently Conclusions Our data suggested that Abc-GC-gemcitabine nanoparticles could have promising potential in treating metastasized and chemoresistant pancreatic cancer by enhancing the drug efficacy and minimizing off target effects. PMID:28366930

  8. Gemcitabine Conjugated Chitosan and Double Antibodies (Abc-GC-Gemcitabine Nanoparticles) Enhanced Cytoplasmic Uptake of Gemcitabine and Inhibit Proliferation and Metastasis In Human SW1990 Pancreatic Cancer Cells.

    PubMed

    Xiao, Jun; Yu, Haibo

    2017-04-03

    BACKGROUND Pancreatic cancer is considered a chemoresistant neoplasm with extremely dismal prognosis and gemcitabine treatment is associated with many side effects and poor overall survival. The study aimed at developing a new nanobioconjugate, which specifically delivered gemcitabine and anti-EGFR antibody into pancreatic cancer cells. MATERIAL AND METHODS The novel nanodrug is based on chitosan platform, which is non-toxic, biocompatibility and biodegradable. We measured the effects of proliferation and metastasis on SW1990 by CCK-8 assay, colony formation assay, wound healing assay and Transwell assay. The expression of related proteins were evaluated by Western blot. RESULTS We synthesized Abc-GC-gemcitabine nanoparticles successfully with the encapsulation rate of nanobioconjugates was 91.63% and the drug loadings was 9.97%. Both GC-gemcitabine microspheres solution (GC group) and Abc-GC-gemcitabine microspheres solution (Abc group) inhibited cells proliferation, colony formation, migration and invasion in SW1990 cells dramatically. Moreover, Abc-GC-gemcitabine microspheres expressed more significant inhibited action than GC-gemcitabine microspheres efficiently CONCLUSIONS Our data suggested that Abc-GC-gemcitabine nanoparticles could have promising potential in treating metastasized and chemoresistant pancreatic cancer by enhancing the drug efficacy and minimizing off target effects.

  9. Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections.

    PubMed

    Veh, R W

    1991-01-02

    For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling.

  10. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    SciTech Connect

    Iwasaki, Ryohei; Kiuchi, Hiroki; Ihara, Masaki; Mori, Toshihiro; Kawakami, Masayuki; Ueda, Hiroshi

    2009-07-03

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  11. Double error shrinkage method for identifying protein binding sites observed by tiling arrays with limited replication

    PubMed Central

    Kim, Youngchul; Bekiranov, Stefan; Lee, Jae K.; Park, Taesung

    2009-01-01

    Motivation: ChIP–chip has been widely used for various genome-wide biological investigations. Given the small number of replicates (typically two to three) per biological sample, methods of analysis that control the variance are desirable but in short supply. We propose a double error shrinkage (DES) method by using moving average statistics based on local-pooled error estimates which effectively control both heterogeneous error variances and correlation structures of an extremely large number of individual probes on tiling arrays. Results: Applying DES to ChIP–chip tiling array study for discovering genome-wide protein-binding sites, we identified 8400 target regions that include highly likely TFIID binding sites. About 33% of these were well matched with the known transcription starting sites on the DBTSS library, while many other newly identified sites have a high chance to be real binding sites based on a high positive predictive value of DES. We also showed the superior performance of DES compared with other commonly used methods for detecting actual protein binding sites. Contact: tspark@snu.ac.kr; jaeklee@virginia.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19667080

  12. Evaluation of the constant potential method in simulating electric double-layer capacitors

    SciTech Connect

    Wang, Zhenxing; Laird, Brian B.; Yang, Yang; Olmsted, David L.; Asta, Mark

    2014-11-14

    A major challenge in the molecular simulation of electric double layer capacitors (EDLCs) is the choice of an appropriate model for the electrode. Typically, in such simulations the electrode surface is modeled using a uniform fixed charge on each of the electrode atoms, which ignores the electrode response to local charge fluctuations in the electrolyte solution. In this work, we evaluate and compare this Fixed Charge Method (FCM) with the more realistic Constant Potential Method (CPM), [S. K. Reed et al., J. Chem. Phys. 126, 084704 (2007)], in which the electrode charges fluctuate in order to maintain constant electric potential in each electrode. For this comparison, we utilize a simplified LiClO{sub 4}-acetonitrile/graphite EDLC. At low potential difference (ΔΨ ⩽ 2 V), the two methods yield essentially identical results for ion and solvent density profiles; however, significant differences appear at higher ΔΨ. At ΔΨ ⩾ 4 V, the CPM ion density profiles show significant enhancement (over FCM) of “inner-sphere adsorbed” Li{sup +} ions very close to the electrode surface. The ability of the CPM electrode to respond to local charge fluctuations in the electrolyte is seen to significantly lower the energy (and barrier) for the approach of Li{sup +} ions to the electrode surface.

  13. Evaluation of the constant potential method in simulating electric double-layer capacitors.

    PubMed

    Wang, Zhenxing; Yang, Yang; Olmsted, David L; Asta, Mark; Laird, Brian B

    2014-11-14

    A major challenge in the molecular simulation of electric double layer capacitors (EDLCs) is the choice of an appropriate model for the electrode. Typically, in such simulations the electrode surface is modeled using a uniform fixed charge on each of the electrode atoms, which ignores the electrode response to local charge fluctuations in the electrolyte solution. In this work, we evaluate and compare this Fixed Charge Method (FCM) with the more realistic Constant Potential Method (CPM), [S. K. Reed et al., J. Chem. Phys. 126, 084704 (2007)], in which the electrode charges fluctuate in order to maintain constant electric potential in each electrode. For this comparison, we utilize a simplified LiClO4-acetonitrile/graphite EDLC. At low potential difference (ΔΨ ⩽ 2 V), the two methods yield essentially identical results for ion and solvent density profiles; however, significant differences appear at higher ΔΨ. At ΔΨ ⩾ 4 V, the CPM ion density profiles show significant enhancement (over FCM) of "inner-sphere adsorbed" Li(+) ions very close to the electrode surface. The ability of the CPM electrode to respond to local charge fluctuations in the electrolyte is seen to significantly lower the energy (and barrier) for the approach of Li(+) ions to the electrode surface.

  14. Evaluation of the constant potential method in simulating electric double-layer capacitors

    NASA Astrophysics Data System (ADS)

    Wang, Zhenxing; Yang, Yang; Olmsted, David L.; Asta, Mark; Laird, Brian B.

    2014-11-01

    A major challenge in the molecular simulation of electric double layer capacitors (EDLCs) is the choice of an appropriate model for the electrode. Typically, in such simulations the electrode surface is modeled using a uniform fixed charge on each of the electrode atoms, which ignores the electrode response to local charge fluctuations in the electrolyte solution. In this work, we evaluate and compare this Fixed Charge Method (FCM) with the more realistic Constant Potential Method (CPM), [S. K. Reed et al., J. Chem. Phys. 126, 084704 (2007)], in which the electrode charges fluctuate in order to maintain constant electric potential in each electrode. For this comparison, we utilize a simplified LiClO4-acetonitrile/graphite EDLC. At low potential difference (ΔΨ ⩽ 2 V), the two methods yield essentially identical results for ion and solvent density profiles; however, significant differences appear at higher ΔΨ. At ΔΨ ⩾ 4 V, the CPM ion density profiles show significant enhancement (over FCM) of "inner-sphere adsorbed" Li+ ions very close to the electrode surface. The ability of the CPM electrode to respond to local charge fluctuations in the electrolyte is seen to significantly lower the energy (and barrier) for the approach of Li+ ions to the electrode surface.

  15. A novel method to get methotrexatum/layered double hydroxides intercalation compounds and their release properties

    NASA Astrophysics Data System (ADS)

    Qi, Fenglin; Zhang, Xiaoqing; Li, Shuping

    2013-08-01

    In this context, the methotrexatum/layered double hydroxides (MTX/LDHs) intercalation compounds have been synthesized by a mechanochemical-hydrothermal method, which involves a grinding process and subsequent hydrothermal treatment. The influence of R (molar ratio of Mg2+ to Al3+ to MTX) values on the structure and morphology of the intercalation compounds and their release properties were investigated systematically. The resulting compounds were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), inductively coupled plasma (ICP), thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis. All the results indicate that R value has significant influence on the intercalation of MTX anions into LDH interlayer and the optimal R value is 2:1:0.5. Furthermore, four dissolution-diffusion kinetic models were used to fit the in vitro release of MTX from LDH layers. The release process can be divided into two stages: firstly surface diffusion and secondly intraparticle diffusion. The study also revealed that the properties of the intercalation compounds is comparable to that obtained from standard methods such as co-precipitation method, but with time, solvent and energy saving.

  16. Comparison of four methods for the biofunctionalization of gold nanorods by the introduction of sulfhydryl groups to antibodies

    PubMed Central

    Mei, Zhong; Wang, Yanyan

    2017-01-01

    Introducing sulfhydryl groups to biomolecules to functionalize gold nanorods (GNRs) is an attractive method that involves the creation of a strong Au–S bond. Previously, we developed a facile method to functionalize GNR surfaces by thiolating antibodies using Traut’s reagent. In the current study, we evaluated several methods for the introduction of thiol groups onto the surface of GNRs by using Traut’s reagent, dithiotreitol (DTT), dithiolaromatic PEG6-CONHNH2, and thiol-polyethylene glycolamine (SH-PEG-NH2) combined with EDC reaction. We showed that the four above-mentioned thiolation methods can efficiently functionalize GNRs and simplify the functionalization procedures. The formed GNR-bioconjugates showed superior stability without compromising the biological activity. The GNR nanochip prepared with these four thiolated antibodies can detect human IgG targets with specificity. However, SH-PEG-NH2 combined with EDC reaction may affect the amount of functionalized GNRs because of the efficiency of thiol moiety linkage to antibodies, thereby affecting the sensitivity of the GNR sensor. The introduction of a thiol group to antibodies by using Traut’s reagent, DTT, and PEG6-CONHNH2 allowed for direct immobilization onto the GNR surface, improved the efficacy of functionalized GNRs, and increased the sensitivity in response to target detection as a biosensor. Given that PEG6-CONHNH2 modification requires glycosylated biomolecules, Traut’s reagent and DTT thiolation are recommended as universal applications of GNR biofunctionalization and can be easily extended to other sensing applications based on other gold nanostructures or new biomolecules. PMID:28326226

  17. Enrollment Forecasting with Double Exponential Smoothing: Two Methods for Objective Weight Factor Selection. AIR Forum 1980 Paper.

    ERIC Educational Resources Information Center

    Gardner, Don E.

    The merits of double exponential smoothing are discussed relative to other types of pattern-based enrollment forecasting methods. The difficulties associated with selecting an appropriate weight factor are discussed, and their potential effects on prediction results are illustrated. Two methods for objectively selecting the "best" weight…

  18. Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins.

    PubMed

    Kavanagh, Owen; Elliott, Christopher T; Campbell, Katrina

    2015-04-01

    Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

  19. A Simple Method for Assessment of Human Anti-Neu5Gc Antibodies Applied to Kawasaki Disease

    PubMed Central

    Padler-Karavani, Vered; Tremoulet, Adriana H.; Yu, Hai; Chen, Xi; Burns, Jane C.; Varki, Ajit

    2013-01-01

    N-Glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin that metabolically incorporates into diverse native glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, though with variable levels and epitope-recognition profiles. These antibodies likely play a role in several inflammation-mediated pathologies including cardiovascular diseases and cancer. In cancer, they have dualistic and opposing roles, either stimulating or repressing disease, as a function of their dose, and some of these antibodies serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of inflammation-mediated diseases, and changes in their levels could potentially be used to monitor disease progression and/or response to therapy. Currently, it is difficult to determine levels of anti-Neu5Gc antibodies in individual human samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we describe a simple and specific method for detection and overall estimation of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah−/− knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use Cmah−/− knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in sera of patients with Kawasaki disease (KD) at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms with risk of thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays

  20. Development of a Time and Cost Benefit Antibody Binding Test-Based Method for Determination of Rabies Vaccine Potency.

    PubMed

    Asgary, Vahid; Mojtabavi, Nazanin; Janani, Alireza; Mousavi, Tahereh; Hadjati, Jamshid; Khosravy, Mohammad Sadeq; Ahangari Cohan, Reza

    2017-04-01

    This study is an improvement on the antibody binding test, known as ABT method, to develop a simple and fast method in comparison with NIH for determination of rabies vaccine potency. In the current study, several commercial human and veterinary vaccines were tested using both modified ABT and NIH methods. The ED50 was calculated using the probit method and the relative potency of each vaccine was measured based on the reference vaccine. The test was repeated four times to calculate the reproducibility of the method. Statistical analysis indicated that there was no significant difference between the result obtained from NIH and modified ABT method for either human or veterinary vaccines (p > 0.05). In addition, the linearity of the method (R(2)) was calculated as 0.94 by serial dilution of a test vaccine. Coefficient variances were determined as less than and more than 10% for the human and veterinary rabies vaccines, respectively. In conclusion, the findings suggest that the modified method could be considered as an alternative approach for rabies vaccine potency determination in in-process quality control tests at industrial scale. It is a time and cost benefit method and accuracy may further be increased by employing monoclonal antibodies against trimeric form of G glycoprotein. However, the use of serum samples may be useful compared with an artificial mix of antibodies because other components from the serum samples could have a positive impact on cell sensitivity and mimic more the complexity of the immune response. Although the modified test has solved a fundamental problem, it is still not sensitive enough for veterinary vaccine assessment and needs further modifications to obtain the acceptability criteria.

  1. Simple hydraulic conductivity estimation by the Kalman filtered double constraint method.

    PubMed

    El-Rawy, M A; Batelaan, O; Zijl, W

    2015-01-01

    This paper presents the Kalman Filtered Double Constraint Method (DCM-KF) as a technique to estimate the hydraulic conductivities in the grid blocks of a groundwater flow model. The DCM is based on two forward runs with the same initial grid block conductivities, but with alternating flux-head conditions specified on parts of the boundary and the wells. These two runs are defined as: (1) the flux run, with specified fluxes (recharge and well abstractions), and (2) the head run, with specified heads (measured in piezometers). Conductivities are then estimated as the initial conductivities multiplied by the fluxes obtained from the flux run and divided by the fluxes obtained from the head run. The DCM is easy to implement in combination with existing models (e.g., MODFLOW). Sufficiently accurate conductivities are obtained after a few iterations. Because of errors in the specified head-flux couples, repeated estimation under varying hydrological conditions results in different conductivities. A time-independent estimate of the conductivities and their inaccuracy can be obtained by a simple linear KF with modest computational requirements. For the Kleine Nete catchment, Belgium, the DCM-KF yields sufficiently accurate calibrated conductivities. The method also results in distinguishing regions where the head-flux observations influence the calibration from areas where it is not able to influence the hydraulic conductivity.

  2. A NUMERICAL METHOD FOR STUDYING SUPER-EDDINGTON MASS TRANSFER IN DOUBLE WHITE DWARF BINARIES

    SciTech Connect

    Marcello, Dominic C.; Tohline, Joel E. E-mail: tohline@phys.lsu.edu

    2012-04-01

    We present a numerical method for the study of double white dwarf (DWD) binary systems at the onset of super-Eddington mass transfer. We incorporate the physics of ideal inviscid hydrodynamical flow, Newtonian self-gravity, and radiation transport on a three-dimensional uniformly rotating cylindrical Eulerian grid. Care has been taken to conserve the key physical quantities such as angular momentum and energy. Our new method conserves total energy to a higher degree of accuracy than other codes that are presently being used to model mass transfer in DWD systems. We present the results of verification tests and simulate the first 20 + orbits of a binary system of mass ratio q 0.7 at the onset of dynamically unstable direct impact mass transfer. The mass transfer rate quickly exceeds the critical Eddington limit by many orders of magnitude, and thus we are unable to model a trans-Eddington phase. It appears that radiation pressure does not significantly affect the accretion flow in the highly super-Eddington regime. An optically thick common envelope forms around the binary within a few orbits. Although this envelope quickly exceeds the spatial domain of the computational grid, the fraction of the common envelope that exceeds zero gravitational binding energy is extremely small, suggesting that radiation-driven mass loss is insignificant in this regime. It remains to be seen whether simulations that capture the trans-Eddington phase of such flows will lead to the same conclusion or show that substantial material gets expelled.

  3. Development of methods for avian oil toxicity studies using the double crested cormorant (Phalacrocorax auritus).

    PubMed

    Cunningham, Fred; Dean, Karen; Hanson-Dorr, Katie; Harr, Kendal; Healy, Kate; Horak, Katherine; Link, Jane; Shriner, Susan; Bursian, Steven; Dorr, Brian

    2017-03-24

    Oral and external dosing methods replicating field exposure were developed using the double crested cormorant (DCCO) to test the toxicity of artificially weathered Deepwater Horizon Mississippi Canyon 252 oil. The majority of previous oil dosing studies conducted on wild-caught birds used gavage methods to dose birds with oil and determine toxicity. However, rapid gut transit time of gavaged oil likely reduces oil absorption. In the present studies, dosing relied on injection of oil into live feeder fish for oral dosing of these piscivorous birds, or applying oil to body contour feathers resulting in transdermal oil exposure and oral exposure through preening. Both oral and external oil dosing studies identified oil-related toxicity endpoints associated with oxidative stress such as hemolytic anemia, liver and kidney damage, and immuno-modulation or compromise. External oil application allowed for controlled study of thermoregulatory stress as well. Infrared thermal images indicated significantly greater surface temperatures and heat loss in treated birds following external oil applications; however, measurements collected by coelomically implanted temperature transmitters showed that internal body temperatures were stable over the course of the study period. Birds exposed to oil externally consumed more fish than control birds, indicating metabolic compensation for thermal stress. Conversely, birds orally dosed with oil experienced hypothermia and consumed less fish compared to control birds.

  4. Determination of optical properties in dental restorative biomaterials using the inverse-adding-doubling method

    NASA Astrophysics Data System (ADS)

    Fernández-Oliveras, Alicia; Rubiño, Manuel; Pérez, María. M.

    2013-11-01

    Light propagation in biological media is characterized by the absorption coefficient, the scattering coefficient, the scattering phase function, the refractive index, and the surface conditions (roughness). By means of the inverse-adding-doubling (IAD) method, transmittance and reflectance measurements lead to the determination of the absorption coefficient and the reduced scattering coefficient. The additional measurement of the phase function performed by goniometry allows the separation of the reduced scattering coefficient into the scattering coefficient and the scattering anisotropy factor. The majority of techniques, such as the one utilized in this work, involve the use of integrating spheres to measure total transmission and reflection. We have employed an integrating sphere setup to measure the total transmittance and reflectance of dental biomaterials used in restorative dentistry. Dental biomaterials are meant to replace dental tissues, such as enamel and dentine, in irreversibly diseased teeth. In previous works we performed goniometric measurements in order to evaluate the scattering anisotropy factor for these kinds of materials. In the present work we have used the IAD method to combine the measurements performed using the integrating sphere setup with the results of the previous goniometric measurements. The aim was to optically characterize the dental biomaterials analyzed, since whole studies to assess the appropriate material properties are required in medical applications. In this context, complete optical characterizations play an important role in achieving the fulfillment of optimal quality and the final success of dental biomaterials used in restorative dentistry.

  5. Methods to uncover an antibody epitope in the KPI domain of Alzheimer's amyloid precursor protein for immunohistochemistry in human brain.

    PubMed

    Campbell, E; Pearson, R C; Parkinson, D

    1999-11-15

    A novel polyclonal antibody (Ab993), specific for a KPI domain epitope of APP, was characterised for use in immunoprecipitation, Western blotting and immunohistochemistry. Conditioned medium from NTera2/D1 cells was used for immunoprecipitation and Western blots. Paraffin-embedded human brain sections were used for immunohistochemistry. The antibody recognised KPI-containing APP on Western blots after standard solubilisation but immunoprecipitation of soluble APP required reduction with 2-mercaptoethanol followed by alkylation of reduced sulphydryl bonds with sodium iodoacetate. Immunohistochemical staining of human brain sections was significantly enhanced by this pre-treatment. Microwaving of sections also increased immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation in 80% formic acid did not confer any enhancement of immunoreactivity. Ab993, applied with the methods reported here, is expected to be valuable in investigations of the pathogenesis of Alzheimer's disease to determine the source of the beta-amyloid peptide.

  6. Rapid method to detect rubella immunoglobulin M and immunoglobulin A antibodies.

    PubMed Central

    Schmitz, H; Shimizu, H; Kampa, D; Doerr, H W

    1975-01-01

    Immunoglobulin (Ig) G was removed from serum specimens by precipitation with gamma chain-specific anti-human IgG of rabbit origin. The remaining rubella virus-specific IgM (and IgA) antibodies were then detected by the rubella hemagglutination-inhibition test. This procedure has proven to be as reliable as estimations carried out with IgM fractions separated on a sucrose density gradient. PMID:1176596

  7. Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

    PubMed Central

    Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders. PMID:27006517

  8. Simple immunohistochemical staining method using large sized gold colloid conjugated secondary antibody.

    PubMed

    Lee, Eun Jung; Kim, Yonggoo; Lim, Jihyang; Kim, Myungshin; Kang, Chang Suk; Lee, Jae Hoon; Mok, Rak Sun; Park, Young Nam; Choi, Ha-Young; Han, Kyungja

    2007-01-01

    To test the feasibility of semiquantitative immunohistochemical staining (IHC) with large sized gold conjugated secondary antibody (gold-2 degrees Ab), we used beads covered with a known amount of primary antibody and a secondary antibody conjugated with gold colloid particles (20 and 40 nm in diameter), and we compared the results to those obtained by enzyme IHC. Beads coated with 6 graded amounts of mouse IgG molecules showed 6 levels of color intensity. The graded color intensities could readily be distinguished. The color developed as soon as we added gold-2 degrees Ab, and the intensities were stable for 1 wk. Enzyme IHC using identical beads showed dregs of pigment after incubation in DAB for 5 min. The large sized gold-2 degrees Ab showed strong signals on cell surfaces; application of the large sized gold-2 degrees Ab to paraffin-embedded tissue sections was also feasible. The color was bright red and was easier to differentiate from hemosiderin pigment than the color developed by enzyme IHC. In conclusion, gold IHC with large sized gold-2 degrees Ab is superior to enzyme IHC for quantification of antigens via IHC. Gold IHC is especially recommended for tissues with many macrophages, such as bone marrow and spleen.

  9. Phase transfer and point-spread function of the human eye determined by a new asymmetric double-pass method.

    PubMed

    Navarro, R; Losada, M A

    1995-11-01

    A recent study has shown that the double-pass method provides a good estimate of the ocular modulation transfer function (MTF) but that it does not yield the phase transfer function (PTF) [J. Opt. Soc. Am. A 12, 195 (1995)]. Therefore, one cannot recover the true retinal point-spread function (PSF). We present a modification of the double-pass method to overcome this problem. The key is to break the symmetry between the two passes. By using an unexpanded Gaussian input beam, we produce a diffraction-limited PSF for the first passes. Then, by using a large exit pupil, we get an aberrated PSF for the second pass. The double-pass aerial image is the cross correlation of both PSF's, so that the Fourier transform of such an aerial image directly provides the true retinal PTF, up to the cutoff frequency of the effective (small), diffraction-limited entrance pupil. The resulting double-pass aerial image is a blurred version of the true retinal PSF. Thus it shows the effect not only of even symmetric aberrations but also of odd and irregular aberrations such as coma. We have explored two different ways to retrieve the true retinal PSF: (a) deblurring of the aerial image and (b) PSF reconstruction combining PTF data with conventional double-pass MTF. We present promising initial results with both artificial and real eyes.

  10. HANFORD DOUBLE SHELL TANK (DST) THERMAL & SEISMIC PROJECT BUCKLING EVALUATION METHODS & RESULTS FOR THE PRIMARY TANKS

    SciTech Connect

    MACKEY, T.C.

    2006-03-17

    This report documents a detailed buckling evaluation of the primary tanks in the Hanford double shell waste tanks. The analysis is part of a comprehensive structural review for the Double-Shell Tank Integrity Project. This work also provides information on tank integrity that specifically responds to concerns raise by the Office of Environment, Safety, and Health (ES&H) Oversight (EH-22) during a review (in April and May 2001) of work being performed on the double-shell tank farms, and the operation of the aging waste facility (AWF) primary tank ventilation system.

  11. Symmetrized complex amplitudes for He double photoionization from the time-dependent close coupling and exterior complex scaling methods

    SciTech Connect

    Horner, D.A.; Colgan, J.; Martin, F.; McCurdy, C.W.; Pindzola, M.S.; Rescigno, T.N.

    2004-06-01

    Symmetrized complex amplitudes for the double photoionization of helium are computed by the time-dependent close-coupling and exterior complex scaling methods, and it is demonstrated that both methods are capable of the direct calculation of these amplitudes. The results are found to be in excellent agreement with each other and in very good agreement with results of other ab initio methods and experiment.

  12. A Facile Method to Fabricate Double Gyroid as A Polymer Template for Nanohybrids

    NASA Astrophysics Data System (ADS)

    Wang, Hsiao-Fang; Ho, Rong-Ming

    2015-03-01

    Here, we suggest a facile method to acquire double gyroid (DG) phase from the self-assembly of chiral block copolymers (BCPs*), polystyrene- b-poly(L-lactide) (PS-PLLA). A wide region for the formation of DG can be found in the phase diagram of the BCPs*, suggesting that helical phase (H*) from the self-assembly of BCPs* can serve as a stepping stone for the formation of the DG due to an easy path for order-order transition from two-dimensional to three-dimensional (network) structure. Moreover, the order-order transition from metastable H* to stable DG can be expedited by blending the PS-PLLA with compatible entity. Moreover, PS-PLLA blends are prepared by using styrene oligomer (S) to fine-tune the morphologies of the blends at which the molecular weight ratio of the S and compatible PS block (r) is less than 0.1. Owing to the use of the low-molecular-weight oligomer, the increase of BCP chain mobility in the blends significantly reduces the transformation time for the order-order transition from H* to DG. Consequently, nanoporous gyroid SiO2 can be fabricated using hydrolyzed PS-PLLA blends as a template for sol-gel reaction followed by removal of the PS matrix.

  13. Relative clock estimation method between two LEO satellites with a double-difference solution constraint

    NASA Astrophysics Data System (ADS)

    Liu, Junhong; Gu, Defeng; Ju, Bing; Lai, Yuwang; Yi, Dongyun

    2015-04-01

    A method of estimating the relative clocks between two spaceborne global positioning system (GPS) receivers based on the single-difference (SD) observations is investigated in this paper. Especially, the advantages of introducing a double-difference (DD) solution constraint, including the orbits and ambiguities, are discussed with the simulated data and the real data of Gravity Recovery And Climate Experiment (GRACE) satellites. The theoretical accuracy analysis shows that the accuracy of the relative clocks is improved and the edge effects are eliminated with a DD solution constraint. The simulations indicate a potential accuracy improvement of at least 30% of the relative clocks with the constraint. Furthermore, one month's real data is processed and the overlapping data arcs are used to validate the accuracy of the relative clock solutions. The average overlapping root mean square (RMS) of the relative clock solutions is approximate 99 ps and 31 ps without and with the DD solution constraint, respectively. Moreover, the jumps of the day boundaries are weakened evidently by adding the DD solution constraint. This paper demonstrates that the accuracy and stability of the estimated relative clocks between two low earth orbit (LEO) satellites from SD observations are improved obviously with the DD solution constraint.

  14. [Development of immunoenzyme methods for detecting antibodies to Aujeszky's disease virus gB glycoprotein in swine serum].

    PubMed

    Morenkov, O S

    2000-01-01

    Four ELISA methods have been developed for detecting antibodies to Aujeszky's disease virus (ADV) glycoprotein gB. Indirect ELISA is based on affinity-purified gB (affi-gB-ELISA); three blocking ELISAs: indirect blocking ELISA (lbgB-ELISA), direct blocking ELISA (db-gB-ELISA), and two-site "sandwich" ELISA (sb-gB-ELISA) are based on monoclonal antibodies to conservative immunodominant epitopes of gB. The specificities and sensitivities of ELISAs were compared with each other and with indirect ELISA based on purified ADV virions (vir-ELISA). Affi-gB ELISA, db-gB-ELISA, and sb-gB-ELISA possess 100% sensitivity, ib-gB-ELISA 98% sensitivity, and vir-ELISA 93% sensitivity. Affi-gB ELISA, ib-gB-ELISA, db-gB-ELISA, and sb-gB-ELISA possess 100% specificity and vir-ELISA 92% specificity. The efficiency of detection of ADV-specific antibodies by affi-gB ELISA, db-gB-ELISA, and sb-gB-ELISA was comparable to that of analogous commercial test. Since db-gB-ELISA is easier to perform than affi-gB-ELISA or sb-gB-ELISA, it is concluded to be the most appropriate test for detecting pigs infected with ADV among non-vaccinated animals.

  15. Lagrange-type modeling of continuous dielectric permittivity variation in double-higher-order volume integral equation method

    NASA Astrophysics Data System (ADS)

    Chobanyan, E.; Ilić, M. M.; Notaroš, B. M.

    2015-05-01

    A novel double-higher-order entire-domain volume integral equation (VIE) technique for efficient analysis of electromagnetic structures with continuously inhomogeneous dielectric materials is presented. The technique takes advantage of large curved hexahedral discretization elements—enabled by double-higher-order modeling (higher-order modeling of both the geometry and the current)—in applications involving highly inhomogeneous dielectric bodies. Lagrange-type modeling of an arbitrary continuous variation of the equivalent complex permittivity of the dielectric throughout each VIE geometrical element is implemented, in place of piecewise homogeneous approximate models of the inhomogeneous structures. The technique combines the features of the previous double-higher-order piecewise homogeneous VIE method and continuously inhomogeneous finite element method (FEM). This appears to be the first implementation and demonstration of a VIE method with double-higher-order discretization elements and conformal modeling of inhomogeneous dielectric materials embedded within elements that are also higher (arbitrary) order (with arbitrary material-representation orders within each curved and large VIE element). The new technique is validated and evaluated by comparisons with a continuously inhomogeneous double-higher-order FEM technique, a piecewise homogeneous version of the double-higher-order VIE technique, and a commercial piecewise homogeneous FEM code. The examples include two real-world applications involving continuously inhomogeneous permittivity profiles: scattering from an egg-shaped melting hailstone and near-field analysis of a Luneburg lens, illuminated by a corrugated horn antenna. The results show that the new technique is more efficient and ensures considerable reductions in the number of unknowns and computational time when compared to the three alternative approaches.

  16. Sifalimumab, an anti-interferon-α monoclonal antibody, in moderate to severe systemic lupus erythematosus: a randomised, double-blind, placebo-controlled study

    PubMed Central

    Khamashta, Munther; Merrill, Joan T; Werth, Victoria P; Furie, Richard; Kalunian, Kenneth; Illei, Gabor G; Drappa, Jorn; Wang, Liangwei; Greth, Warren

    2016-01-01

    Objectives The efficacy and safety of sifalimumab were assessed in a phase IIb, randomised, double-blind, placebo-controlled study (NCT01283139) of adults with moderate to severe active systemic lupus erythematosus (SLE). Methods 431 patients were randomised and received monthly intravenous sifalimumab (200 mg, 600 mg or 1200 mg) or placebo in addition to standard-of-care medications. Patients were stratified by disease activity, interferon gene-signature test (high vs low based on the expression of four genes) and geographical region. The primary efficacy end point was the percentage of patients achieving an SLE responder index response at week 52. Results Compared with placebo, a greater percentage of patients who received sifalimumab (all dosages) met the primary end point (placebo: 45.4%; 200 mg: 58.3%; 600 mg: 56.5%; 1200 mg 59.8%). Other improvements were seen in Cutaneous Lupus Erythematosus Disease Area and Severity Index score (200 mg and 1200 mg monthly), Physician's Global Assessment (600 mg and 1200 mg monthly), British Isles Lupus Assessment Group-based Composite Lupus Assessment (1200 mg monthly), 4-point reductions in the SLE Disease Activity Index−2000 score and reductions in counts of swollen joints and tender joints. Serious adverse events occurred in 17.6% of patients on placebo and 18.3% of patients on sifalimumab. Herpes zoster infections were more frequent with sifalimumab treatment. Conclusions Sifalimumab is a promising treatment for adults with SLE. Improvement was consistent across various clinical end points, including global and organ-specific measures of disease activity. Trial registration number NCT01283139; Results. PMID:27009916

  17. Application of the nonlinear, double-dynamic Taguchi method to the precision positioning device using combined piezo-VCM actuator.

    PubMed

    Liu, Yung-Tien; Fung, Rong-Fong; Wang, Chun-Chao

    2007-02-01

    In this research, the nonlinear, double-dynamic Taguchi method was used as design and analysis methods for a high-precision positioning device using the combined piezo-voice-coil motor (VCM) actuator. An experimental investigation into the effects of two input signals and three control factors were carried out to determine the optimum parametric configuration of the positioning device. The double-dynamic Taguchi method, which permits optimization of several control factors concurrently, is particularly suitable for optimizing the performance of a positioning device with multiple actuators. In this study, matrix experiments were conducted with L9(3(4)) orthogonal arrays (OAs). The two most critical processes for the optimization of positioning device are the identification of the nonlinear ideal function and the combination of the double-dynamic signal factors for the ideal function's response. The driving voltage of the VCM and the waveform amplitude of the PZT actuator are combined into a single quality characteristic to evaluate the positioning response. The application of the double-dynamic Taguchi method, with dynamic signal-to-noise ratio (SNR) and L9(3(4)) OAs, reduced the number of necessary experiments. The analysis of variance (ANOVA) was applied to set the optimum parameters based on the high-precision positioning process.

  18. Sparse constrained wavelet-based double-difference seismic tomography method and its applications

    NASA Astrophysics Data System (ADS)

    Fang, H.; Zhang, H.

    2013-12-01

    resolution matrix simultaneously. We have successfully incorporated the methods of wavelet transform, sparse constraint, and general cross validation into the double-difference tomography code tomoDD. We have applied the new code to both synthetic and real data sets. Compared with the original tomoDD code, the new code can resolve the model contrasts more clearly and is also data adaptive.

  19. Imaging of 3-D seismic velocity structure of Southern Sumatra region using double difference tomographic method

    NASA Astrophysics Data System (ADS)

    Lestari, Titik; Nugraha, Andri Dian

    2015-04-01

    Southern Sumatra region has a high level of seismicity due to the influence of the subduction system, Sumatra fault, Mentawai fault and stretching zone activities. The seismic activities of Southern Sumatra region are recorded by Meteorological Climatological and Geophysical Agency (MCGA's) Seismograph network. In this study, we used earthquake data catalog compiled by MCGA for 3013 events from 10 seismic stations around Southern Sumatra region for time periods of April 2009 - April 2014 in order to invert for the 3-D seismic velocities structure (Vp, Vs, and Vp/Vs ratio). We applied double-difference seismic tomography method (tomoDD) to determine Vp, Vs and Vp/Vs ratio with hypocenter adjustment. For the inversion procedure, we started from the initial 1-D seismic velocity model of AK135 and constant Vp/Vs of 1.73. The synthetic travel time from source to receiver was calculated using ray pseudo-bending technique, while the main tomographic inversion was applied using LSQR method. The resolution model was evaluated using checkerboard test and Derivative Weigh Sum (DWS). Our preliminary results show low Vp and Vs anomalies region along Bukit Barisan which is may be associated with weak zone of Sumatran fault and migration of partial melted material. Low velocity anomalies at 30-50 km depth in the fore arc region may indicated the hydrous material circulation because the slab dehydration. We detected low seismic seismicity in the fore arc region that may be indicated as seismic gap. It is coincides contact zone of high and low velocity anomalies. And two large earthquakes (Jambi and Mentawai) also occurred at the contact of contrast velocity.

  20. Imaging of 3-D seismic velocity structure of Southern Sumatra region using double difference tomographic method

    SciTech Connect

    Lestari, Titik; Nugraha, Andri Dian

    2015-04-24

    Southern Sumatra region has a high level of seismicity due to the influence of the subduction system, Sumatra fault, Mentawai fault and stretching zone activities. The seismic activities of Southern Sumatra region are recorded by Meteorological Climatological and Geophysical Agency (MCGA’s) Seismograph network. In this study, we used earthquake data catalog compiled by MCGA for 3013 events from 10 seismic stations around Southern Sumatra region for time periods of April 2009 – April 2014 in order to invert for the 3-D seismic velocities structure (Vp, Vs, and Vp/Vs ratio). We applied double-difference seismic tomography method (tomoDD) to determine Vp, Vs and Vp/Vs ratio with hypocenter adjustment. For the inversion procedure, we started from the initial 1-D seismic velocity model of AK135 and constant Vp/Vs of 1.73. The synthetic travel time from source to receiver was calculated using ray pseudo-bending technique, while the main tomographic inversion was applied using LSQR method. The resolution model was evaluated using checkerboard test and Derivative Weigh Sum (DWS). Our preliminary results show low Vp and Vs anomalies region along Bukit Barisan which is may be associated with weak zone of Sumatran fault and migration of partial melted material. Low velocity anomalies at 30-50 km depth in the fore arc region may indicated the hydrous material circulation because the slab dehydration. We detected low seismic seismicity in the fore arc region that may be indicated as seismic gap. It is coincides contact zone of high and low velocity anomalies. And two large earthquakes (Jambi and Mentawai) also occurred at the contact of contrast velocity.

  1. Immunochemical method for detection of antibody against HTLV-III core protein based upon recombinant HTLV-III gag gene encoded protein

    SciTech Connect

    Chang, N.T.; Ghrayeb, J.

    1989-02-28

    A method is described of detecting antibody against HTLV-III core protein in a biological fluid, comprising the steps of: a. providing an antigen immunoadsorbent comprising a solid phase to which is attached a HTLV-III core antigen which is a chimeric antigen comprising an amino acid sequence beginning at amino acid number 1 through 99, and extending to amino acid number 228, the chimeric antigen being immunoreactive with antibody against HTLV-III core protein; b. incubating the immunoadsorbent with a sample of the biological fluid to be tested under conditions which allow antibody in the sample to complex with the antigen immunoadsorbent; c. separating the immmunoadsorbent from the sample; and d. determining antibody bound to the iuumoadsorbent as an indication of antibody against HTLV-III core protein in the sample.

  2. Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

    PubMed Central

    Lee, Hyung-Kook (Peter); Wright, Ashley P.; Zinn, Kai

    2009-01-01

    Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens. PMID:20040910

  3. Looking for ugly ducklings: The role of the stability of BrdU-antibody complex and the improved method of the detection of DNA replication

    PubMed Central

    Konečný, Petr; Frydrych, Ivo; Koberna, Karel

    2017-01-01

    5-Bromo-2′-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation. Whereas highly efficient antibodies created stable complexes with BrdU, the low efficiency antibodies were unstable. A substantial loss of the signal occurred within several minutes. The increase of the complex stability can be achieved by i) formaldehyde fixation or ii) a quick reaction with a secondary antibody. These steps allowed the same or even higher signal/background ratio to be reached as in the highly efficient antibodies. Based on our findings, we optimised an approach for the fully enzymatic detection of BrdU enabling the fast detection of replicational activity without a significant effect on the tested proteins or the fluorescence of the fluorescent proteins. The method was successfully applied for image and flow cytometry. The speed of the method is comparable to the approach based on 5-ethynyl-2′-deoxyuridine. Moreover, in the case of short labelling pulses, the optimised method is even more sensitive. The approach is also applicable for the detection of 5-trifluoromethyl-2'-deoxyuridine. PMID:28358913

  4. Schrödinger method as N-body double and UV completion of dust

    NASA Astrophysics Data System (ADS)

    Uhlemann, Cora; Kopp, Michael; Haugg, Thomas

    2014-07-01

    We investigate large-scale structure formation of collisionless dark matter in the phase space description based on the Vlasov (or collisionless Boltzmann) equation whose nonlinearity is induced solely by gravitational interaction according to the Poisson equation. Determining the time evolution of density and peculiar velocity demands solving the full Vlasov hierarchy for the moments of the phase space distribution function. In the presence of long-range interaction no consistent truncation of the hierarchy is known apart from the pressureless fluid (dust) model, which is incapable of describing virialization due to the occurrence of shell-crossing singularities and the inability to generate vorticity and higher cumulants like velocity dispersion. Our goal is to find a simple ansatz for the phase space distribution function that approximates the full Vlasov distribution function without pathologies in a controlled way and therefore can serve as theoretical N-body double and as a replacement for the dust model. We argue that the coarse-grained Wigner probability distribution obtained from a wave function fulfilling the Schrödinger-Poisson equation (SPE) is the sought-after function. We show that its evolution equation approximates the Vlasov equation and therefore also the dust fluid equations before shell crossing, but cures the shell-crossing singularities and is able to describe regions of multistreaming and virialization. This feature was already employed in cosmological simulations of large-scale structure formation by Widrow and Kaiser (1993). The coarse-grained Wigner ansatz allows us to calculate all higher moments from density and velocity analytically, thereby incorporating nonzero higher cumulants in a self-consistent manner. On this basis we are able to show that the Schrödinger method automatically closes the corresponding hierarchy such that it suffices to solve the SPE in order to directly determine density and velocity and all higher cumulants.

  5. An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates

    PubMed Central

    Li, Yi; Gu, Christine; Gruenhagen, Jason; Yehl, Peter; Chetwyn, Nik P.; Medley, Colin D.

    2016-01-01

    ABSTRACT Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs. PMID:26891281

  6. MTRX1011A, a humanized anti-CD4 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: a Phase I randomized, double-blind, placebo-controlled study incorporating pharmacodynamic biomarker assessments

    PubMed Central

    2011-01-01

    Introduction The purpose of this study was to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of the humanized anti-CD4 monoclonal antibody MTRX1011A in a randomized, double-blind placebo-controlled Phase 1 study in patients with rheumatoid arthritis (RA). Methods In the single ascending dose (SAD) portion of the study, patients received single doses of a placebo or MTRX1011A at 0.3, 1.0, 3.5 and 7.0 mg/kg intravenously (IV) or 1.0 and 3.5 mg/kg subcutaneously (SC), followed by five weeks of evaluation. In the multi-dose (MD) portion of the study, placebo or MTRX1011A was administered weekly for eight doses at 1.5 or 3.5 mg/kg SC, or 5 mg/kg IV, followed by eight weeks of evaluation. Results MTRX1011A was well tolerated in the SAD phase up to 7 mg/kg IV and in the MD phase up to 1.5 mg/kg SC. At weekly doses of 3.5 mg/kg SC and 5 mg/kg IV, a moderate pruritic papular rash was observed in some MTRX1011A-treated patients, which was considered a dose-limiting toxicity for this clinical indication. No serious adverse events occurred in any cohort. Reduction in disease activity was modest. PD assessments demonstrated that MTRX1011A induced a dose-dependent down-modulation of CD4 expression on peripheral blood CD4 T cells, CD4 receptor occupancy, increases in serum sCD4-MTRX1011A complexes and up-regulation of CD69 on T cells, but was non-depleting. Conclusions The maximum tolerated dose of MTRX1011A was 1.5 mg/kg SC administered weekly. At this dose MTRX1011A did not achieve maximum PD activity expected to be required for reduction in disease activity. PMID:22029963

  7. Single vs. double layer suturing method repair of the urethral plate in the rabbit model of hypospadias

    PubMed Central

    Shirazi, Mehdi; Rahimi, Mohammad

    2016-01-01

    Introduction There are different methods of urethroplasty in hypospadias. The present study aimed to compare the repair of the urethral plate by single vs. double layer suturing. Material and methods Fifteen male rabbits were assigned to the control, single layer, and double layer urethral plate suturing groups (n = 5). Experimental hypospadias was induced in the second and third groups and the urethral plates were sutured. After two weeks, the penis was dissected out and underwent histopathological processing. Stereological studies were applied to obtain quantitative histological data regarding the structure of the urethra and the related part of the corpus spongiosum. Results Volume density of the urethral epithelium (the fraction of unit volume of the urethra occupied by its epithelium) was higher in the single layer suturing group when compared to the double layer or control groups (p <0.01). Additionally, the volume density of the urethral lumen (the fraction of the corpus spongiosum that is occupied by the urethral lumen) in the single versus the double layer suturing groups was respectively 2.4 and 2 folds higher than that in the control group (p <0.01). Besides, the volume density of the lumen was significantly higher in the single layer suturing when compared to the double layer suturing group (p <0.01). However, no significant difference was observed among the study groups regarding the volume density of the collagen and vessels in the incised site of the penis which implied that the fraction of the urethra and surrounding corpus spongiosum was occupied by collagen and vessels. Conclusions Urethral plate repair by the single layer suturing method could be accompanied by higher epithelialization and wider lumen in the rabbit model of hypospadias. PMID:28127462

  8. Chicken egg antibodies for immunohistochemical labeling of growth hormone and prolactin in bovine pituitary gland.

    PubMed

    Schmidt, P; Erhard, M H; Schams, D; Hafner, A; Folger, S; Lösch, U

    1993-09-01

    We describe the production of polyclonal chicken antibodies specific for bovine growth hormone (bGH) and prolactin (PRL). Antibodies were generated by immunization of laying hens with recombinant bGH (rbGH), pituitary derived bGH (pbGH), and ovine PRL (oPRL). After the lipoprotein fraction was removed by dextran sulfate precipitation the antibodies were isolated from the egg yolks by ammonium sulfate precipitation. Immunization with rbGH and oPRL generated large amounts of specific antibodies, as revealed by ELISA and Western blot analysis. Antibodies against pbGH showed pronounced crossreactions with oPRL. The antibodies against rbGH and oPRL were well suited for sensitive and specific labeling of the GH- and PRL-synthesizing cells in bovine pituitary glands by immunohistochemistry. In addition, a quick and sensitive procedure for demonstration of both bGH- and PRL-synthesizing cells in a single paraffin section by double immunohistochemistry is presented. The chicken anti-bGH antibodies showed excellent results in combination with rabbit anti-PRL antibodies. The main advantage of avian antibodies in double immunostaining methods is the lack of crossreactions between avian antibodies and mammalian immunoglobulins and receptors which bind to the crystalline fragment of mammalian immunoglobulins (Fc receptors).

  9. Antimitochondrial antibody

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  10. An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

    PubMed

    Maheshwari, S C; Sharma, S B; Kumar, A; Sokhey, J

    1998-09-01

    Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

  11. Small-scale screening method for low-viscosity antibody solutions using small-angle X-ray scattering.

    PubMed

    Fukuda, Masakazu; Watanabe, Atsushi; Hayasaka, Akira; Muraoka, Masaru; Hori, Yuji; Yamazaki, Tadao; Imaeda, Yoshimi; Koga, Akiko

    2017-03-01

    In this study, we investigated the concentration range in which self-association starts to form in humanized IgG monoclonal antibody (mAb) solutions. Furthermore, on the basis of the results, we developed a practical method of screening for low-viscosity antibody solutions by using small-angle X-ray scattering (SAXS) measurements utilizing small quantities of samples. With lower-viscosity mAb3, self-association was not detected in the range of 1-80mg/mL. With higher-viscosity mAb1, on the other hand, self-association was detected in the range of 10-20mg/mL and was clearly enhanced by a decrease in temperature. The viscosities of mAb solutions at 160, 180, and 200mg/mL at 25°C quantitatively correlated very well with the particle size parameters obtained by SAXS measurements of mAb solutions at 15mg/mL at 5°C. The quantity of mAb sample required for the SAXS measurements was only 0.15mg, which is about one-hundredth of that required for actual viscosity measurements at a high concentration, and such quantities could be available even at an early stage of development. In conclusion, the SAXS analysis method proposed in this study is a valuable tool for the development of concentrated mAb therapeutics with high manufacturability and high usability for subcutaneous injection.

  12. Development of a Double Glass Mounting Method Using Formaldehyde Alcohol Azocarmine Lactophenol (FAAL) and its Evaluation for Permanent Mounting of Small Nematodes

    PubMed Central

    ZAHABIUN, Farzaneh; SADJJADI, Seyed Mahmoud; ESFANDIARI, Farideh

    2015-01-01

    Background: Permanent slide preparation of nematodes especially small ones is time consuming, difficult and they become scarious margins. Regarding this problem, a modified double glass mounting method was developed and compared with classic method. Methods: A total of 209 nematode samples from human and animal origin were fixed and stained with Formaldehyde Alcohol Azocarmine Lactophenol (FAAL) followed by double glass mounting and classic dehydration method using Canada balsam as their mounting media. The slides were evaluated in different dates and times, more than four years. Different photos were made with different magnification during the evaluation time. Results: The double glass mounting method was stable during this time and comparable with classic method. There were no changes in morphologic structures of nematodes using double glass mounting method with well-defined and clear differentiation between different organs of nematodes in this method. Conclusion: Using this method is cost effective and fast for mounting of small nematodes comparing to classic method. PMID:26811729

  13. Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer: Application to the determination of adiponectin cytokine in serum.

    PubMed

    Ojeda, Irene; Barrejón, Myriam; Arellano, Luis M; González-Cortés, Araceli; Yáñez-Sedeño, Paloma; Langa, Fernando; Pingarrón, José M

    2015-12-15

    An electrochemical immunosensor for adiponectin (APN) using screen printed carbon electrodes (SPCEs) modified with functionalized double-walled carbon nanotubes (DWCNTs) as platforms for immobilization of the specific antibodies is reported. DWCNTs were functionalized by treatment with 4-aminobenzoic acid (HOOC-Phe) in the presence of isoamylnitrite resulting in the formation of 4-carboxyphenyl-DWCNTs. The oriented binding of specific antibodies toward adiponectin was accomplished by using the metallic-complex chelating polymer Mix&Go™. The HOOC-Phe-DWCNTs-modified SPCEs were characterized by cyclic voltammetry and compared with HOOC-Phe-SWCNTs/SPCE. The different variables affecting the performance of the developed immunosensor were optimized. Under the selected conditions, a calibration plot for APN was constructed showing a range of linearity extending between 0.05 and 10.0 μg/mL which is adequate for the determination of the cytokine in real samples. A detection limit of 14.5 ng/mL was achieved. The so prepared immunosensor exhibited a good reproducibility for the APN measurements, excellent storage stability and selectivity, and a much shorter assay time than the available ELISA kits. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing human serum from female or male healthy patients.

  14. Two Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single-Ascending-Dose Studies To Investigate the Safety, Tolerability, and Pharmacokinetics of an Anti-Influenza A Virus Monoclonal Antibody, MHAA4549A, in Healthy Volunteers

    PubMed Central

    Deng, Rong; Derby, Michael A.; Larouche, Richard; Horn, Priscilla; Anderson, Malia; Maia, Mauricio; Carrier, Stephanie; Pelletier, Isabelle; Burgess, Tracy; Kulkarni, Priya; Newton, Elizabeth; Tavel, Jorge A.

    2016-01-01

    Hospitalized patients with severe influenza are at significant risk for morbidity and mortality. MHAA4549A is a human monoclonal immunoglobulin (Ig) G1 antibody that binds to a highly conserved stalk region of the influenza A virus hemagglutinin protein and neutralizes all tested seasonal human influenza A virus strains. Two phase 1 trials examined the safety, tolerability, and pharmacokinetics of MHAA4549A in healthy volunteers. Both single ascending-dose trials were randomized, double blinded, and placebo controlled. Trial 1 randomized 21 healthy adults into four cohorts receiving a single intravenous dose of 1.5, 5, 15, or 45 mg/kg MHAA4549A or placebo. Trial 2 randomized 14 healthy adults into two cohorts receiving a single intravenous fixed dose of 8,400 mg or 10,800 mg of MHAA4549A or placebo. Subjects were followed for 120 days after dosing. No subject was discontinued in either trial, and no serious adverse events were reported. The most common adverse event in both studies was mild headache (trial 1, 4/16 subjects receiving MHAA4549A and 1/5 receiving placebo; trial 2, 4/8 subjects receiving MHAA4549A and 2/6 receiving placebo). MHAA4549A produced no relevant time- or dose-related changes in laboratory values or vital signs compared to those with placebo. No subjects developed an antitherapeutic antibody response following MHAA4549A administration. MHAA4549A showed linear serum pharmacokinetics, with a mean half-life of 22.5 to 23.7 days. MHAA4549A is safe and well tolerated in healthy volunteers up to a single intravenous dose of 10,800 mg and demonstrates linear serum pharmacokinetics consistent with those of a human IgG1 antibody lacking known endogenous targets in humans. (These trials have been registered at ClinicalTrials.gov under registration no. NCT01877785 and NCT02284607). PMID:27381392

  15. Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies.

    PubMed

    Smith, Michael T; Zhang, Shu; Adams, Troy; DiPaolo, Byron; Dally, Jennifer

    2017-02-17

    Capillary and microfluidic chip electrophoresis technologies are heavily utilized for development, characterization, release, and stability testing of biopharmaceuticals. Within the biopharmaceutical industry, CE-SDS and M-CGE are commonly used for purity determination by separation and quantitation of size-based variants. M-CGE is used primarily as a R&D tool for product and process development, while cGMP release and stability testing applications are commonly reserved for CE-SDS. This paper describes the establishment of a M-CGE platform method to be used for R&D and cGMP applications, including release and stability testing, for monoclonal antibodies. The M-CGE platform method enables testing for product development support and cGMP release and stability using the same method, and utilization of one CE technology for the entire lifecycle of a biopharmaceutical product. Critical method parameters were identified, and the analytical design space of those critical parameters was defined using design of experiments (DOE) studies. Once defined through DOE studies, the method design space was validated according to ICH Q2 (R1) guidelines. Additional molecules of the same validated class were verified for use in the method by experimental confirmation of accuracy, specificity, and stability indicating capabilities. The platform method model facilitates rapid utilization of the method in development and GMP testing environments, and eliminates the need for individual validations for assets of the same class entering early stage development. This article is protected by copyright. All rights reserved.

  16. CD138 and CD31 Double-Positive Cells Comprise the Functional Antibody-Secreting Plasma Cell Compartment in Primate Bone Marrow

    PubMed Central

    Martinez-Murillo, Paola; Pramanik, Lotta; Sundling, Christopher; Hultenby, Kjell; Wretenberg, Per; Spångberg, Mats; Karlsson Hedestam, Gunilla B.

    2016-01-01

    Plasma cells (PCs) are defined as terminally differentiated B cells that secrete large amounts of immunoglobulin (Ig). PCs that reside in the bone marrow (BM) are responsible for maintaining long-term antibody (Ab) responses after infection and vaccination, while PCs present in the blood are generally short-lived. In rhesus macaques, a species frequently used for the evaluation of human vaccines, B cells resemble those found in humans. However, a detailed characterization of BM-resident rhesus PC phenotype and function is lacking. Here, we examined Ig secretion of distinct rhesus CD138+ populations by B cell ELISpot analysis to couple phenotype with function. We demonstrate that the CD20low/−CD138+CD31+ BM population was highly enriched for antibody-secreting cells with IgG being the predominant isotype (60%), followed by IgA (33%) and IgM (7%). Transmission electron microscopy analysis confirmed PC enrichment in the CD20low/−CD138+CD31+ population with cells containing nuclei with “spokes of a wheel” chromatin structure and prominent rough endoplasmic reticulum. This panel also stained human BM PCs and allowed a clear distinction between BM PCs and short-lived peripheral PCs, providing an improved strategy to isolate PCs from rhesus BM for further analysis. PMID:27446073

  17. Efficacy and Toxicity Assessment of Different Antibody Based Antiangiogenic Drugs by Computational Docking Method

    PubMed Central

    Mukherjee, Sayan; Chatterjee, Gopa; Ghosh, Moumita; Das, Bishwajit

    2016-01-01

    Bevacizumab and trastuzumab are two antibody based antiangiogenic drugs that are in clinical practice for the treatment of different cancers. Presently applications of these drugs are based on the empirical choice of clinical experts that follow towards population based clinical trials and, hence, their molecular efficacies in terms of quantitative estimates are not being explored. Moreover, different clinical trials with these drugs showed different toxicity symptoms in patients. Here, using molecular docking study, we made an attempt to reveal the molecular rationale regarding their efficacy and off-target toxicity. Though our study reinforces their antiangiogenic potentiality and, among the two, trastuzumab has much higher efficacy; however, this study also reveals that compared to bevacizumab, trastuzumab has higher toxicity effect, specially on the cardiovascular system. This study also reveals the molecular rationale of ocular dysfunction by antiangiogenic drugs. The molecular rationale of toxicity as revealed in this study may help in the judicious choice as well as therapeutic scheduling of these drugs in different cancers. PMID:27047544

  18. Structural models of antibody variable fragments: A method for investigating binding mechanisms

    NASA Astrophysics Data System (ADS)

    Petit, Samuel; Brard, Frédéric; Coquerel, Gérard; Perez, Guy; Tron, François

    1998-03-01

    The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that anti-nucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nculeosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

  19. A proficiency testing method for detecting antibodies against Brucella abortus in quantitative and qualitative serological tests.

    PubMed

    Gall, D; Nielsen, K; Nicola, A; Renteria, T

    2008-12-01

    A proficiency testing panel for detecting antibodies against Brucella abortus was developed and evaluated by both primary binding and conventional serological tests, using the guidelines of the World Organisation for Animal Health and the International Organization for Standardization Guide 43-1. All serological tests were judged satisfactory. Among the primary binding tests, the competitive enzyme-linked immunosorbent assay (ELISA 2) and the indirect enzyme-linked immunosorbent assay (ELISA 1), with standard deviation indices (z-scores) of -0.06 and 0.10, respectively, performed best. Similarly, E(n) numbers (i.e. a way of comparing different measurements of performance) of 0 for both the competitive ELISA 2 and the indirect ELISA 1 indicated that these tests performed best in the initial round of proficiency testing. The conventional serological tests all passed the panel. Comparing data from both the quantitative and qualitative tests demonstrated that this proficiency testing scheme was fit for the purpose for which it was designed.

  20. [Construction of a recombinant baculovirus transfer vector with two promoters expressing the anti-human CD28 chimeric antibody by using TP-PCR method].

    PubMed

    Zhu, Yan; Chen, Yong-Jing; Qiu, Yu-Hua; Zheng, Feng-Feng; Zhu, Jiang

    2005-09-01

    CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.

  1. Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers

    PubMed Central

    Gorris, María Teresa; Alarcon, Benito; Lopez, María M.; Cambra, Mariano

    1994-01-01

    Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants. PMID:16349293

  2. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  3. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  4. A Quick and Parallel Analytical Method Based on Quantum Dots Labeling for ToRCH-Related Antibodies

    NASA Astrophysics Data System (ADS)

    Yang, Hao; Guo, Qing; He, Rong; Li, Ding; Zhang, Xueqing; Bao, Chenchen; Hu, Hengyao; Cui, Daxiang

    2009-12-01

    Quantum dot is a special kind of nanomaterial composed of periodic groups of II-VI, III-V or IV-VI materials. Their high quantum yield, broad absorption with narrow photoluminescence spectra and high resistance to photobleaching, make them become a promising labeling substance in biological analysis. Here, we report a quick and parallel analytical method based on quantum dots for ToRCH-related antibodies including Toxoplasma gondii, Rubella virus, Cytomegalovirus and Herpes simplex virus type 1 (HSV1) and 2 (HSV2). Firstly, we fabricated the microarrays with the five kinds of ToRCH-related antigens and used CdTe quantum dots to label secondary antibody and then analyzed 100 specimens of randomly selected clinical sera from obstetric outpatients. The currently prevalent enzyme-linked immunosorbent assay (ELISA) kits were considered as “golden standard” for comparison. The results show that the quantum dots labeling-based ToRCH microarrays have comparable sensitivity and specificity with ELISA. Besides, the microarrays hold distinct advantages over ELISA test format in detection time, cost, operation and signal stability. Validated by the clinical assay, our quantum dots-based ToRCH microarrays have great potential in the detection of ToRCH-related pathogens.

  5. High-field double-pancake superconducting coils and a method of winding

    DOEpatents

    Materna, Peter A.

    1985-01-01

    A double-pancake coil having first and second pancakes may comprise a plurality of conductor means, each conductor means having a different grade and having one or more conductors, wherein each pancake of said double-pancake coil is comprised of inner and outer turns; wherein said inner turns are comprised of at least one of said conductor means wound about an axis and nested within one another; wherein said outer turns are comprised of said inner conductor means and at least one other conductor means co-wound about said inner turns and nested within one another; wherein each of said conductor means is wound along said axis from said first pancake to said second pancake at a different turn.

  6. High-field double-pancake superconducting coils and a method of winding

    DOEpatents

    Materna, P.A.

    1984-01-31

    A double-pancake coil having first and second pancakes may comprise a plurality of conductor means, each conductor means having a different grade and having one or more conductors, wherein each pancake of said double-pancake coil is comprised of inner and outer turns; wherein said inner turns are comprised of at least one of said conductor means wound about an axis and nested within one another; wherein said outer turns are comprised of said inner conductor means and at least one other conductor means co-wound about said inner turns and nested within one another; wherein each of said conductor means is wound along said axis from said first pancake to said second pancake at a different turn.

  7. Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

    PubMed Central

    Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

    2002-01-01

    We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

  8. Comparing two methods of plastination and glycerin preservation to study skeletal system after Alizarin red-Alcian blue double staining

    PubMed Central

    Mohsen, Setayesh M.; Esfandiari, Ebrahim; Rabiei, Abbas A.; Hanaei, Mahsa S.; Rashidi, Bahman

    2013-01-01

    Background: Plastination is a new method of preserving tissue samples for a long time. This study aimed to compare the new plastination technique with the conventional preservative method in glycerin for fetus skeleton tissues and young rats dyed by Alizarin red- Alcian blue double staining. Materials and Methods: In this study, 4 groups of 1-day, 3-day, 12-day and mature rats were selected and, after being anesthetized and slaughtered, their skin was completely removed. In Alizarin red- Alcian blue double staining method, first the samples were fixed in 95% ethanol and then their cartilages were dyed by 0.225% Alcian blue solution; after that, they were cleared in 1% KOH. Then, the bones were dyed in 0.003% Alizarin red solution and finally the tissue was decolorized in 95% ethanol. In each group, half of the samples were preserved by the conventional method in a glycerin container and the other half were plastinated. Results: In the present study, the samples preserved by plastination technique were dry, odorless, indecomposable and tangible. Quality of coloring had an inverse relationship with rats’ age. Transparency of the plastinated samples had also an inverse relationship with rats’ age. Therefore, skeletal tissue of younger rats had higher quality and transparency in both preservation methods (glycerin and plastination). Conclusion: This study showed that plastination technique was an appropriate method in comparison with glycerin preservation, which conserved skeletal tissue of fetus and young rats colored by Alizarin red- Alcian blue double staining. And the final result was that plastination technique can generate dry, odorless, indecomposable and tangible samples. PMID:23930264

  9. High anti–desmoglein 3 antibody ELISA index and negative indirect immunofluorescence result in a patient with pemphigus vulgaris in remission: evaluation of the antibody profile by newly developed methods.

    PubMed

    Nakahara, Tomoko; Takagi, Atsushi; Yamagami, Jun; Kamiya, Koji; Aoyama, Yumi; Iwatsuki, Keiji; Ikeda, Shigaku

    2014-12-01

    IMPORTANCE Pemphigus vulgaris (PV) is a disease that features blistering of the skin and mucous membranes caused by autoantibodies directed against desmoglein 3 (Dsg3) and/or desmoglein 1(Dsg1). Indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) are 2 methods that are widely used to measure Dsg3 orDsg1 antibody titers in PV. Although the titers of these autoantibodies are generally correlated with disease activity, some patients with a high ELISA index do not have severe symptoms.We encountered a patient with PV in remission,who had a high anti-Dsg3 antibody ELISA index while the IIF result was negative.OBSERVATIONS The anti-Dsg3 antibodies of our patient mainly recognized Ca2+-dependent conformational epitopes and targeted mature Dsg3 protein.We report this case focusing on the discrepancy between ELISA and IIF findings, as well as on the specific characteristics of the patient's autoantibodies evaluated by newly developed methods.CONCLUSIONS AND RELEVANCE This case emphasizes that a discrepancy between disease activity, the ELISA index for Dsg3, and/or IIF findings can occur in PV. Further research on similar patients will be required to elucidate the pathogenic mechanisms in patients with PV who have nonpathogenic antibodies and show a discrepancy between ELISA and IIF.

  10. Double-peak elution profile of a monoclonal antibody in cation exchange chromatography is caused by histidine-protonation-based charge variants.

    PubMed

    Luo, Haibin; Cao, Mingyan; Newell, Kelcy; Afdahl, Christopher; Wang, Jihong; Wang, William K; Li, Yuling

    2015-12-11

    We have systemically investigated unusual elution behaviors of an IgG4 (mAb A) in cation exchange chromatography (CEX). This mAb A exhibited two elution peaks under certain conditions when being purified by several strong CEX columns. When either of the two peaks was isolated and re-injected on the same column, the similar pattern was observed again during elution. The protein distribution between the two peaks could be altered by NaCl concentration in the feed, or NaCl concentration in wash buffer, or elution pH, suggesting two pH-associated strong-and-weak binding configurations. The protein distributions under different pH values showed good correlation with protonated/un-protonated fractions of a histidine residue. These results suggest that the double-peak elution profile associates with histidine-protonation-based charge variants. By conducting pepsin digestion, amino-acid specific chemical modifications, peptide mapping, and measuring the effects of elution residence time, a histidine in the variable fragment (Fab) was identified to be the root cause. Besides double-peak pattern, mAb A can also exhibit peak-shouldering or single elution peak on different CEX resins, reflecting different resins' resolving capability on protonated/un-protonated forms. This work characterizes a novel cause for unusual elution behaviors in CEX and also provides alternative avenues of purification development for mAbs with similar behaviors.

  11. Grid-based methods for diatomic quantum scattering problems III: Double photoionization of molecular hydrogen in prolate spheroidal coordinates

    SciTech Connect

    Tao, Liang; McCurdy, Bill; Rescigno, Tom

    2010-06-10

    Our previously developed finite-element/ discrete variable representation in prolate spheroidal coordinates is extended to two-electron systems with a study of double ionization of H$_2$ with fixed-nuclei. Particular attention is paid to the development of fast and accurate methods for treating the electron-electron interaction. The use of exterior complex scaling in the implementation offers a simple way of enforcing Coulomb boundary conditions for the electronic double continuum. While the angular distributions calculated in this study are found to be completely consistent with our earlier treatments that employed single-center expansions in spherical coordinates, we find that the magnitude of the integrated cross sections are sensitive to small changes in the initial-state wave function. The present formulation offers significant advantages with respect to convergence and efficiency and opens the way to calculations on more complicated diatomic targets.

  12. Macrocyclic polyaminocarboxylates for stable radiometal antibody conjugates for therapy, SPECT and PET imaging

    DOEpatents

    Mease, R.C.; Mausner, L.F.; Srivastava, S.C.

    1997-06-17

    A simple method for the synthesis of 1,4,7, 10-tetraazacyclododecane N,N{prime}N{double_prime},N{prime}{double_prime}-tetraacetic acid and 1,4,8,11-tetraazacyclotetradecane N,N{prime},N{double_prime},N{prime}{double_prime}-tetraacetic acid involves cyanomethylating 1,4,7,10-tetraazacyclododecane or 1,4,8,11-tetraazacyclotetradecane to form a tetranitrile and hydrolyzing the tetranitrile. These macrocyclic compounds are functionalized through one of the carboxylates and then conjugated to various biological molecules including monoclonal antibodies. The resulting conjugated molecules are labeled with radiometals for SPECT and PET imaging and for radiotherapy. 4 figs.

  13. Quantitative estimation of diphtheria and tetanus toxoids. 6. Use of different antibody titration methods for evaluation of immunogenicity in animals during potency assay of diphtheria toxoid.

    PubMed

    Lyng, J; Heron, I

    1992-06-01

    Two diphtheria toxoid preparations were compared in potency assays in guinea-pigs using different methods for evaluation of the responses to vaccination. The methods used were the direct skin challenge (Schick test) and ELISA and VERO cell titration of antibodies. The different evaluation methods resulted in the same relative potencies between the toxoids. It was observed that when first-vaccination sera were compared with a second-vaccination serum, the relative antibody concentration depended on whether ELISA or VERO cell titration was used.

  14. Semi-monolithic cavity for external resonant frequency doubling and method of performing the same

    NASA Technical Reports Server (NTRS)

    Hemmati, Hamid (Inventor)

    1999-01-01

    The fabrication of an optical cavity for use in a laser, in a frequency doubling external cavity, or any other type of nonlinear optical device, can be simplified by providing the nonlinear crystal in combination with a surrounding glass having an index of refraction substantially equal to that of the nonlinear crystal. The closed optical path in this cavity is formed in the surrounding glass and through the nonlinear crystal which lies in one of the optical segments of the light path. The light is transmitted through interfaces between the surrounding glass in the nonlinear crystal through interfaces which are formed at the Brewster-angle to minimize or eliminate reflection.

  15. Linearized electro-optic racetrack modulator based on double injection method in silicon.

    PubMed

    Cohen, Roei Aviram; Amrani, Ofer; Ruschin, Shlomo

    2015-02-09

    Racetrack-based modulator of increased linearity for optical links is presented and analyzed. The modulator is referred to as FLAME - Finer Linearity Amplitude Modulation Element. Linearity is improved via the introduction of a Double Injection approach. Large spurious-free-dynamic-range (SFDR) of 132dB·Hz(4/5) can thus be theoretically obtained. The FLAME is studied for silicon platform and requires small footprint size (100 × 50µm2) and low operation voltage, 2.5V. This makes the FLAME an appealing candidate for large scale integration in RF photonics.

  16. A noniterative asymmetric triple excitation correction for the density-fitted coupled-cluster singles and doubles method: Preliminary applications

    NASA Astrophysics Data System (ADS)

    Bozkaya, Uǧur

    2016-04-01

    An efficient implementation of the asymmetric triples correction for the coupled-cluster singles and doubles [ΛCCSD(T)] method [S. A. Kucharski and R. J. Bartlett, J. Chem. Phys. 108, 5243 (1998); T. D. Crawford and J. F. Stanton, Int. J. Quantum Chem. 70, 601 (1998)] with the density-fitting [DF-ΛCCSD(T)] approach is presented. The computational time for the DF-ΛCCSD(T) method is compared with that of ΛCCSD(T). Our results demonstrate that the DF-ΛCCSD(T) method provide substantially lower computational costs than ΛCCSD(T). Further application results show that the ΛCCSD(T) and DF-ΛCCSD(T) methods are very beneficial for the study of single bond breaking problems as well as noncovalent interactions and transition states. We conclude that ΛCCSD(T) and DF-ΛCCSD(T) are very promising for the study of challenging chemical systems, where the coupled-cluster singles and doubles with perturbative triples method fails.

  17. A Novel Method for Calculation of Strain Energy Release Rate of Asymmetric Double Cantilever Laminated Composite Beams

    NASA Astrophysics Data System (ADS)

    Shokrieh, M. M.; Zeinedini, A.

    2014-06-01

    In this research, a novel data reduction method for calculation of the strain energy release rate ( SERR) of asymmetric double cantilever beams ( ADCB) is presented. For this purpose the elastic beam theory ( EBT) is modified and the new method is called as the modified elastic beam theory ( MEBT). Also, the ADCB specimens are modeled using ABAQUS/Standard software. Then, the initiation of delamination of ADCB specimens is modeled using the virtual crack closure technique ( VCCT). Furthermore, magnitudes of the SERR for different samples are also calculated by an available data reduction method, called modified beam theory ( MBT). Using the hand lay-up method, different laminated composite samples are manufactured by E-glass/epoxy unidirectional plies. In order to measure the SERR, all samples are tested using an experimental setup. The results determined by the new data reduction method ( MEBT) show good agreements with the results of the VCCT and the MBT.

  18. iSeq: A New Double-Barcode Method for Detecting Dynamic Genetic Interactions in Yeast

    PubMed Central

    Jaffe, Mia; Sherlock, Gavin; Levy, Sasha F.

    2016-01-01

    Systematic screens for genetic interactions are a cornerstone of both network and systems biology. However, most screens have been limited to characterizing interaction networks in a single environment. Moving beyond this static view of the cell requires a major technological advance to increase the throughput and ease of replication in these assays. Here, we introduce iSeq—a platform to build large double barcode libraries and rapidly assay genetic interactions across environments. We use iSeq in yeast to measure fitness in three conditions of nearly 400 clonal strains, representing 45 possible single or double gene deletions, including multiple replicate strains per genotype. We show that iSeq fitness and interaction scores are highly reproducible for the same clonal strain across replicate cultures. However, consistent with previous work, we find that replicates with the same putative genotype have highly variable genetic interaction scores. By whole-genome sequencing 102 of our strains, we find that segregating variation and de novo mutations, including aneuploidy, occur frequently during strain construction, and can have large effects on genetic interaction scores. Additionally, we uncover several new environment-dependent genetic interactions, suggesting that barcode-based genetic interaction assays have the potential to significantly expand our knowledge of genetic interaction networks. PMID:27821633

  19. Facile method of quantification for oxidized tryptophan degradants of monoclonal antibody by mixed mode ultra performance liquid chromatography.

    PubMed

    Wong, Cintyu; Strachan-Mills, Camille; Burman, Sudhir

    2012-12-28

    Oxidation in therapeutic monoclonal antibody is a common occurrence and it may affect potency. Thus controlling and monitoring the amount of oxidized variant in the drug product sample is important since it may impact the purity. Here, we present the development of a fast and easy method utilizing size exclusion - ultra performance liquid chromatography (SE-UPLC) run under moderate hydrophobic conditions (mixed mode) to monitor the heterogeneity in drug product samples. The best separation was obtained using Waters Acquity BEH200 size exclusion column along with a mobile phase consisting of sodium acetate and sodium sulfate that separates IgG into aggregate, monomer, and fragment. The moderate salt concentration resulted in a second mode of separation based on hydrophobicity, resolving a monomer pre-peak from the monomer main peak. Multi-angle light scattering (MALS) determined the pre-peak has a similar mass as the IgG monomer. Characterization of the purified pre-peak fraction using mass spectrometry (MS), and bioactivity revealed this degradant to be a Trp-oxidized IgG monomer with significantly reduced bioactivity. Method qualification of the mixed mode UPLC method showed good recovery for the spiked monomer pre-peak and Fab fragment. However, the recovery of spiked dimer was low. This method is suitable for determining the relative distribution of the oxidized monomer and the native monomer species.

  20. Analytical QbD: development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates.

    PubMed

    Pathak, Mili; Dutta, Debayon; Rathore, Anurag

    2014-08-01

    This paper presents a quality by design (QbD) based development of a novel native PAGE (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N-PAGE relevant to those academicians and other small companies involved in early-stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD-based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.

  1. Organic double layer element driven by triboelectric nanogenerator: Study of carrier behavior by non-contact optical method

    NASA Astrophysics Data System (ADS)

    Chen, Xiangyu; Taguchi, Dai; Manaka, Takaaki; Iwamoto, Mitsumasa

    2016-02-01

    By using optical electric-field-induced second-harmonic generation (EFISHG) technique, we studied carrier behavior caused by contact electrification (CE) in an organic double-layer element. This double-layer sample was half suspended in the open air, where one electrode (anode or cathode) was connected with a Cu foil for electrification while the other electrode was floated. Results showed two distinct carrier behaviors, depending on the (anode or cathode) connections to the Cu foil, and these carrier behaviors were analyzed based on the Maxwell-Wagner model. The double-layer sample works as a simple solar cell device. The photovoltaic effect and CE process have been proved to be two paralleled effects without strong interaction with each other, while photoconductivity changing in the sample can enhance the relaxation of CE induced charges. By probing the carrier behavior in this half-suspended device, the EFISHG technique has been demonstrated to be an effective non-contact method for clarifying the CE effect on related energy harvesting devices and electronics devices. Meanwhile, the related physical analysis in this letter is also useful for elucidating the fundamental characteristic of hybrid energy system based on solar cell and triboelectric nanogenerator.

  2. 'That's OK. He's a guy': a mixed-methods study of gender double-standards for alcohol use.

    PubMed

    de Visser, Richard O; McDonnell, Elizabeth J

    2012-01-01

    Although drinking and drunkenness have traditionally been considered masculine behaviours, young women's alcohol consumption has increased in recent years. This mixed methods study was conducted to examine the extent to which young people endorse gender double-standards for alcohol use--i.e., less acceptance of drinking and drunkenness in women than men--and how these influence men's and women's alcohol consumption. A sample of 731 English university students completed an online survey of gender role attitudes, beliefs about the gendered nature of alcohol use and recent alcohol consumption. Sixteen participants were then purposively selected for individual interviews: eight women and men with the most egalitarian gender role beliefs, and eight women and men with the least egalitarian beliefs. The two sets of data revealed that although there were few sex differences in actual levels of drinking or drunkenness, gender double-standards for alcohol use persist: beer drinking, binge drinking and public drunkenness tended to be perceived as masculine, and even the most egalitarian respondents were more judgemental of women's drinking. Participants modified their drinking style so as to maintain a desired gender identity. Although gender double-standards could be a focus of interventions to encourage moderate drinking, such approaches could reinforce gender inequalities.

  3. Development of a streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Reagent development.

    PubMed

    Karacay, H; Sharkey, R M; Govindan, S V; McBride, W J; Goldenberg, D M; Hansen, H J; Griffiths, G L

    1997-01-01

    With pretargeting, radioisotope delivery to tumor is decoupled from the long antibody localization process, and this can increase tumor:blood ratios dramatically. Several reagents were prepared for each step of a "two-step" pretargeting method, and their properties were investigated. For pretargeting tumor, streptavidin-monoclonal antibody (StAv-mab) conjugates were prepared by cross-linking sulfo-SMCC-derivatized streptavidin to a free thiol (SH) group on MN-14 [a high-affinity anti-carcinoembryonic antigen (CEA) mab]. Thiolated mabs were generated either by reaction of 2-iminothiolane (2-IT) with mab lysine residues or by reduction of mab disulfide bonds with (2-mercaptoethyl)amine (MEA). Both procedures gave protein-protein conjugates isolated in relatively low yields (20-25%) after preparative size-exclusion (SE) chromatography purification with conservative peak collection. Both StAv-MN-14 conjugates retained their ability to bind to CEA, to an anti-idiotypic antibody to MN-14 (WI2), and to biotin, as demonstrated by SE-HPLC. Two clearing agents, WI2 mab and a biotin-human serum albumin (biotin-HSA) conjugate, were developed to remove excess circulating StAv-MN-14 conjugates in animals. Both clearing proteins were also modified with galactose residues, introduced using an activated thioimidate derivative, to produce clearing agents which would clear rapidly and clear primary mab rapidly. At least 14 galactose residues on WI2 were required to reduce blood levels to 5.9 +/- 0.7% ID/g in 1 h. Faster blood clearance (0.7 +/- 0.2% ID/g) was observed in 1 h using 44 galactose units per WI2. For the delivery of radioisotope to tumor, several biotinylated conjugates consisting of biotin, a linker, and a chelate were prepared. Conjugates showed good in vitro and in vivo stability when D-amino acid peptides were used as linkers, biotin-peptide-DOTA-indium-111 had a slightly longer blood circulation time (0.09 +/- 0.02% ID/g in 1 h) than biotin-peptide-DTPA-indium-111 (0

  4. The use of ammonium sulphate precipitation method for the determination of antigen-binding capacity and affinity of anti-tetanus antibodies in human serum.

    PubMed

    Alausa, O K

    1975-01-01

    Radioimmunoassay of anti-tetanus antibodies produced in human serum in response to tetanus toxoid immunization was carried out by employing the non-specific precipitation effect of ammonium sulphate described by Farr (1958). The results showed that the method was sensitive and was able to differentiate between immune and non-immune persons. The effects of booster dose injection on the quantity and quality of antibodies produced during immunization were discussed, and the possible use of the method to predict the amount of immunogen, the timing and number of injections required for optimal host response in immunization schedules was suggested.

  5. HANFORD DOUBLE SHELL TANK (DST) THERMAL & SEISMIC PROJECT BUCKLING EVALUATION METHODS & RESULTS FOR THE PRIMARY TANKS

    SciTech Connect

    MACKEY TC; JOHNSON KI; DEIBLER JE; PILLI SP; RINKER MW; KARRI NK

    2007-02-14

    This report documents a detailed buckling evaluation of the primary tanks in the Hanford double-shell waste tanks (DSTs), which is part of a comprehensive structural review for the Double-Shell Tank Integrity Project. This work also provides information on tank integrity that specifically responds to concerns raised by the Office of Environment, Safety, and Health (ES&H) Oversight (EH-22) during a review of work performed on the double-shell tank farms and the operation of the aging waste facility (AWF) primary tank ventilation system. The current buckling review focuses on the following tasks: (1) Evaluate the potential for progressive I-bolt failure and the appropriateness of the safety factors that were used for evaluating local and global buckling. The analysis will specifically answer the following questions: (a) Can the EH-22 scenario develop if the vacuum is limited to -6.6-inch water gage (w.g.) by a relief valve? (b) What is the appropriate factor of safety required to protect against buckling if the EH-22 scenario can develop? (c) What is the appropriate factor of safety required to protect against buckling if the EH-22 scenario cannot develop? (2) Develop influence functions to estimate the axial stresses in the primary tanks for all reasonable combinations of tank loads, based on detailed finite element analysis. The analysis must account for the variation in design details and operating conditions between the different DSTs. The analysis must also address the imperfection sensitivity of the primary tank to buckling. (3) Perform a detailed buckling analysis to determine the maximum allowable differential pressure for each of the DST primary tanks at the current specified limits on waste temperature, height, and specific gravity. Based on the I-bolt loads analysis and the small deformations that are predicted at the unfactored limits on vacuum and axial loads, it is very unlikely that the EH-22 scenario (i.e., progressive I-bolt failure leading to global

  6. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    PubMed

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods.

  7. Normalization method of highly forward-peaked scattering phase function using the double exponential formula for radiative transfer

    NASA Astrophysics Data System (ADS)

    Fujii, Hiroyuki; Okawa, Shinpei; Yamada, Yukio; Hoshi, Yoko; Watanabe, Masao

    2016-12-01

    Numerical calculation of photon migration in biological tissue using the radiative transfer equation (RTE) has attracted great interests in biomedical optics and imaging. Because biological tissue is a highly forward-peaked scattering medium, a normalization of scattering phase function in the RTE is crucial. This paper proposes a simple way of normalizing the phase function by the double exponential formula, which is heuristically modified from the original one. The proposed method is validated by the agreement between the numerical solution of the RTE with the proposed method and analytical solution of the RTE for the case of a highly forward-peaked scattering medium, while the numerical solutions with conventional normalization methods disagree with the analytical solution. This result suggests the proposed method is accurate in numerical calculation of the RTE.

  8. Fracture Toughness of Thin Plates by the Double-Torsion Test Method

    NASA Technical Reports Server (NTRS)

    Salem, Jonathan A.; Radovic, Miladin; Lara-Curzio, Edgar; Nelson, George

    2006-01-01

    Double torsion testing can produce fracture toughness values without crack length measurement that are comparable to those measured via standardized techniques such as the chevron-notch, surface-crack-in-flexure and precracked beam if the appropriate geometry is employed, and the material does not exhibit increasing crack growth resistance. Results to date indicate that 8 < W/d < 80 and L/W > 2 are required if crack length is not considered in stress intensity calculations. At L/W = 2, the normalized crack length should be 0.35 < a/L < 0.65; whereas for L/W = 3, 0.2 < a/L < 0.75 is acceptable. In addition, the load-points need to roll to reduce friction. For an alumina exhibiting increasing crack growth resistance, values corresponding to the plateau of the R-curve were measured. For very thin plates (W/d > 80) nonlinear effects were encountered.

  9. An investigation into the mechanics of double-sided incremental forming using finite element methods

    NASA Astrophysics Data System (ADS)

    Moser, Newell; Zhang, Zixuan; Ren, Huaqing; Ehmann, Kornel; Cao, Jian

    2016-10-01

    Double-Sided Incremental Forming (DSIF) is a developing sheet metal manufacturing process that has gained a lot of attention in recent years due to its inherent flexibility, low-overhead cost, and die-less nature. However, it can be challenging to define the tool gap so as to achieve a desired pressure through the sheet thickness since one must first predict sheet thinning. In this investigation, a novel part design is proposed which varies in-plane curvature as a function of depth. A finite element model for DSIF is developed and the strain histories in various regions are extracted. It was concluded that if the supporting tool loses contact with the sheet, localized necking can occur prior to part failure. Additionally, part geometry can have significant effects on the tool contact area which, consequently, affects the evolution of strain.

  10. Standardizing the double-observer survey method for estimating mountain ungulate prey of the endangered snow leopard.

    PubMed

    Suryawanshi, Kulbhushansingh R; Bhatnagar, Yash Veer; Mishra, Charudutt

    2012-07-01

    Mountain ungulates around the world have been threatened by illegal hunting, habitat modification, increased livestock grazing, disease and development. Mountain ungulates play an important functional role in grasslands as primary consumers and as prey for wild carnivores, and monitoring of their populations is important for conservation purposes. However, most of the several currently available methods of estimating wild ungulate abundance are either difficult to implement or too expensive for mountainous terrain. A rigorous method of sampling ungulate abundance in mountainous areas that can allow for some measure of sampling error is therefore much needed. To this end, we used a combination of field data and computer simulations to test the critical assumptions associated with double-observer technique based on capture-recapture theory. The technique was modified and adapted to estimate the populations of bharal (Pseudois nayaur) and ibex (Capra sibirica) at five different sites. Conducting the two double-observer surveys simultaneously led to underestimation of the population by 15%. We therefore recommend separating the surveys in space or time. The overall detection probability for the two observers was 0.74 and 0.79. Our surveys estimated mountain ungulate populations (± 95% confidence interval) of 735 (± 44), 580 (± 46), 509 (± 53), 184 (± 40) and 30 (± 14) individuals at the five sites, respectively. A detection probability of 0.75 was found to be sufficient to detect a change of 20% in populations of >420 individuals. Based on these results, we believe that this method is sufficiently precise for scientific and conservation purposes and therefore recommend the use of the double-observer approach (with the two surveys separated in time or space) for the estimation and monitoring of mountain ungulate populations.

  11. Common-mode differential-mode (CMDM) method for double-nuclear MR signal excitation and reception at ultrahigh fields.

    PubMed

    Pang, Yong; Zhang, Xiaoliang; Xie, Zhentian; Wang, Chunsheng; Vigneron, Daniel B

    2011-11-01

    Double-tuned radio-frequency (RF) coils for heteronuclear mangentic resonance (MR) require sufficient electromagnetic isolation between the two resonators operating at two Larmor frequencies and independent tuning in order to attain highly efficient signal acquisition at each frequency. In this work, a novel method for double-tuned coil design at 7T based on the concept of common-mode differential-mode (CMDM) was developed and tested. Common mode (CM) and differential mode (DM) currents exist within two coupled parallel transmission lines, e.g., microstrip lines, yielding two different current distributions. The electromagnetic (EM) fields of the CM and DM are orthogonal to each other, and thus, the two modes are intrinsically EM decoupled. The modes can be tuned independently to desired frequencies, thus satisfying the requirement of dual-frequency MR applications. To demonstrate the feasibility and efficiency of the proposed CMDM technique, CMDM surface coils and volume coils using microstrip transmission line for (1)H and (13)C MRI/MRSI were designed, constructed, and tested at 7T. Bench test results showed that the isolations between the two frequency channels of the CMDM surface coil and volume coil were better than -30 and -25 dB, respectively. High quality MR phantom images were also obtained using the CMDM coils. The performance of the CMDM technique was validated through a comparison with the conventional two-pole design method at 7T. The proposed CMDM technique can be also implemented by using other coil techniques such as lumped element method, and can be applied to designing double-tuned parallel imaging coil arrays. Furthermore, if the two resonant modes of a CMDM coil were tuned to the same frequency, the CMDM coil becomes a quadrature coil due to the intrinsic orthogonal field distribution of CM and DM.

  12. [Comparison of two types of double-lined simulated landfill leakage detection based on high voltage DC method].

    PubMed

    Yang, Ping; Nai, Chang-Xin; Dong, Lu; Wang, Qi; Wang, Yan-Wen

    2006-01-01

    Two types of double high density polyethylene (HDPE) liners landfill that clay or geogrid was added between the two HDPE liners. The general resistance of the second mode is 15% larger than the general resistance of the first mode in the primary HDPE liner detection, and 20% larger than that of the first one in the secondary HDPE liner detection. High voltage DC method can accomplish the leakage detection and location of these two types of landfill and the error of leakage location is less than 10cm when electrode space is 1m.

  13. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  14. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  15. Membrane filter-fluorescent-antibody method for detection and enumeration of bacteria in water.

    PubMed

    Guthrie, R K; Reeder, D J

    1969-03-01

    A technique which employs nonfluorescing membrane filters and specific fluoresceinisothiocynate-labeled antiserum has been successfully used in the identification and enumeration of known species of Escherichia coli which have been added to natural populations of bacteria found in water. The quantitative results compared favorably with those of standard tests. The use of a dissecting microscope with an external lighting arrangement provided a simple requirement for equipment. This method may be useful in monitoring specific bacterial types from waters which were being monitored for specific pollution.

  16. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations.

    PubMed

    Rea, Jennifer C; Moreno, G Tony; Lou, Yun; Farnan, Dell

    2011-01-25

    Ion-exchange chromatography is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies. Despite good resolving power and robustness, ionic strength-based ion-exchange separations are product-specific and time-consuming to develop. We have previously reported a novel pH-based separation of proteins by cation exchange chromatography that was multi-product, high-resolution, and robust against variations in sample matrix salt concentration and pH. In this study, a pH gradient-based separation method using cation exchange chromatography was evaluated in a mock validation. This method was shown to be robust for monoclonal antibodies and suitable for its intended purpose of charge heterogeneity analysis. Simple mixtures of defined buffer components were used to generate the pH gradients that separated closely related antibody species. Validation characteristics, such as precision and linearity, were evaluated. Robustness to changes in protein load, buffer pH and column oven temperature was demonstrated. The stability-indicating capability of this method was determined using thermally stressed antibody samples. In addition, intermediate precision was demonstrated using multiple instruments, multiple analysts, multiple column lots, and different column manufacturers. Finally, the precision for this method was compared to conventional ion-exchange chromatography and imaged capillary isoelectric focusing. These results demonstrate the superior precision and robustness of this multi-product method, which can be used for the high-throughput evaluation of in-process and final product samples.

  17. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  18. Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody.

    PubMed Central

    Patel, T P; Parekh, R B; Moellering, B J; Prior, C P

    1992-01-01

    A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and 'profile' analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it. Images Fig. 1. PMID:1497622

  19. Investigation of Crack Growth Process in Dense Hydroxyapatite Using the Double Torsion Method

    NASA Astrophysics Data System (ADS)

    Benaqqa, C.; Chevalier, J.; Saâdaoui, M.; Fantozzi, G.

    In this work, double torsion tests were performed to investigate slow crack growth behavior of dense hydroxyapatite materials. Crack rate, V, versus stress intensity factor, K I , laws were obtained for different environments and processing conditions. Stress assisted corrosion by water molecules in oxide ceramics is generally responsible for slow crack growth. The different propagation stages obtained here could be analyzed in relation to this process. The presence of a threshold defining a safety range of use was also observed. Hydroxyapatite ceramics appear to be very sensitive to slow crack growth, crack propagation occurring even at very low K I . This can be explained by the fact that their contain hydroxyl groups (HAP: Ca10(PO4)6(OH)2), favoring water adsorption on the crack surface and thus a strong decrease of surface energy in the presence of water. This study demonstrates that processing conditions must be carefully controlled, specially sintering temperature, which plays a key role on V-K I laws. Sintering at 50°C above or below the optimal temperature, for example, may shift the V-K I law towards very low stress intensity factors. The influence of ageing is finally discussed.

  20. A double-index method to classify Kuroshio intrusion paths in the Luzon Strait

    NASA Astrophysics Data System (ADS)

    Huang, Zhida; Liu, Hailong; Hu, Jianyu; Lin, Pengfei

    2016-06-01

    A double index (DI), which is made up of two sub-indices, is proposed to describe the spatial patterns of the Kuroshio intrusion and mesoscale eddies west to the Luzon Strait, based on satellite altimeter data. The area-integrated negative and positive geostrophic vorticities are defined as the Kuroshio warm eddy index (KWI) and the Kuroshio cold eddy index (KCI), respectively. Three typical spatial patterns are identified by the DI: the Kuroshio warm eddy path (KWEP), the Kuroshio cold eddy path (KCEP), and the leaking path. The primary features of the DI and three patterns are further investigated and compared with previous indices. The effects of the integrated area and the algorithm of the integration are investigated in detail. In general, the DI can overcome the problem of previously used indices in which the positive and negative geostrophic vorticities cancel each other out. Thus, the proportions of missing and misjudged events are greatly reduced using the DI. The DI, as compared with previously used indices, can better distinguish the paths of the Kuroshio intrusion and can be used for further research.

  1. Double-salting out assisted liquid-liquid extraction (SALLE) HPLC method for estimation of temozolomide from biological samples.

    PubMed

    Jain, Darshana; Athawale, Rajani; Bajaj, Amrita; Shrikhande, Shruti

    2014-11-01

    The role of temozolomide (TMZ) in treatment of high grade gliomas, melanomas and other malignancies is being defined by the current clinical developmental trials. Temozolomide belongs to the group of alkylating agents and is prescribed to patients suffering from most aggressive forms of brain tumors. The estimation techniques for temozolomide from the extracted plasma or biological samples includes high-performance liquid chromatography with UV detection (HPLC-UV), micellar electrokinetic capillary chromatography (MKEC) and liquid chromatography coupled to mass spectroscopy (LC-MS). These methods suffer from disadvantages like low resolution, low sensitivity, low recovery or cost involvement. An analytical method possessing capacity to estimate low quantities of TMZ in plasma samples with high extraction efficiency (%) and high resolution with cost effectiveness needs to be developed. Cost effective, robust and low plasma component interfering HPLC method using salting out liquid-liquid extraction (SALLE) technique was developed and validated for estimation of drug from plasma samples. The extraction efficiency (%) with conventional LLE technique with methanol, ethyl acetate, dichloromethane and acetonitrile was found to be 5.99±2.45, 45.39±4.56, 46.04±1.14 and 46.23±3.67 respectively. Extraction efficiency (%) improved with SALLE where sodium chloride was used as an electrolyte and was found to be 6.80±5.56, 52.01±3.13, 62.69±2.11 and 69.20±1.18 with methanol, ethyl acetate, dichloromethane and acetonitrile as organic solvent. Upon utilization of two salts for extraction (double salting liquid-liquid extraction) the extraction efficiency (%) was further improved and was twice of LLE. It was found that double salting liquid-liquid extraction technique yielded extraction efficiency (%) of 11.71±5.66, 55.62±3.44, 77.28±2.89 and 87.75±0.89. Hence a method based on double SALLE was developed for quantification of TMZ demonstrating linearity in the range of

  2. Variable-cell double-ended surface walking method for fast transition state location of solid phase transitions.

    PubMed

    Zhang, Xiao-Jie; Liu, Zhi-Pan

    2015-10-13

    To identify the low energy pathway for solid-to-solid phase transition has been a great challenge in physics and material science. This work develops a new theoretical method, namely, variable-cell double-ended surface walking (VC-DESW) to locate the transition state (TS) and deduce the pathway in solid phase transition. Inherited from the DESW method ( J. Chem. Theory Comput. 2013 , 9 , 5745 ) for molecular systems, the VC-DESW method implements an efficient mechanism to couple the lattice and atom degrees of freedom. The method features with fast pseudopathway building and accurate TS location for solid phase transition systems without requiring expensive Hessian computation and iterative pathway optimization. A generalized coordinate, consisting of the lattice vectors and the scaled atomic coordinates, is designed for describing the crystal potential energy surface (PES), which is able to capture the anisotropic behavior in phase transition. By comparing with the existing method for solid phase transition in different systems, we show that the VC-DESW method can be much more efficient for finding the TS in crystal phase transition. With the combination of the recently developed unbiased stochastic surface walking pathway sampling method, the VC-DESW is further utilized to resolve the lowest energy pathway of SiO2 α-quartz to quartz-II phase transition from many likely reaction pathways. These new methods provide a powerful platform for understanding and predicting the solid phase transition mechanism and kinetics.

  3. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from s...

  4. Immune Infertility Should Be Positively Diagnosed Using an Accurate Method by Monitoring the Level of Anti-ACTL7a Antibody

    PubMed Central

    Fu, Jun; Yao, Rongyan; Luo, Yanyun; Yang, Dantong; Cao, Yang; Qiu, Yi; Song, Wei; Miao, Shiying; Gu, Yiqun; Wang, Linfang

    2016-01-01

    Infertility is currently a major public health problem. Anti-sperm antibodies (ASAs) markedly reduce sperm quality, which can subsequently lead to male and/or female infertility. The accurate detection of ASAs derived from specific spermatozoa is, therefore, clinically useful. We have focused on the spermatozoa-specific expression protein ACTL7a for many years and have developed an enzyme-linked immunosorbent assay (ELISA) to detect the concentration of anti-ACTL7a antibodies in fertile sera (n = 267) and infertile sera (n = 193). Infertile sera were collected from the positive sera of tray agglutination tests (TAT), which is a routine ASA screening methodology. We found that the concentration of anti-ACTL7a antibodies was significantly higher in the infertile sera (than in the fertile sera, P < 0.0001) and much higher in the TAT ≥ 16 infertile sera. The ELISA was much better for male sera detection (AUC = 0.9899). If we set the standard at a strongly positive value (calculated by ROC curve), the positive predictive value of the antibody detection reached 100 percent, with a false positive rate of zero. The developed ELISA method for anti-ACTL7a antibody detection is therefore sensitive, accurate, and easy to perform, making it an excellent potential tool for future clinical use. PMID:26957350

  5. Evaluation of indirect fluorescent antibody test (IFAT) for the diagnosis and screening of lumpy skin disease using Bayesian method.

    PubMed

    Gari, G; Biteau-Coroller, F; LeGoff, C; Caufour, P; Roger, F

    2008-06-22

    The performance of indirect fluorescence antibody test (IFAT) for serological diagnosis and screening of lumpy skin disease (LSD) was evaluated using methods without gold standard. Virus neutralization test (VNT) was used as the second test and the study sites were selected from two different geographical places in Ethiopia to get different disease prevalence. The analysis of conditional dependent Bayesian model for the accuracy of IFAT showed that sensitivity, specificity, prevalence of the population Pi(1) and the population Pi(2) were 0.92 (0.89-0.95), 0.88 (0.85-0.91), 0.28 (0.25-0.32) and 0.06 (0.048-0.075), respectively. The posterior inferences obtained for VNT sensitivity, specificity and conditional correlation between the tests for sensitivity (rhoD) and specificity (rhoDc) were 0.78 (0.74-0.83), 0.97 (0.95-0.99), 0.052 (-0.03-0.15) and 0.019 (-0.01-0.06), respectively. The interval estimation of conditional correlation for both sensitivity and specificity clusters around zero and thus conditional dependence between the two tests was not significant. Although accuracy measure would not be the only basis for test selection, the result of our study demonstrated that IFAT has a reasonable high accuracy to be used for the diagnosis and sero-surveillance analysis of LSD in the target population.

  6. Interfacial width in polymer bilayer films prepared by double-spin-coating and flotation methods.

    PubMed

    Fujii, Yoshihisa; Atarashi, Hironori; Hino, Masahiro; Nagamura, Toshihiko; Tanaka, Keiji

    2009-09-01

    A spin-coating method with the aid of selective solvents has been used to construct multilayer structures for organic devices under the assumption that the solvents do not invade a preformed structure. To confirm the assumption, we examined the interfacial width (lambda(i)) of model polymer bilayers, composed of polystyrene and perdeuterated poly(methyl methacrylate), prepared by spin-coating and flotation methods. Neutron reflectivity measurements revealed that the lambda(i) value was larger for the spin-coating method than for the flotation method. These results cast doubt on the validity of the assumption. This knowledge should be kept in mind when this method is applied to construct multilayer structures.

  7. Layered double hydroxide/polyethylene terephthalate nanocomposites. Influence of the intercalated LDH anion and the type of polymerization heating method

    SciTech Connect

    Herrero, M.; Martinez-Gallegos, S.; Labajos, F.M.; Rives, V.

    2011-11-15

    Conventional and microwave heating routes have been used to prepare PET-LDH (polyethylene terephthalate-layered double hydroxide) composites with 1-10 wt% LDH by in situ polymerization. To enhance the compatibility between PET and the LDH, terephthalate or dodecyl sulphate had been previously intercalated in the LDH. PXRD and TEM were used to detect the degree of dispersion of the filler and the type of the polymeric composites obtained, and FTIR spectroscopy confirmed that the polymerization process had taken place. The thermal stability of these composites, as studied by thermogravimetric analysis, was enhanced when the microwave heating method was applied. Dodecyl sulphate was more effective than terephthalate to exfoliate the samples, which only occurred for the terephthalate ones under microwave irradiation. - Graphical abstract: Conventional and microwave heating routes were used to prepare PET-LDH (polyethylene terephthalate-layered double hydroxide) composites with 1-10 wt% LDH by in situ polymerization. To enhance the compatibility between PET and the LDH, terephthalate or dodecyl sulphate was previously intercalated into the LDH. The microwave process improves the dispersion and the thermal stability of nanocomposites due to the interaction of the microwave radiation and the dipolar properties of EG and the homogeneous heating. Highlights: > LDH-PET compatibility is enhanced by preintercalation of organic anions. > Dodecylsulphate performance is much better than that of terephthalate. > Microwave heating improves the thermal stability of the composites. > Microwave heating improves as well the dispersion of the inorganic phase.

  8. Rapid recovery of polycrystalline silicon from kerf loss slurry using double-layer organic solvent sedimentation method

    NASA Astrophysics Data System (ADS)

    Xing, Peng-fei; Guo, Jing; Zhuang, Yan-xin; Li, Feng; Tu, Gan-feng

    2013-10-01

    The rapid development of photovoltaic (PV) industries has led to a shortage of silicon feedstock. However, more than 40% silicon goes into slurry wastes due to the kerf loss in the wafer slicing process. To effectively recycle polycrystalline silicon from the kerf loss slurry, an innovative double-layer organic solvent sedimentation process was presented in the paper. The sedimentation velocities of Si and SiC particles in some organic solvents were investigated. Considering the polarity, viscosity, and density of solvents, the chloroepoxy propane and carbon tetrachloride were selected to separate Si and SiC particles. It is found that Si and SiC particles in the slurry waste can be successfully separated by the double-layer organic solvent sedimentation method, which can greatly reduce the sedimentation time and improve the purity of obtained Si-rich and SiC-rich powders. The obtained Si-rich powders consist of 95.04% Si, and the cast Si ingot has 99.06% Si.

  9. Antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, J.F.

    1988-06-28

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be about 5.0 nm. Methods and reagents are disclosed in which antibodies or Fab' fragments thereof are covalently bound to a stable cluster of gold atoms. 2 figs.

  10. A comparison study of the EF-18 agar/Hydrophobic Grid Membrane Filter (HGMF) method and the enzyme linked antibody (ELA)/HGMF method to the HPB standard method in the isolation of Salmonella.

    PubMed

    Warburton, D W; Arling, V; Worobec, S; Mackenzie, J; Todd, E C; Lacasse, P; Lamontagne, G; Plante, R; Shaw, S; Bowen, B

    1994-09-01

    As part of a comparative and collaborative study of rapid methods for the detection of Salmonella and the standard Health Protection Branch (HPB) method, six Federal and Provincial Laboratories compared the EF-18 agar/Hydrophobic Grid Membrane Filters (HGMF) method to the standard HPB method. Two Federal Laboratories also compared the enzyme linked antibody (ELA)/HGMF method (which is a further development of the EF-18 agar/HGMF method) to the standard method. During this study the false-negative rates ranged from 0% to 15% for the standard HPB method, from 5.88% to 43.5% for the EF-18 agar/HGMF method, and from 0% to 10.5% for the ELA/HGMF method. The EF-18 agar/HGMF method did not compare well with the standard HPB method due to the number of false-negatives. Problems with this method resulted from the inability to isolate colonies of Salmonella on the HGMF due to the small colony size, abnormal colony coloration, and overgrowth by competitors. The ELA/HGMF method, however, was shown to be comparable to the standard HPB method. The main advantages of this method are that the antibody-staining step is independent of colony coloration and carbohydrate utilization on the plating media; the ability to detect some unusual strains of Salmonella irrespective of their atypical reactions on the media; and the ELA staining can indicate the presence of Salmonella even when the HGMF is overgrown by competitors. Also, cultural confirmation can proceed simultaneously yet independently of the ELA staining procedure. The data presented here indicate that this method is worth further study.

  11. Synthesis of macrocyclic polyaminocarboxylates and their use for preparing stable radiometal antibody immunoconjugates for therapy, SPECT and PET imaging

    DOEpatents

    Mease, R.C.; Mausner, L.F.; Srivastava, S.C.

    1995-06-27

    A simple method for the synthesis of 1,4,7,10-tetraazacyclododecane N,N{prime}N{double_prime},N{prime}{double_prime}-tetraacetic acid and 1,4,8,11-tetraazacyclotetradecane N,N{prime},N{double_prime},N{prime}{double_prime}-tetraacetic acid involves cyanomethylating 1,4,7,10-tetraazacyclododecane or 1,4,8,11-tetraazacyclotetradecane to form a tetranitrile and hydrolyzing the tetranitrile. These macrocyclic compounds are functionalized through one of the carboxylates and then conjugated to various biological molecules including monoclonal antibodies. The resulting conjugated molecules are labeled with radiometals for SPECT and PET imaging and for radiotherapy. 4 figs.

  12. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

    PubMed Central

    Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  13. The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method

    PubMed Central

    Clargo, Alison M; Hudson, Ashley R; Ndlovu, Welcome; Wootton, Rebecca J; Cremin, Louise A; O'Dowd, Victoria L; Nowosad, Carla R; Starkie, Dale O; Shaw, Sophie P; Compson, Joanne E; White, Dominic P; MacKenzie, Brendon; Snowden, James R; Newnham, Laura E; Wright, Michael; Stephens, Paul E; Griffiths, Meryn R; Lawson, Alastair DG; Lightwood, Daniel J

    2014-01-01

    Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe. PMID:24423622

  14. Quality Improvement Method using Double Error Correction in Burst Transmission Systems

    NASA Astrophysics Data System (ADS)

    Tsuchiya, Naosuke; Tomiyama, Shigenori; Tanaka, Kimio

    Recently, it has a tendency to reduce an error correction and flow control in order to realize a high speed transmission in a burst transmission systems such as ATM network, IP (Internet Protocol) network, frame relay and so on. Therefore a degradation of network quality, an information loss caused by buffer overflow and decrease of average bit error rate, are occurred, especially for high speed information such as high definition television signals, it is necessary to improve these degradations. This paper proposes one of the typical reconstruction methods of lost information and an improvement of average bit error rate. In order to analyse the degradation phenomena, the Gilbert model is introduced for burst errors and the Fluid flow model for buffer overflow. This method is applied to ATM network which mainly transmit a video signals and it makes clear that proposed method is useful for high speed transmission.

  15. A method for polycrystalline silicon delineation applicable to a double-diffused MOS transistor

    NASA Technical Reports Server (NTRS)

    Halsor, J. L.; Lin, H. C.

    1974-01-01

    Method is simple and eliminates requirement for unreliable special etchants. Structure is graded in resistivity to prevent punch-through and has very narrow channel length to increase frequency response. Contacts are on top to permit planar integrated circuit structure. Polycrystalline shield will prevent creation of inversion layer in isolated region.

  16. Sorting of Double-Walled Carbon Nanotubes According to Their Outer Wall Electronic Type via a Gel Permeation Method.

    PubMed

    Moore, Katherine E; Pfohl, Moritz; Tune, Daniel D; Hennrich, Frank; Dehm, Simone; Chakradhanula, Venkata Sai K; Kübel, Christian; Krupke, Ralph; Flavel, Benjamin S

    2015-04-28

    In this work, we demonstrate the application of the gel permeation technique to the sorting of double-walled carbon nanotubes (DWCNTs) according to their outer wall electronic type. Our method uses Sephacryl S-200 gel and yields sorted fractions of DWCNTs with impurities removed and highly enriched in nanotubes with either metallic (M) or semiconducting (S) outer walls. The prepared fractions are fully characterized using optical absorption spectroscopy, transmission electron microscopy, and atomic force microscopy, and the entire procedure is monitored in real time using process Raman analysis. The sorted DWCNTs are then integrated into single nanotube field effect transistors, allowing detailed electronic measurement of the transconductance properties of the four unique inner@outer wall combinations of S@S, S@M, M@S, and M@M.

  17. A Two-Step Double Filter Method to Extract Open Water Surfaces from Landsat ETM+ Imagery

    NASA Astrophysics Data System (ADS)

    Wang, Haijing; Kinzelbach, Wolfgang

    2010-05-01

    In arid and semi-arid areas, lakes and temporal ponds play a significant role in agriculture and livelihood of local communities as well as in ecology. Monitoring the changes of these open water bodies allows to draw conclusions on water use as well as climatic impacts and can assist in the formulation of a sustainable resource management strategy. The simultaneous monitoring of larger numbers of water bodies with respect to their stage and area is feasible with the aid of remote sensing. Here the monitoring of lake surface areas is discussed. Landsat TM and ETM+ images provide a medium resolution of 30m, and offer an easily available data source to monitor the long term changes of water surfaces in arid and semi-arid regions. In the past great effort was put into developing simple indices to extract water surfaces from satellite images. However, there is a common problem in achieving accurate results with these indices: How to select a threshold value for water pixels without introducing excessive subjective judgment. The threshold value would also have to vary with location, land features and seasons, allowing for inherent uncertainty. A new method was developed using Landsat ETM+ imaginary (30 meter resolution) to extract open water surfaces. This method uses the Normalized Difference of Vegetation Index (NDVI) as the basis for an objective way of selecting threshold values of Modified Normalized Difference of Water Index (MNDWI) and Stress Degree Days (SDD), which were used as a combined filter to extract open water surfaces. We choose two study areas to verify the method. One study area is in Northeast China, where bigger lakes, smaller muddy ponds and wetlands are interspersed with agricultural land and salt crusts. The other one is Kafue Flats in Zambia, where seasonal floods of the Zambezi River create seasonal wetlands in addition to the more permanent water ponds and river channels. For both sites digital globe images of 0.5 meter resolution are available

  18. A comparison between the Farr radioimmunoassay and a new automated fluorescence immunoassay for the detection of antibodies against double stranded DNA in serum

    PubMed Central

    Derksen, R; Bast, E; Strooisma, T; Jacobs, J

    2002-01-01

    Methods: A cross sectional study comprising 440 samples from 440 patients, sent to the laboratory over a three month period for anti-dsDNA testing. Chart review was performed, blinded for test results, to count for each patient the number of American College of Rheumatology criteria for the classification of SLE that were fulfilled. At least four criteria were met by 248 (56%) patients (SLE), one to three criteria by 77 (18%) (lupus-like disease, LLD), and no criterion by 115 (26%) (non-SLE/non-LLD). Results from serum samples from the non-SLE/non-LLD and SLE groups were used to calculate receiver operating characteristic curves. Results: For the Farr assay, specificities of 95% and 99% corresponded to sensitivities of 72% and 56% respectively. For the ELIA dsDNA test these levels of specificity corresponded to sensitivities of 44% and 17% respectively. Conclusions: The Farr radioimmunoassay is superior to the ELIA dsDNA test for identifying patients with SLE. PMID:12429543

  19. 2D double interaction method for modeling small particles contaminating microstructures located on substrates

    NASA Astrophysics Data System (ADS)

    Albella, P.; Moreno, F.; Saiz, J. M.; González, F.

    2007-07-01

    An interaction model developed in previous research [de la Peña JL, González F, Saiz JM, Moreno F, Valle PJ. Sizing particles on substrates. A general method for oblique incidence. J Appl Phys 1999; 85:432] is extended to the study of two-scaled systems consisting of particles located on larger structures. Far-field scattering patterns produced by these systems can be obtained by coherent addition of different electromagnetic contributions, each one obtained from an independent isolated particle calculation. Results are performed on a 2D scheme, where they can be easily compared with those given by an exact method. This analysis shows some features of the scattering patterns that can be obtained with high reliability. Research on this kind of systems can be applied to 3D situations like particle substrate contamination and particle particle contamination.

  20. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1991-01-01

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

  1. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1991-07-02

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

  2. Estimation of the effective phase function of bulk diffusing materials with the inverse adding-doubling method.

    PubMed

    Leyre, Sven; Meuret, Youri; Durinck, Guy; Hofkens, Johan; Deconinck, Geert; Hanselaer, Peter

    2014-04-01

    The accuracy of optical simulations including bulk diffusors is heavily dependent on the accuracy of the bulk scattering properties. If no knowledge on the physical scattering effects is available, an iterative procedure is usually used to obtain the scattering properties, such as the inverse Monte Carlo method or the inverse adding-doubling (AD) method. In these methods, a predefined phase function with one free parameter is usually used to limit the number of free parameters. In this work, three predefined phase functions (Henyey-Greenstein, two-term Henyey-Greenstein, and Gegenbauer kernel (GK) phase function) are implemented in the inverse AD method to determine the optical properties of two strongly diffusing materials: low-density polyethylene and TiO₂ particles. Using the presented approach, an estimation of the effective phase function was made. It was found that the use of the GK phase function resulted in the best agreement between calculated and experimental transmittance, reflectance, and scattered radiant intensity distribution for the LDPE sample. For the TiO₂ sample, a good agreement was obtained with both the two-term Henyey-Greenstein and the GK phase function.

  3. A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination.

    PubMed

    Arndt, Patricia A; Garratty, George

    2010-07-01

    Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected).

  4. Method for generation of peptide-specific IgY antibodies directed to Staphylococcus aureus extracellular fibrinogen binding protein epitope.

    PubMed

    Walczak, Maciej; Grzywa, Renata; Łupicka-Słowik, Agnieszka; Skoreński, Marcin; Bobrek, Kamila; Nowak, Daria; Boivin, Stephane; Brown, Eric L; Oleksyszyn, Józef; Sieńczyk, Marcin

    2015-09-01

    The IgY antibodies offer an attractive alternative to mammalian IgGs in research, diagnosis and medicine. The isolation of immunoglobulin Y from the egg yolks is efficient and economical, causing minimal suffering to animals. Here we present the methodology for the production of IgY antibodies specific to Staphylococcus aureus fibrinogen binding protein (Efb) and its peptidyl epitope (spanning residues 127-140). The Efb is an extracellular, adhesion protein which binds both human fibrinogen and complement C3 protein thus contributing to the high infectious potential of this pathogen. The selected epitope of Efb protein is responsible for the interaction with C3. The immunochemical characterization of both anti-Efb and epitope-specific IgY antibodies revealed their similar avidity, titer, and reactivity profile, although some differences in the hen's immune response to administered antigens is discussed.

  5. Peptide composition as antigen for detection of antibodies to HTLV-I, as a vaccine for ATL and methods therefore

    SciTech Connect

    Wang, C.Y.; Wang, J.J.G.; Walters, D.W

    1989-05-23

    This patent describes a peptide composition having specific immunoreactivity to antibodies to HTLV-1 comprising a peptide selected from the group consisting of: (I) GLDLLFWEQGGLCKALQEQC-X; (II) QNRRGLDLLFWEQGGLCKALQEQC-X; (III) NRRGLDLLFWEQGGLC-X, wherein X is -OH or -NH/sub 2/, analogues therefore wherein the amino acids in the sequence are substituted as long as the immunoreactivity to antibodies to HTLV-I derived from the three dimensional conformation to the sequences are substantially preserved; and mixtures and polymers of the peptides.

  6. A versatile method for protein-based antigen bioanalysis in non-clinical pharmacokinetics studies of a human monoclonal antibody drug by an immunoaffinity liquid chromatography-tandem mass spectrometry.

    PubMed

    Onami, Ichio; Ayabe, Miho; Murao, Naoaki; Ishigai, Masaki

    2014-03-21

    A versatile immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC-MS/MS. Although a typical immunoassay or an immunoaffinity LC-MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (<115% interbatch accuracies) and precise (<15%, interbatch coefficient of variation) across a range of 3.13-200ng/mL RANKL. Commercial enzyme-linked immunosorbent assay (ELISA) kit was not able to determine the total RANKL because interference by denosumab decreased recovery. In contrast, the antibody drug had less effect on the LC-MS/MS method. The method now provides a bioanalytical platform for developing other protein-based antigen assays in the early drug stage.

  7. Accuracy of effective dose estimation in personal dosimetry: a comparison between single-badge and double-badge methods and the MOSFET method.

    PubMed

    Januzis, Natalie; Belley, Matthew D; Nguyen, Giao; Toncheva, Greta; Lowry, Carolyn; Miller, Michael J; Smith, Tony P; Yoshizumi, Terry T

    2014-05-01

    The purpose of this study was three-fold: (1) to measure the transmission properties of various lead shielding materials, (2) to benchmark the accuracy of commercial film badge readings, and (3) to compare the accuracy of effective dose (ED) conversion factors (CF) of the U.S. Nuclear Regulatory Commission methods to the MOSFET method. The transmission properties of lead aprons and the accuracy of film badges were studied using an ion chamber and monitor. ED was determined using an adult male anthropomorphic phantom that was loaded with 20 diagnostic MOSFET detectors and scanned with a whole body CT protocol at 80, 100, and 120 kVp. One commercial film badge was placed at the collar and one at the waist. Individual organ doses and waist badge readings were corrected for lead apron attenuation. ED was computed using ICRP 103 tissue weighting factors, and ED CFs were calculated by taking the ratio of ED and badge reading. The measured single badge CFs were 0.01 (±14.9%), 0.02 (±9.49%), and 0.04 (±15.7%) for 80, 100, and 120 kVp, respectively. Current regulatory ED CF for the single badge method is 0.3; for the double-badge system, they are 0.04 (collar) and 1.5 (under lead apron at the waist). The double-badge system provides a better coefficient for the collar at 0.04; however, exposure readings under the apron are usually negligible to zero. Based on these findings, the authors recommend the use of ED CF of 0.01 for the single badge system from 80 kVp (effective energy 50.4 keV) data.

  8. Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

    PubMed

    Panda, Rakhi; Fiedler, Katherine L; Cho, Chung Y; Cheng, Raymond; Stutts, Whitney L; Jackson, Lauren S; Garber, Eric A E

    2015-12-09

    The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

  9. Label-free and real-time detection of antigen-antibody interactions by Oblique-incidence Reflectivity Difference (OIRD) method

    NASA Astrophysics Data System (ADS)

    He, LiPing; Sun, Yue; Dai, Jun; Wang, JingYi; Lü, HuiBin; Wang, ShuFang; Jin, KuiJuan; Zhou, YueLiang; Yang, GuoZhen

    2012-09-01

    We label-free and real-time detected three interaction processes of antigen-antibodies, Human Immunoglobulin G (IgG), Rabbit IgG, and Mouse IgG as the targets, and Goat Anti-human IgG, Goat Anti-rabbit IgG, and Goat Anti-mouse IgG as the probe, by the Oblique-incidence Reflectivity Difference (OIRD) method. The interaction dynamic curves of the OIRD signal, corresponding to the interaction processes of antigen-antibodies, are generated. The reaction times from beginning to equilibrium state are about 1800, 900, and 1200 s for Human IgG, Rabbit IgG, and Mouse IgG, respectively. The experimental results demonstrate that the OIRD method not only can distinguish biomolecular interactions, but also can be used in real-time detection of interactions and dynamic processes of biomolecules.

  10. Method and system including a double rotary kiln pyrolysis or gasification of waste material

    SciTech Connect

    McIntosh, M.J.; Arzoumanidis, G.G.

    1997-09-02

    A method is described for destructively distilling an organic material in particulate form wherein the particulates are introduced through an inlet into one end of an inner rotating kiln ganged to and coaxial with an outer rotating kiln. The inner and outer kilns define a cylindrical annular space with the inlet being positioned in registry with the axis of rotation of the ganged kilns. During operation, the temperature of the wall of the inner rotary kiln at the inlet is not less than about 500 C to heat the particulate material to a temperature in the range of from about 200 C to about 900 C in a pyrolyzing atmosphere to reduce the particulate material as it moves from the one end toward the other end. The reduced particulates including char are transferred to the annular space between the inner and the outer rotating kilns near the other end of the inner rotating kiln and moved longitudinally in the annular space from near the other end toward the one end in the presence of oxygen to combust the char at an elevated temperature to produce a waste material including ash. Also, heat is provided which is transferred to the inner kiln. The waste material including ash leaves the outer rotating kiln near the one end and the pyrolysis vapor leaves through the particulate material inlet. 5 figs.

  11. Method and system including a double rotary kiln pyrolysis or gasification of waste material

    DOEpatents

    McIntosh, Michael J.; Arzoumanidis, Gregory G.

    1997-01-01

    A method of destructively distilling an organic material in particulate form wherein the particulates are introduced through an inlet into one end of an inner rotating kiln ganged to and coaxial with an outer rotating kiln. The inner and outer kilns define a cylindrical annular space with the inlet being positioned in registry with the axis of rotation of the ganged kilns. During operation, the temperature of the wall of the inner rotary kiln at the inlet is not less than about 500.degree. C. to heat the particulate material to a temperature in the range of from about 200.degree. C. to about 900.degree. C. in a pyrolyzing atmosphere to reduce the particulate material as it moves from the one end toward the other end. The reduced particulates including char are transferred to the annular space between the inner and the outer rotating kilns near the other end of the inner rotating kiln and moved longitudinally in the annular space from near the other end toward the one end in the presence of oxygen to combust the char at an elevated temperature to produce a waste material including ash. Also, heat is provided which is transferred to the inner kiln. The waste material including ash leaves the outer rotating kiln near the one end and the pyrolysis vapor leaves through the particulate material inlet.

  12. A method and system including a double rotary kiln pyrolysis or gasification of waste material

    SciTech Connect

    McIntosh, M.J.; Arzoumanidis, G.G.

    1995-12-31

    A method is described for destructively distilling an organic material in particulate form wherein the particulates are introduced through an inlet into one end of an inner rotating kiln ganged to and coaxial with an outer rotating kiln. The inner and outer kilns define a cylindrical annular space with the inlet being positioned in registry with the axis of rotation of the ganged kilns. During operation, the temperature of the wall of the inner rotary kiln at the inlet is not less than about 500 C to heat the particulate material to a temperature in the range of from about 200 C to about 900 C in a pyrolyzing atmosphere to reduce the particulate material as it moves from the one end toward the other end. The reduced particulates including char are transferred to the annular space between the inner and the outer rotating kilns near the other end of the inner rotating kiln and moved longitudinally in the annular space from near the other end toward the one end in the presence of oxygen to combust the char at an elevated temperature to produce a waste material including ash. Also, heat is provided which is transferred to the inner kiln. The waste material including ash leaves the outer rotating kiln near the one end and the pyrolysis vapor leaves through the particulate material inlet.

  13. Mining human antibody repertoires

    PubMed Central

    2010-01-01

    Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties. PMID:20505349

  14. The Double Solid Reactant Method for modeling the release of trace elements from dissolving solid phases: I. Outline and limitations

    NASA Astrophysics Data System (ADS)

    Accornero, Marina; Marini, Luigi

    2008-10-01

    A Double Solid Reactant Method was elaborated from a suggestion of Marini (Geological sequestration of carbon dioxide: Thermodynamics, kinetics, and reaction path modeling. Developments in Geochemistry, Elsevier, Amsterdam, 2007) to simulate the release of trace elements during the progressive dissolution of solid phases. The method is based on the definition, for each dissolving solid, of both an entity whose thermodynamic and kinetic properties are known (either a pure mineral or a solid mixture) and a special reactant, that is, a material of known stoichiometry and unknown thermodynamic and kinetic properties. The special reactant is utilised to take into account the concentrations of trace elements in the dissolving solid phase. In this communication, the influence of several trace elements on the Δ G f o, Δ G r o and log K of the minerals considered by Lelli et al. (Environ Geol, 2007) and Accornero and Marini (Geobasi, 2007a; Proceedings of IMWA symposium, Cagliari, 27 31 May 2007b) was evaluated assuming ideal mixing in the solid state. These effects were found to be negligible for albite and the leucite latitic glass, limited for muscovites and chlorites, and slightly more important for apatites. These influences become progressively higher with increasing concentration of trace elements in these minerals. Based on these deviations in thermodynamic parameters, special reactants should not include oxide components with molar fractions higher than 0.003.

  15. How the method of synthesis governs the local and global structure of zinc aluminum layered double hydroxides

    SciTech Connect

    Pushparaj, Suraj Shiv Charan; Forano, Claude; Prevot, Vanessa; Lipton, Andrew S.; Rees, Gregory; Hanna, John V.; Nielsen, Ulla Gro

    2015-11-10

    A series of zinc aluminum layered double hydroxides (ZnAl LDHs), [Zn1-xAlx (OH)2Ax,nH2O with A = NO3-, Cl- or CO3] were prepared by the urea and co-precipitation synthesis methods, which allowed for a detailed investigation on how synthesis parameters such as pH, metal ion concentration and post synthesis treatment influence the local and global structure of the LDH product. Information about sample composition, purity, defects and other structural aspects of the LDH products were obtained from powder X-ray diffraction, transmission electron microscopy, micro-Raman, and elemental analysis, as well as solid state 1H, 27Al and 67Zn NMR spectroscopy. Our results show that the urea method results in LDHs, which on the global scale are highly crystalline LDHs, whereas solid state NMR shows the different local environments indicating local disorder most likely linked to the presence of Al-rich phases. However, these Alrich phases are not detected by global range techniques, as they either defects within the LDH particles or separate phase(s) associated with LDHs. In contrast, samples prepared by coprecipitation especially synthesized under careful pH control and subsequently hydrothermal treated have high local order and good crystallinity (particle size). Our results show that both molecular level and macroscopic techniques are needed to assess the composition of LDHs, as the conventional PXRD and TEM analysis of LDHs failed to identify the many structural defects and/or amorphous phases.

  16. A Novel Method for Characterizing Fatigue Delamination Growth Under Mode I Using the Double Cantilever Beam Specimen

    NASA Technical Reports Server (NTRS)

    Carvalho, Nelson; Murri, G.

    2014-01-01

    A novel method is proposed to obtain Mode I delamination growth rate from a Double Cantilever Beam (DCB) specimen. In the proposed method, Unidirectional (UD) DCB specimens are tested in fatigue at different initial maximum energy release rates levels. The growth rate data obtained in the first increments of crack growth at each maximum energy release rate level are used to generate a Paris Law equation, which characterizes delamination growth rate without fiber-bridging, and can also be used to determine a delamination onset curve. The remaining delamination growth rate data from each test are used to determine a modified Paris law, which characterizes the delamination growth rate in a DCB specimen, explicitly accounting for fiber-bridging. The proposed expression captures well the scatter in experimental data obtained using the DCB specimens, suggesting its adequacy. The Paris Law characterizing delamination growth rate without fiber-bridging predicts higher delamination growth rates for the same maximum energy release rate applied, leading to a conservative estimate for delamination growth. This is particularly relevant, since in generic ply interfaces, fiber-bridging is less predominant than in UD DCB specimens. Failing to account for fiber-bridging in UD DCB specimens may underestimate the delamination growth rate, yielding non-conservative predictions.

  17. An improved method for constructing and selectively silanizing double-barreled, neutral liquid-carrier, ion-selective microelectrodes

    PubMed Central

    Deveau, Jason S.T.; Grodzinski, Bernard

    2005-01-01

    We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ~150 μm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix. PMID:16136222

  18. Analytical model for random dopant fluctuation in double-gate MOSFET in the subthreshold region using macroscopic modeling method

    NASA Astrophysics Data System (ADS)

    Shin, Yong Hyeon; Yun, Ilgu

    2016-12-01

    An analytical model is proposed for the random dopant fluctuation (RDF) in a symmetric double-gate metal-oxidesemiconductor field-effect-transistor (DG MOSFET) in the subthreshold region. Unintended impurity dopants cannot be absolutely prevented during the device fabrication; hence, it is important to analytically model the fluctuations in the electrical characteristics caused by these impurity dopants. Therefore, a macroscopic modeling method is applied to represent the impurity dopants in DG MOSFETs. With this method, the two-dimensional (2D) Poisson equation is separated into a basic analytical DG MOSFET model with channel doping concentration NA and an impurity-dopant-related term with local doping concentration NRD confined in a specific rectangular area. To solve the second term, the manually solvable 2D Green's function for DG MOSFETs is used. Through calculation of the channel potential (ϕ(x, y)), the variations in the drive current (IDS) and threshold voltage (Vth) are extracted from the analytical model. All results from the analytical model for an impurity dopant in a DG MOSFET are examined by comparisons with the commercially available 2D numerical simulation results, with respect to various oxide thicknesses (tox), channel lengths (L), and location of impurity dopants.

  19. A fusion of the closed-shell coupled cluster singles and doubles method and valence-bond theory for bond breaking.

    PubMed

    Small, David W; Head-Gordon, Martin

    2012-09-21

    Closed-shell coupled cluster singles and doubles (CCSD) is among the most important of electronic-structure methods. However, it fails qualitatively when applied to molecular systems with more than two strongly correlated electrons, such as those with stretched or broken covalent bonds. We show that it is possible to modify the doubles amplitudes to obtain a closed-shell CCSD method that retains the computational cost and desirable features of standard closed-shell CCSD, e.g., correct spin symmetry, size extensivity, orbital invariance, etc., but produces greatly improved energies upon bond dissociation of multiple electron pairs; indeed, under certain conditions the dissociation energies are exact.

  20. Double calibration: an accurate, reliable and easy-to-use method for 3D scapular motion analysis.

    PubMed

    Brochard, Sylvain; Lempereur, Mathieu; Rémy-Néris, Olivier

    2011-02-24

    The most recent non-invasive methods for the recording of scapular motion are based on an acromion marker (AM) set and a single calibration (SC) of the scapula in a resting position. However, this method fails to accurately measure scapular kinematics above 90° of arm elevation, due to soft tissue artifacts of the skin and muscles covering the acromion. The aim of this study was to evaluate the accuracy, and inter-trial and inter-session repeatability of a double calibration method (DC) in comparison with SC. The SC and DC data were measured with an optoelectronic system during arm flexion and abduction at different angles of elevation (0-180°). They were compared with palpation of the scapula using a scapula locator. DC data was not significantly different from palpation for 5/6 axes of rotation tested (Y, X, and Z in abduction and flexion), where as SC showed significant differences for 5/6 axes. The root mean square errors ranged from 2.96° to 4.48° for DC and from 6° to 9.19° for SC. The inter-trial repeatability was good to excellent for SC and DC. The inter-session repeatability was moderate to excellent for SC and moderate to good for DC. Coupling AM and DC is an easy-to-use method, which yields accurate and reliable measurements of scapular kinematics for the complete range of arm motion. It can be applied to the measurement of shoulder motion in many fields (sports, orthopaedics, and rehabilitation), especially when large ranges of arm motion are required.

  1. Monoclonal Antibodies.

    PubMed

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner.

  2. NMDA receptor antibodies

    PubMed Central

    Ramberger, Melanie; Bsteh, Gabriel; Schanda, Kathrin; Höftberger, Romana; Rostásy, Kevin; Baumann, Matthias; Aboulenein-Djamshidian, Fahmy; Lutterotti, Andreas; Deisenhammer, Florian; Berger, Thomas

    2015-01-01

    Objectives: To analyze the frequency of NMDA receptor (NMDAR) antibodies in patients with various inflammatory demyelinating diseases of the CNS and to determine their clinical correlates. Methods: Retrospective case-control study from 2005 to 2014 with the detection of serum IgG antibodies to NMDAR, aquaporin-4, and myelin oligodendrocyte glycoprotein by recombinant live cell-based immunofluorescence assays. Fifty-one patients with acute disseminated encephalomyelitis, 41 with neuromyelitis optica spectrum disorders, 34 with clinically isolated syndrome, and 89 with multiple sclerosis (MS) were included. Due to a known association of NMDAR antibodies with seizures and behavioral symptoms, patients with those clinical manifestations were preferentially included and are therefore overrepresented in our cohort. Nine patients with NMDAR encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were used as controls. Results: NMDAR antibodies were found in all 9 patients with NMDAR encephalitis but in only 1 of 215 (0.5%) patients with inflammatory demyelination and in none of the controls. This patient had relapsing-remitting MS with NMDAR antibodies present at disease onset, with an increase in NMDAR antibody titer with the onset of psychiatric symptoms and cognitive deficits. Conclusion: In demyelinating disorders, NMDAR antibodies are uncommon, even in those with symptoms seen in NMDAR encephalitis. PMID:26309901

  3. Reconstruction of the esophagojejunostomy by double stapling method using EEA™ OrVil™ in laparoscopic total gastrectomy and proximal gastrectomy

    PubMed Central

    2011-01-01

    Here we report the method of anastomosis based on double stapling technique (hereinafter, DST) using a trans-oral anvil delivery system (EEATM OrVilTM) for reconstructing the esophagus and lifted jejunum following laparoscopic total gastrectomy or proximal gastric resection. As a basic technique, laparoscopic total gastrectomy employed Roux-en-Y reconstruction, laparoscopic proximal gastrectomy employed double tract reconstruction, and end-to-side anastomosis was used for the cut-off stump of the esophagus and lifted jejunum. We used EEATM OrVilTM as a device that permitted mechanical purse-string suture similarly to conventional EEA, and endo-Surgitie. After the gastric lymph node dissection, the esophagus was cut off using an automated stapler. EEATM OrVilTM was orally and slowly inserted from the valve tip, and a small hole was created at the tip of the obliquely cut-off stump with scissors to let the valve tip pass through. Yarn was cut to disconnect the anvil from a tube and the anvil head was retained in the esophagus. The end-Surgitie was inserted at the right subcostal margin, and after the looped-shaped thread was wrapped around the esophageal stump opening, assisting Maryland forceps inserted at the left subcostal and left abdomen were used to grasp the left and right esophageal stump. The surgeon inserted anvil grasping forceps into the right abdomen, and after grasping the esophagus with the forceps, tightened the end Surgitie, thereby completing the purse-string suture on the esophageal stump. The main unit of the automated stapler was inserted from the cut-off stump of the lifted jejunum, and a trocar was made to pass through. To prevent dropout of the small intestines from the automated stapler, the automated stapler and the lifted jejunum were fastened with silk thread, the abdomen was again inflated, and the lifted jejunum was led into the abdominal cavity. When it was confirmed that the automated stapler and center rod were made completely linear

  4. Homoclinic behaviors and chaotic motions of double layered viscoelastic nanoplates based on nonlocal theory and extended Melnikov method

    SciTech Connect

    Wang, Yu; Wang, Yi-Ze; Li, Feng-Ming

    2015-06-15

    The nonlinear dynamical equations are established for the double layered viscoelastic nanoplates (DLNP) subjected to in-plane excitation based on the nonlocal theory and von Kármán large deformation theory. The extended high dimensional homoclinic Melnikov method is employed to study the homoclinic phenomena and chaotic motions for the parametrically excited DLNP system. The criteria for the homoclinic transverse intersection for both the asynchronous and synchronous buckling cases are proposed. Lyapunov exponents and phase portraits are obtained to verify the Melnikov-type analysis. The influences of structural parameters on the transverse homoclinic orbits and homoclinic bifurcation sets are discussed for the two buckling cases. Some novel phenomena are observed in the investigation. It should be noticed that the nonlocal effect on the homoclinic behaviors and chaotic motions is quite remarkable. Hence, the small scale effect should be taken into account for homoclinic and chaotic analysis for nanostructures. It is significant that the nonlocal effect on the homoclinic phenomena for the asynchronous buckling case is quite different from that for the synchronous buckling case. Moreover, due to the van der Walls interaction between the layers, the nonlocal effect on the homoclinic behaviors and chaotic motions for high order mode is rather tiny under the asynchronous buckling condition.

  5. Self-consistent double-hybrid density-functional theory using the optimized-effective-potential method

    NASA Astrophysics Data System (ADS)

    Śmiga, Szymon; Franck, Odile; Mussard, Bastien; Buksztel, Adam; Grabowski, Ireneusz; Luppi, Eleonora; Toulouse, Julien

    2016-10-01

    We introduce an orbital-optimized double-hybrid (DH) scheme using the optimized-effective-potential (OEP) method. The orbitals are optimized using a local potential corresponding to the complete exchange-correlation energy expression including the second-order Møller-Plesset correlation contribution. We have implemented a one-parameter version of this OEP-based self-consistent DH scheme using the BLYP density-functional approximation and compared it to the corresponding non-self-consistent DH scheme for calculations on a few closed-shell atoms and molecules. While the OEP-based self-consistency does not provide any improvement for the calculations of ground-state total energies and ionization potentials, it does improve the accuracy of electron affinities and restores the meaning of the LUMO orbital energy as being connected to a neutral excitation energy. Moreover, the OEP-based self-consistent DH scheme provides reasonably accurate exchange-correlation potentials and correlated densities.

  6. Electrode Mass Balancing as an Inexpensive and Simple Method to Increase the Capacitance of Electric Double-Layer Capacitors.

    PubMed

    Andres, Britta; Engström, Ann-Christine; Blomquist, Nicklas; Forsberg, Sven; Dahlström, Christina; Olin, Håkan

    Symmetric electric double-layer capacitors (EDLCs) have equal masses of the same active material in both electrodes. However, having equal electrode masses may prevent the EDLC to have the largest possible specific capacitance if the sizes of the hydrated anions and cations in the electrolyte differ because the electrodes and the electrolyte may not be completely utilized. Here we demonstrate how this issue can be resolved by mass balancing. If the electrode masses are adjusted according to the size of the ions, one can easily increase an EDLC's specific capacitance. To that end, we performed galvanostatic cycling to measure the capacitances of symmetric EDLCs with different electrode mass ratios using four aqueous electrolytes- Na2SO4, H2SO4, NaOH, and KOH (all with a concentration of 1 M)-and compared these to the theoretical optimal electrode mass ratio that we calculated using the sizes of the hydrated ions. Both the theoretical and experimental values revealed lower-than-1 optimal electrode ratios for all electrolytes except KOH. The largest increase in capacitance was obtained for EDLCs with NaOH as electrolyte. Specifically, we demonstrate an increase of the specific capacitance by 8.6% by adjusting the electrode mass ratio from 1 to 0.86. Our findings demonstrate that electrode mass balancing is a simple and inexpensive method to increase the capacitance of EDLCs. Furthermore, our results imply that one can reduce the amount of unused material in EDLCs and thus decrease their weight, volume and cost.

  7. Searching for Dual AGNs in Galaxy Mergers: Understanding Double-Peaked [O III] and Ultra Hard X-rays as Selection Method

    NASA Astrophysics Data System (ADS)

    McGurk, Rosalie C.; Max, Claire E.; Medling, Anne; Shields, Gregory A.

    2015-01-01

    When galaxies merge, gas accretes onto both central supermassive black holes. Thus, one expects to see close pairs of active galactic nuclei (AGNs), or dual AGNs, in a fraction of galaxy mergers. However, finding them remains a challenge. The presence of double-peaked [O III] or of ultra hard X-rays have been proposed as techniques to select dual AGNs efficiently. We studied a sample of double-peaked narrow [O III] emitting AGNs from SDSS DR7. By obtaining new and archival high spatial resolution images taken with the Keck 2 Laser Guide Star Adaptive Optics system and the near-infrared (IR) camera NIRC2, we showed that 30% of double-peaked [O III] emission line SDSS AGNs have two spatial components within a 3' radius. However, spatially resolved spectroscopy or X-ray observations are needed to confirm these galaxy pairs as systems containing two AGNs. We followed up these spatially-double candidate dual AGNs with integral field spectroscopy from Keck OSIRIS and Gemini GMOS and with long-slit spectroscopy from Keck NIRSPEC and Shane Kast Double Spectrograph. We find double-peaked emitters are caused sometimes by dual AGN and sometimes by outflows or narrow line kinematics. We also performed Chandra X-ray ACIS-S observations on 12 double-peaked candidate dual AGNs. Using our observations and 8 archival observations, we compare the distribution of X-ray photons to our spatially double near-IR images, measure X-ray luminosities and hardness ratios, and estimate column densities. By assessing what fraction of double-peaked emission line SDSS AGNs are true dual AGNs, we can better determine whether double-peaked [O III] is an efficient dual AGN indicator and constrain the statistics of dual AGNs. A second technique to find dual AGN is the detection of ultra hard X-rays by the Swift Burst Alert Telescope. We use CARMA observations to measure and map the CO(1-0) present in nearby ultra-hard X-ray Active Galactic Nuclei (AGNs) merging with either a quiescent companion

  8. Distance-Guided Forward and Backward Chain-Growth Monte Carlo Method for Conformational Sampling and Structural Prediction of Antibody CDR-H3 Loops.

    PubMed

    Tang, Ke; Zhang, Jinfeng; Liang, Jie

    2017-01-10

    Antibodies recognize antigens through the complementary determining regions (CDR) formed by six-loop hypervariable regions crucial for the diversity of antigen specificities. Among the six CDR loops, the H3 loop is the most challenging to predict because of its much higher variation in sequence length and identity, resulting in much larger and complex structural space, compared to the other five loops. We developed a novel method based on a chain-growth sequential Monte Carlo method, called distance-guided sequential chain-growth Monte Carlo for H3 loops (DiSGro-H3). The new method samples protein chains in both forward and backward directions. It can efficiently generate low energy, near-native H3 loop structures using the conformation types predicted from the sequences of H3 loops. DiSGro-H3 performs significantly better than another ab initio method, RosettaAntibody, in both sampling and prediction, while taking less computational time. It performs comparably to template-based methods. As an ab initio method, DiSGro-H3 offers satisfactory accuracy while being able to predict any H3 loops without templates.

  9. A double-lyophilization method for the preparation of CS/GO-COOH scaffold and its application in blood detoxification.

    PubMed

    Zhou, Gang; Wang, Lei; Li, Jianchao; Tai, Jun; Su, Haisheng; Zhang, Jing; Xi, Yuan; Fan, Yubo

    2016-10-10

    The accumulation of uremic toxins in blood might induce chronic renal failure (CRF). The incidence of CRF was as high as 10%. The traditional therapy for CRF was hemodialysis, which was more effective to remove small molecules, such as urea and creatinine. However, this detoxification method ignored the tissue functional adaption due to the retention of macromolecule uremic toxins. To solve this problem, this paper developed a new kind of chitosan/carboxyl graphite oxide (CS/GO-COOH) scaffold via a double-lyophilization method. Then, this synthetic scaffold was characterized by Fourier transform infrared spectroscopy, scanning electron microscope, hydrophilic test, mechanical property, and in vitro detoxification test. Covalent bonding and hydrogen bonding were formed, indicating the strong interactions between CS and GO-COOH. There were interconnected networks in the synthesized scaffold. The mechanical test suggested that the GO-2500 scaffold had excellent mechanical strength, which was 7.41 ± 0.82 MPa with 25% shrink. What is more, GO-2500 could totally rebound within 1s, after compressed to 90% shrink. The rates of GO-2500 were 1587 ± 60 and 246 ± 10% according to the water uptake and retention data, respectively. Furthermore, the detoxification of GO-2500 to urea, creatinine, VB12, and β2-m were 67.59 ± 2.31, 39.67 ± 2.95, 31.51 ± 2.62, and 83.82 ± 7.76 mg/g, respectively. The resulting CS/GO-COOH scaffold held great potential for the detoxification of uremic toxins.

  10. N-Methyl-D-aspartate receptor antibody could be a cause of catatonic symptoms in psychiatric patients: case reports and methods for detection

    PubMed Central

    Tsutsui, Ko; Kanbayashi, Takashi; Takaki, Manabu; Omori, Yuki; Imai, Yumiko; Nishino, Seiji; Tanaka, Keiko; Shimizu, Tetsuo

    2017-01-01

    The symptoms of catatonia have been reported to be similar to the initial symptoms of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Subsequently, this autoimmune limbic encephalitis has been noticed by many psychiatrists. For a differential diagnosis of catatonic state, it is important to detect anti-NMDAR encephalitis. This encephalitis is expected to be in remission by early detection and treatment. We should be more cautious about catatonic symptoms of schizophrenia. When a patient is suspected of having encephalitis, we should screen for anti-NMDAR antibodies in cerebrospinal fluid samples using a cell-based assay. We describe the methods of NMDAR antibody detection and the etiology of this encephalitis with case reports. Two representative cases with catatonia and non-catatonia (brief psychotic disorder) were reported. Schizophrenia is a general, heterogeneous, and complicated disorder, and its pathophysiology is unknown. There is an established evidence of NMDAR hypofunction, which is the functional disconnection of the central component; this is one of the most recognized models for schizophrenia. Furthermore, it is said that autoimmune mechanisms have been involved, at least in subgroups of schizophrenia patients. Further study of anti-NMDAR antibody and its related encephalitis would give essential clues for the research of schizophrenia, catatonia, and atypical psychosis. PMID:28223808

  11. N-Methyl-D-aspartate receptor antibody could be a cause of catatonic symptoms in psychiatric patients: case reports and methods for detection.

    PubMed

    Tsutsui, Ko; Kanbayashi, Takashi; Takaki, Manabu; Omori, Yuki; Imai, Yumiko; Nishino, Seiji; Tanaka, Keiko; Shimizu, Tetsuo

    2017-01-01

    The symptoms of catatonia have been reported to be similar to the initial symptoms of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Subsequently, this autoimmune limbic encephalitis has been noticed by many psychiatrists. For a differential diagnosis of catatonic state, it is important to detect anti-NMDAR encephalitis. This encephalitis is expected to be in remission by early detection and treatment. We should be more cautious about catatonic symptoms of schizophrenia. When a patient is suspected of having encephalitis, we should screen for anti-NMDAR antibodies in cerebrospinal fluid samples using a cell-based assay. We describe the methods of NMDAR antibody detection and the etiology of this encephalitis with case reports. Two representative cases with catatonia and non-catatonia (brief psychotic disorder) were reported. Schizophrenia is a general, heterogeneous, and complicated disorder, and its pathophysiology is unknown. There is an established evidence of NMDAR hypofunction, which is the functional disconnection of the central component; this is one of the most recognized models for schizophrenia. Furthermore, it is said that autoimmune mechanisms have been involved, at least in subgroups of schizophrenia patients. Further study of anti-NMDAR antibody and its related encephalitis would give essential clues for the research of schizophrenia, catatonia, and atypical psychosis.

  12. Immunostaining with EGFR mutation-specific antibodies: a reliable screening method for lung adenocarcinomas harboring EGFR mutation in biopsy and resection samples.

    PubMed

    Fan, Xiangshan; Liu, Biao; Xu, Haodong; Yu, Bo; Shi, Shanshan; Zhang, Jin; Wang, Xuan; Wang, Jiandong; Lu, Zhenfeng; Ma, Henghui; Zhou, Xiaojun

    2013-08-01

    Mutation analysis of epidermal growth factor receptor (EGFR) is essential in determining the therapeutic strategy for lung adenocarcinoma. Immunohistochemical (IHC) staining with EGFR mutation-specific antibodies of del E746-A750 in exon 19 and L858R in exon 21 has been evaluated in resection specimens in a few studies but rarely in biopsy samples. A total of 169 cases (78 biopsies and 91 resected specimens) of lung adenocarcinoma with EGFR mutation status predefined by direct DNA sequencing were histologically examined, and IHC was performed using EGFR mutation-specific antibodies of del E746-A750 and L858R. The cases with positive results by IHC but negative results by direct DNA sequencing were examined by amplified refractory mutation system. Our results showed that the frequency of EGFR mutations for both E746-A750 deletion and L858R mutation was 38.5% (65/169) by DNA sequencing or amplified refractory mutation system and 34.3% (58/169) by IHC in lung adenocarcinomas. Based on molecular test results, the overall sensitivity, specificity, positive predictive value, and negative predictive value of IHC using these 2 antibodies in all (biopsy/resection) cases were 87.7% (80%/94.3%), 99.0% (97.9%/100%), 98.3% (96%/100%), and 92.8% (88.7%/96.6%), respectively. Lung adenocarcinomas with a predominant acinar, papillary, lepidic, or solid growth pattern more often harbor EGFR mutation of del E746-A750 or L858R. In conclusion, the immunostaining with EGFR del E746-A750 and L858R mutation antibodies is a reliable screening method with high specificity and sensitivity for identifying the EGFR mutation in both resected and biopsied lung adenocarcinomas.

  13. Longitudinal analysis of pneumococcal antibodies during community-acquired pneumonia reveals a much higher involvement of Streptococcus pneumoniae than estimated by conventional methods alone.

    PubMed

    van Mens, Suzan P; Meijvis, Sabine C A; Endeman, Henrik; van Velzen-Blad, Heleen; Biesma, Douwe H; Grutters, Jan C; Vlaminckx, Bart J M; Rijkers, Ger T

    2011-05-01

    In up to half of all cases of community-acquired pneumonia (CAP), no pathogen can be identified with conventional diagnostic methods. The most common identified causative agent is Streptococcus pneumoniae. In this study, pneumococcal antibody responses during CAP were analyzed to estimate the contribution of the pneumococcus to all cases of CAP for epidemiological purposes. Pneumococcal antibodies against 14 different serotypes were measured in serum of hospitalized CAP patients. Patients participated in one of two consecutive clinical trials in a general 600-bed teaching hospital in the Netherlands (between October 2004 and June 2009). A significant pneumococcal immune response was defined as at least a 2-fold increase in antibody concentrations against a single serotype between an early (day 1) and a late (day 30) serum sample of each patient with an end concentration above 0.35 μg/ml. A total of 349 adult CAP patients participated in two consecutive clinical trials. For 200 patients, sufficient serum samples were available to determine antibody responses: 62 pneumococcal pneumonia patients, 57 nonpneumococcal pneumonia patients, and 81 patients with an unidentified causative agent. A significant immune response was detected in 45% (28/62 patients) of pneumococcal pneumonia patients, in 5% (3/57) of nonpneumococcal pneumonia patients, and in 28% (23/81) of patients with an unidentified causative agent. The estimated contribution of pneumococci in patients with an unidentified causative agent was calculated to be 57% (95% confidence interval, 36 to 86%). A substantial fraction of pneumococcal pneumonia patients do not elicit a serotype-specific immune response.

  14. Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine — The Method for the Effective Suppression of This Cross-Reactivity

    PubMed Central

    Strunin, Dmytro; Rosenberg, Ivan; Koberna, Karel

    2012-01-01

    5-Bromo-2′-deoxyuridine (BrdU) and 2′-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed. PMID:23272138

  15. Use of monoclonal antibodies in an epidemiological marker system: a retrospective study of lung specimens from the 1976 outbreak of Legionnaires disease in Philadelphia by indirect fluorescent-antibody and enzyme-linked immunosorbent assay methods.

    PubMed Central

    Brown, S L; Bibb, W F; McKinney, R M

    1985-01-01

    Autopsy specimens of lung tissues from 15 patients that contracted legionellosis during the 1976 Philadelphia outbreak of Legionnaires disease were examined for the presence of Legionella organisms and soluble antigens by indirect fluorescent-antibody (IFA) testing and by an enzyme-linked immunosorbent assay (ELISA) with both polyclonal and monoclonal antibodies. In all 15 cases, at least one specimen was positive for Legionella pneumophila serogroup 1 (Lp-1) antigens by a polyclonal antibody ELISA system. Of the 15 cases tested for Lp-1, 9 were positive by a polyclonal antibody IFA test. Nine mouse monoclonal antibodies to Lp-1 gave essentially the same reactivity pattern with extracts from lung tissue homogenates as that obtained with a Philadelphia 1 culture extract by using a monoclonal antibody ELISA system. The same monoclonal antibody panel gave similar results when used in the IFA system with tissue homogenates. Monoclonal antibodies can be used as epidemiological marker systems with both IFA and ELISA testing. This study provides evidence that the 1976 common source outbreak in Philadelphia was probably caused by a single Lp-1 strain. ELISA testing of the soluble antigen of Lp-1 from lung tissue homogenate supernatants was more sensitive than IFA testing of the homogenates and should be extremely useful as either a primary test or as an adjunct to fluorescent antibody testing for legionellosis. Images PMID:3881470

  16. From rabbit antibody repertoires to rabbit monoclonal antibodies

    PubMed Central

    Weber, Justus; Peng, Haiyong; Rader, Christoph

    2017-01-01

    In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies. PMID:28336958

  17. Electrode Mass Balancing as an Inexpensive and Simple Method to Increase the Capacitance of Electric Double-Layer Capacitors

    PubMed Central

    Andres, Britta; Engström, Ann-Christine; Blomquist, Nicklas; Forsberg, Sven; Dahlström, Christina; Olin, Håkan

    2016-01-01

    Symmetric electric double-layer capacitors (EDLCs) have equal masses of the same active material in both electrodes. However, having equal electrode masses may prevent the EDLC to have the largest possible specific capacitance if the sizes of the hydrated anions and cations in the electrolyte differ because the electrodes and the electrolyte may not be completely utilized. Here we demonstrate how this issue can be resolved by mass balancing. If the electrode masses are adjusted according to the size of the ions, one can easily increase an EDLC’s specific capacitance. To that end, we performed galvanostatic cycling to measure the capacitances of symmetric EDLCs with different electrode mass ratios using four aqueous electrolytes— Na2SO4, H2SO4, NaOH, and KOH (all with a concentration of 1 M)—and compared these to the theoretical optimal electrode mass ratio that we calculated using the sizes of the hydrated ions. Both the theoretical and experimental values revealed lower-than-1 optimal electrode ratios for all electrolytes except KOH. The largest increase in capacitance was obtained for EDLCs with NaOH as electrolyte. Specifically, we demonstrate an increase of the specific capacitance by 8.6% by adjusting the electrode mass ratio from 1 to 0.86. Our findings demonstrate that electrode mass balancing is a simple and inexpensive method to increase the capacitance of EDLCs. Furthermore, our results imply that one can reduce the amount of unused material in EDLCs and thus decrease their weight, volume and cost. PMID:27658253

  18. Investigation of Anti-Toxocara Antibodies in Epileptic Patients and Comparison of Two Methods: ELISA and Western Blotting

    PubMed Central

    Zibaei, Mohammad; Firoozeh, Farzaneh; Bahrami, Parviz; Sadjjadi, Seyed Mahmoud

    2013-01-01

    The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA) using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8%) and 3 (3.5%) persons exhibited Toxocara immunoglobulin G (IgG) antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (n = 85), 3 (3.5%) persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy. PMID:23710354

  19. Antibody-Mediated Rejection: A Review

    PubMed Central

    Garces, Jorge Carlos; Giusti, Sixto; Staffeld-Coit, Catherine; Bohorquez, Humberto; Cohen, Ari J.; Loss, George E.

    2017-01-01

    Background: Chronic antibody injury is a serious threat to allograft outcomes and is therefore the center of active research. In the continuum of allograft rejection, the development of antibodies plays a critical role. In recent years, an increased recognition of molecular and histologic changes has provided a better understanding of antibody-mediated rejection (AMR), as well as potential therapeutic interventions. However, several pathways are still unknown, which accounts for the lack of efficacy of some of the currently available agents that are used to treat rejection. Methods: We review the current diagnostic criteria for AMR; AMR paradigms; and desensitization, treatment, and prevention strategies. Results: Chronic antibody-mediated endothelial injury results in transplant glomerulopathy, manifested as glomerular basement membrane duplication, double contouring, or splitting. Clinical manifestations of AMR include proteinuria and a rise in serum creatinine. Current strategies for the treatment of AMR include antibody depletion with plasmapheresis (PLEX), immunoadsorption (IA), immunomodulation with intravenous immunoglobulin (IVIG), and T cell– or B cell–depleting agents. Some treatment benefits have been found in using PLEX and IA, and some small nonrandomized trials have identified some benefits in using rituximab and the proteasome inhibitor-based therapy bortezomib. More recent histologic follow-ups of patients treated with bortezomib have not shown significant benefits in terms of allograft outcomes. Furthermore, no specific treatment approaches have been approved by the US Food and Drug Administration. Other agents used for more difficult rejections include bortezomib and eculizumab (an anti-C5 monoclonal antibody). Conclusion: AMR is a fascinating field with ample opportunities for research and progress in the future. Despite the use of advanced techniques for the detection of human leukocyte antigen (HLA) or non-HLA donor-specific antibodies

  20. CEBPA-double-mutated acute myeloid leukemia displays a unique phenotypic profile: a reliable screening method and insight into biological features.

    PubMed

    Mannelli, Francesco; Ponziani, Vanessa; Bencini, Sara; Bonetti, Maria Ida; Benelli, Matteo; Cutini, Ilaria; Gianfaldoni, Giacomo; Scappini, Barbara; Pancani, Fabiana; Piccini, Matteo; Rondelli, Tommaso; Caporale, Roberto; Gelli, Anna Maria Grazia; Peruzzi, Benedetta; Chiarini, Marco; Borlenghi, Erika; Spinelli, Orietta; Giupponi, Damiano; Zanghì, Pamela; Bassan, Renato; Rambaldi, Alessandro; Rossi, Giuseppe; Bosi, Alberto

    2017-03-01

    Mutations in CCAAT/enhancer binding protein α (CEBPA) occur in 5-10% of cases of acute myeloid leukemia. CEBPA-double-mutated cases usually bear biallelic N- and C-terminal mutations and are associated with a favorable clinical outcome. Identification of CEBPA mutants is challenging because of the variety of mutations, intrinsic characteristics of the gene and technical issues. Several screening methods (fragment-length analysis, gene expression array) have been proposed especially for large-scale clinical use; although efficient, they are limited by specific concerns. We investigated the phenotypic profile of blast and maturing bone marrow cell compartments at diagnosis in 251 cases of acute myeloid leukemia. In this cohort, 16 (6.4%) patients had two CEBPA mutations, whereas ten (4.0%) had a single mutation. First, we highlighted that the CEBPA-double-mutated subset displays recurrent phenotypic abnormalities in all cell compartments. By mutational analysis after cell sorting, we demonstrated that this common phenotypic signature depends on CEBPA-double-mutated multi-lineage involvement. From a multidimensional study of phenotypic data, we developed a classifier including ten core and widely available parameters. The selected markers on blasts (CD34, CD117, CD7, CD15, CD65), neutrophil (SSC, CD64), monocytic (CD14, CD64) and erythroid (CD117) compartments were able to cluster CEBPA-double-mutated cases. In a validation set of 259 AML cases from three independent centers, our classifier showed excellent performance with 100% specificity and 100% sensitivity. We have, therefore, established a reliable screening method, based upon multidimensional analysis of widely available phenotypic parameters. This method provides early results and is suitable for large-scale detection of CEBPA-double-mutated status, allowing gene sequencing to be focused in selected cases.

  1. Microwave-assisted melt reaction method for the intercalation of carboxylic acid anions into layered double hydroxides.

    PubMed

    Rosa, Roberto; Leonelli, Cristina; Villa, Carla; Priarone, Giulia

    2013-01-01

    Carboxylic acid anions intercalated layered double hydroxides are currently gaining increasing interest due to their potential applications in pharmaceutical field for controlled drug release in novel tunable drug delivery systems. In this work different aliphatic carboxylic acid anions were intercalated into the interlayers of commercial as well as synthetically prepared layered double hydroxides, through a novel microwave mediated melt reaction approach. The volumetric nature of microwave dielectric heating was exploited in order to rapidly heat the intimate mixture of the lamellar inorganic precursor and the appropriate organic acid, at the melting temperature of the particular mono- or dicarboxylic acid used, reaching the intercalation in approximately two hours treatment.

  2. Precise characterization method of antibody-conjugated magnetic nanoparticles for pathogen detection using stuffer-free multiplex ligation-dependent probe amplification.

    PubMed

    Chung, Boram; Shin, Gi Won; Choi, Woong; Joo, Jinmyoung; Jeon, Sangmin; Jung, Gyoo Yeol

    2014-12-01

    Antibody-conjugated magnetic nanoparticles (Ab-MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab-MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab-MNPs as a pathogen detection method requires accurate characterization of Ab-MNP capture ability and standardization of all handling processes. In this study, we used high-resolution CE-single strand conformational polymorphism coupled with a stuffer-free multiplex ligation-dependent probe amplification system to characterize Ab-MNPs. The capture ability of Ab-MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab-MNPs was also assessed. The results showed that the stuffer-free multiplex ligation-dependent probe amplification system has the potential to serve as a standard characterization method for Ab-MNPs. Moreover, the precise characterization of Ab-MNPs facilitated robust pathogen detection in various applications.

  3. Mapping of change in cerebral glucose utilization using fluorine-18 fluorodeoxyglucose double injection and the constrained weighted-integration method

    SciTech Connect

    Murase, K. |; Kuwabara, Hiroto; Yasuhara, Yoshifumi; Evans, A.C.; Gjedde, A.

    1996-12-01

    The authors developed a method for mapping the change in cerebral glucose utilization at two different physiological states using [{sup 18}F]fluorodeoxyglucose (FDG) double injection and the constrained weighted-integration method. They studied young normal subjects without (baseline-baseline group, n = 5) and with (baseline-stimulation group, n = 5) vibrotactile stimulation of the fingertips of the right hand. Dynamic scans were performed using positron emission tomography (PET) following an initial dose (the first session, 0--30 min) and an additional dose (the second session, 30--60 min). The parametric images of the net clearance of FDG from blood to brain (K*), unidirectional blood-to-brain clearance (K*{sub 1}) and cerebral metabolic rate of glucose (CMR{sub glc}) of the two sessions were generated. The averaged subtraction (second minus first session) and t-statistic images were generated, which were rendered into Talairach`s sterotaxic coordinates and merged with the averaged magnetic resonance imaging (MRI) image. In the baseline-baseline group, regional K*, K*{sub 1}, and CMR{sub glc} in the first and second sessions were strongly correlated (r{sup 2} = 0.953, 0.935, and 0.951, respectively, n = 340). In the baseline-stimulation group, significant increases in these estimates were obtained in the contralateral primary somatosensory cortex (SI) (from 3.43 {+-} 0.78 to 4.02 {+-} 1.01 ml/100 g/min for K*, 7.85 {+-} 1.88 to 9.09 {+-} 1.71 ml/100 g/min for K*{sub 1}, and 28.0 {+-} 5.9 to 32.3 {+-} 5.5 {micro}mol/100 g/min for CMR{sub glc}), while there were no significant changes in the ipsilateral SI (from 3.45 {+-} 0.84 to 3.39 {+-} 0.72 ml/100 g/min for K*, 8.17 {+-} 2.33 to 8.37 {+-} 1.75 ml/100 g/min for K*{sub 1}, and 29.5 {+-} 8.1 to 29.1 {+-} 8.2 {micro}mol/100 g/min for CMR{sub glc}). Significant increases in K* and CMR{sub glc} in the contralateral SI were clearly demonstrated in the t-statistic image.

  4. [Mg/Al layered double hydroxides prepared by microwave-assisted co-precipitation method for the removal of bromate].

    PubMed

    Zhong, Qiong; Li, Huan

    2014-04-01

    In this paper, Mg/Al layered double hydroxides (Mg/Al LDHs) were prepared by the microwave-assisted co-precipitation method and the conventional co-precipitation method. The samples were labeled as Mg/Al LDHs-MW and Mg/Al LDHs-H, respectively. Mg/Al LDHs were characterized by X-ray diffractometer (XRD), Brunauer-Emmett-Teller (BET), scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR). The results showed that the application of microwave in the preparation process promoted the formation of smaller pore diameter and higher crystallinity particles. The pore size and particle size of Mg/Al LDHs-MW were 41.13 nm and 427.08 nm, respectively. Batch experiments were investigated to evaluate the effect of dosage, initial pH and regeneration frequencies for bromate removal. The conclusion showed that the process of bromate removal on Mg/Al LDHs could be described by the pseudo-second kinetic model. The Langmuir isotherm well described the experimental data, and the Mg/Al LDHs-MW has a stronger adsorption capacity while the maximum adsorption capacity (q(0)) of Mg/Al LDHs-MW for bromate was 321.26 microg x g(-1) which was larger than the q(0) (288.74 microg x g(-1)) of Mg/Al LDHs-H. For the continuous fixed-bed column, model simulations using the Thomas model showed that the experimental data obtained at three different columns packed with Mg/Al LDHs-MW were able to predict breakthrough curves. Simulating the maximum adsorption capacity of adsorption column for bromate removal was 288.81 microg x g(-1). When the bed depth was 10 cm, inlet concentration was 800 microg x L(-1) and flow rate was 4.0 mL x min, the correlation coefficient of model was 0.92, indicating that the experimental data was described well by the Thomas model.

  5. Antineutrophil cytoplasmic antibodies.

    PubMed

    Bosch, Xavier; Guilabert, Antonio; Font, Josep

    2006-07-29

    Much like other autoantibodies (eg, anti-double stranded DNA in systemic lupus erythematosus or antiglomerular basement membrane antibodies in Goodpasture's syndrome), antineutrophil cytoplasmic antibodies (ANCA) have provided doctors with a useful serological test to assist in diagnosis of small-vessel vasculitides, including Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and their localised forms (eg, pauci-immune necrotising and crescentic glomerulonephritis). 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. Present treatments for ANCA-associated vasculitis are not free from side-effects and as many as 50% of patients relapse within 5 years. Accurate understanding of the key pathogenic points of ANCA-associated vasculitis can undoubtedly provide a more rational therapeutic approach.

  6. A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection

    PubMed Central

    Birdsall, Robert E.; McCarthy, Sean M.; Janin-Bussat, Marie Claire; Perez, Michel; Haeuw, Jean-François; Chen, Weibin; Beck, Alain

    2016-01-01

    abstract Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows. PMID:26651262

  7. Combustion method for assay of biological materials labeled with carbon-14 or tritium, or double-labeled

    NASA Technical Reports Server (NTRS)

    Huebner, L. G.; Kisieleski, W. E.

    1969-01-01

    Dry catalytic combustion at high temperatures is used for assaying biological materials labeled carbon-14 and tritium, or double-labeled. A modified oxygen-flask technique is combined with standard vacuum-line techniques and includes convenience of direct in-vial collection of final combustion products, giving quantitative recovery of tritium and carbon-14.

  8. Double checking: a second look

    PubMed Central

    Chreim, Samia; Forster, Alan

    2015-01-01

    Abstract Rationale, aims and objectives Double checking is a standard practice in many areas of health care, notwithstanding the lack of evidence supporting its efficacy. We ask in this study: ‘How do front line practitioners conceptualize double checking? What are the weaknesses of double checking? What alternate views of double checking could render it a more robust process?’ Method This is part of a larger qualitative study based on 85 semi‐structured interviews of health care practitioners in general internal medicine and obstetrics and neonatology; thematic analysis of the transcribed interviews was undertaken. Inductive and deductive themes are reported. Results Weaknesses in the double checking process include inconsistent conceptualization of double checking, double (or more) checking as a costly and time‐consuming procedure, double checking trusted as an accepted and stand‐alone process, and double checking as preventing reporting of near misses. Alternate views of double checking that would render it a more robust process include recognizing that double checking requires training and a dedicated environment, Introducing automated double checking, and expanding double checking beyond error detection. These results are linked with the concepts of collective efficiency thoroughness trade off (ETTO), an in‐family approach, and resilience. Conclusion(s) Double checking deserves more questioning, as there are limitations to the process. Practitioners could view double checking through alternate lenses, and thus help strengthen this ubiquitous practice that is rarely challenged. PMID:26568537

  9. The use of the adding-doubling method for the optical optimization of planar luminescent down shifting layers for solar cells.

    PubMed

    Leyre, Sven; Cappelle, Jan; Durinck, Guy; Abass, Aimi; Hofkens, Johan; Deconinck, Geert; Hanselaer, Peter

    2014-05-05

    To enhance the efficiency of solar cells, a luminescent down shifting layer can be applied in order to adapt the solar spectrum to the spectral internal quantum efficiency of the semiconductor. Optimization of such luminescent down shifting layers benefits from quick and direct evaluation methods. In this paper, the potential of the adding-doubling method is investigated to simulate the optical behavior of an encapsulated solar cell including a planar luminescent down shifting layer. The results of the adding-doubling method are compared with traditional Monte Carlo ray tracing simulations. The average relative deviation is found to be less than 1.5% for the absorptance in the active layer and the reflectance from the encapsulated cell, while the computation time can be decreased with a factor 52. Furthermore, the adding-doubling method is adopted to investigate the suitability of the SrB4O7:5%Sm2 + ,5%Eu2 + phosphor as a luminescent down shifting material in combination with a Copper Indium Gallium Selenide solar cell. A maximum increase of 9.0% in the short-circuit current can be expected if precautions are taken to reduce the scattering by matching the refractive index of host material to the phosphor particles. To be useful as luminescent down shifting material, the minimal value of the quantum yield of the phosphor is determined to be 0.64.

  10. A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads.

    PubMed

    Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying

    2016-08-03

    A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%.

  11. Comparison of inguinal hernia repairs performed with lichtenstein, rutkow-robbins, and gilbert double layer graft methods.

    PubMed

    Karaca, A Serdar; Ersoy, Omer Faik; Ozkan, Namik; Yerdel, Mehmet Ali

    2015-02-01

    Tension-free repairs are performed commonly in inguinal hernia operations. The objective of the present study is to compare the outcomes of three different tension-free repair methods known as Lichtenstein, Rutkow-Robbins, and Gilbert double layer. One-hundred and fifty patients diagnosed with inguinal hernia were randomly split into three groups. The comparisons across groups were carried out in terms of operation length, postoperative pain, femoral vein flow velocity, early and late complications, recurrence rates, length of hospital stay, time required to return to work, and cost analysis. No difference was found between the groups regarding age, gender, type and classification of hernia, postoperative pain, and late complications (p > 0.05). Operation length was 53.70 ± 12.32 min in the Lichtenstein group, 44.29 ± 12.37 min in the Rutkow-Robbins group, and 45.21 ± 14.36 min in the Gilbert group (p < 0.05). Mean preoperative and postoperative femoral vein flow velocity values were 13.88 ± 2.237 and 13.42 ± 2.239 cm/s for Lichtenstein group, 12.64 ± 2.98 and 12.16 ± 2.736 cm/s for Rutkow-Robbins group, and 16.02 ± 3.19 and 15.52 ± 3.358 cm/s for the Gilbert group, respectively. Statistical difference was found between all the groups (p < 0.001). However, no difference was determined between the groups regarding the decrease rates (p = 0.977). Among early complications, hematoma was observed in one (2 %) patient of Lichtenstein group, five (10 %) patients of Rutkow-Robbins group, and three (6 %) patients of Gilbert group (p = 0.033). Cost analysis produced the following results for Lichtenstein, Rutkow-Robbins, and Gilbert groups: US $157.94 ± 50.05, $481.57 ± 11.32, and $501.51 ± 73.59, respectively (p < 0.001). Lichtenstein operation was found to be more advantageous compared with the other techniques in terms of cost analysis as well as having unaffected femoral blood

  12. Generalized amplitude-phase retrieval algorithm attack on 'double images encryption method with resistance against the special attack based on an asymmetric algorithm'

    NASA Astrophysics Data System (ADS)

    Zhang, Chenggong; He, Wenqi; Cai, Zewei; Peng, Xiang

    2016-09-01

    A generalized amplitude-phase retrieval algorithm (GAPRA) attack on `double images encryption method with resistance against the special attack based on an asymmetric algorithm' (DIEM) is presented in this paper. The analysis shows that the DIEM is a cascaded cryptosystem, which consist of a joint transform correlator architecture and a phasetruncated Fourier transform scheme. A GAPRA attack is proposed and the potential risk of the cascaded cryptosystems is discussed. By using our method, an attacker could crack high-quality results of the plaintexts. A set of simulation results demonstrate the validity and feasibility of the proposed method.

  13. A novel method for dengue virus detection and antibody screening using a graphene-polymer based electrochemical biosensor.

    PubMed

    Navakul, Krongkaew; Warakulwit, Chompunuch; Yenchitsomanus, Pa-Thai; Panya, Aussara; Lieberzeit, Peter A; Sangma, Chak

    2017-02-01

    Dengue fever is a major disease that kills many people in the developing world every year. During early infection, a patient displays a high temperature without other signs. After this stage, and without proper treatment, serious damage to internal organs can happen, which occasionally leads to death. A rapid technique for the early detection of dengue virus (DENV) could reduce the number of fatalities. This study presents a new technique for the detection, classification and antibody screening of DENV based on electrochemical impedance spectroscopy (EIS). We found that the charge transfer resistance (Rct) of a gold electrode coated with graphene oxide reinforced polymer was influenced by virus type and quantity exposed on the surface. Molecular recognition capability established during the GO-polymer composite preparation was used to explain this observation. The linear dependence of Rct versus virus concentrations ranged from 1 to 2×10(3)pfu/mL DENV with a 0.12 pfu/mL detection limit.

  14. Autologous antibodies that bind neuroblastoma cells.

    PubMed

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  15. Voltage—clamp analysis of the early current in frog skeletal muscle fibre using the double sucrose-gap method

    PubMed Central

    Ildefonse, Michèle; Rougier, Oger

    1972-01-01

    1. The early membrane currents in skeletal muscle fibres have been studied with the double sucrose-gap method, according to the experimental procedure used for the study of the giant axon of the squid. 2. The earliest observable current is a capacitative transient. An equivalent circuit with two capacity-resistance systems can account for such a current. The components of the electrical circuit are estimated from the records. 3. The capacitative transient current is followed by an initial current, the time and voltage dependence of which are analogous to those of nerve fibres. During this phase of current, the membrane behaves like a sodium electrode for modifications of the external sodium concentration. 4. Using tetrodotoxin, a specific inhibitor of the sodium permeability, it is possible to separate two current components, (i) a transient component, suppressed by tetrodotoxin, which reflects the modifications, with time and voltage, of the membrane permeability towards sodium ions; the larger the depolarization, the shorter the time to reach peak current. This current is inward for depolarizations (V) of about +30 mV to about +130 mV (VNa). There was some variation in these values between individual fibres. Beyond VNa, the current is outward. This current shows a small rectification — inward going — for high depolarizations which is comparable to the behaviour of the Ranvier node membrane. (ii) A quasi-instantaneous outward component, only voltage dependent. The current—voltage relation of this current shows an outward going rectification for high depolarizations. This current can be considered as a `leak' current, in which chloride ions could play an important role. 5. A sodium tail current is observed when the imposed potential fall occurs during the time course of the initial current. It corresponds to the deactivation of the sodium current previously activated. Because of the large contemporary capacitative currents, it is difficult to measure

  16. Technology Review of Nondestructive Methods for Examination of Water Intrusion Areas on Hanford’s Double-Shell Waste Tanks

    SciTech Connect

    Watkins, Michael L.; Pardini, Allan F.

    2008-05-09

    Under a contract with CH2M Hill Hanford Group, Inc., PNNL has performed a review of the NDE technology and methods for examination of the concrete dome structure of Hanford’s double-shell tanks. The objective was to provide a matrix of methodologies that could be evaluated based on applicability, ease of deployment, and results that could provide information that could be used in the ongoing structural analysis of the tank dome. PNNL performed a technology evaluation with the objective of providing a critical literature review for all applicable technologies based on constraints provided by CH2M HILL. These constraints were not mandatory, but were desired. These constraints included performing the evaluation without removing any soil from the top of the tank, or if necessary, requesting that the hole diameter needed to gain access to evaluate the top of the tank structure to be no greater than approximately 12-in. in diameter. PNNL did not address the details of statistical sampling requirements as they depend on an unspecified risk tolerance. PNNL considered these during the technology evaluation and have reported the results in the remainder of this document. Many of the basic approaches to concrete inspection that were reviewed in previous efforts are still in use. These include electromagnetic, acoustic, radiographic, etc. The primary improvements in these tools have focused on providing quantitative image reconstruction, thus providing inspectors and analysts with three-dimensional data sets that allow for operator visualization of relevant abnormalities and analytical integration into structural performance models. Available instruments, such as radar used for bridge deck inspections, rely on post-processing algorithms and do not provide real-time visualization. Commercially available equipment only provides qualitative indications of relative concrete damage. It cannot be used as direct input for structural analysis to assess fitness for use and if

  17. [Comparison of the methenamine silver staining, direct fluorescent antibody and nested-polymerase chain reaction methods in the diagnosis of Pneumocystis carinii pneumonia].

    PubMed

    Güneş, Ilkay; Kalkanci, Ayşe; Kuştimur, Semra; Ergüven, Sibel; Ozet, Gülsüm; Ekim, Numan

    2004-01-01

    Pneumocystis carinii is one of the most common causative agents of pneumonia in immunocompromised patients, but the problems in the laboratory diagnosis of the disease frequently leads to diagnosis according to the response to medical treatment. In this study, the presence of P. carinii was investigated in immunocompromised patients who were presenting with the clinical symptoms of atypical pneumonia, by Gomori methenamine silver staining (GMS), direct fluorescent antibody (DFA) test and nested-polymerase chain reaction (nPCR) methods. Fifty-three samples of 49 patients were included in the study. Twelve of the samples (22.6%) were found to be positive by nPCR, 6 of them (11.3%) were found to be positive by DFA, while only one of them (1.8%) was positive by GMS staining method. As a result, for the appropriate treatment and prophylaxis of P. carinii infections, PCR which is a rapid and reliable diagnostic test should be used for diagnosis.

  18. Molecular-specific urokinase antibodies

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  19. A novel method for the detection of antibodies to adalimumab in the presence of drug reveals "hidden" immunogenicity in rheumatoid arthritis patients.

    PubMed

    van Schouwenburg, Pauline A; Bartelds, Geertje M; Hart, Margreet H; Aarden, Lucien; Wolbink, Gerrit Jan; Wouters, Diana

    2010-10-31

    Production of anti drug antibodies (ADA) in adalimumab treated RA patients is associated with reduced serum adalimumab levels and less clinical response. However, most current assays to measure ADA are unable to detect ADA in complex with adalimumab. Thus, ADA is only measured if antibody production exceeds drug levels in the serum, meaning that ADA formation is underestimated. The aim of this study is to develop a method to detect ADA in the presence of drug. A pH-shift-anti-idiotype Antigen binding test (PIA) was used to enable ADA measurement in the presence of adalimumab. ADA-adalimumab complexes were dissociated by acid treatment and addition of excess rabbit anti-idiotype-F(ab) before neutralization. Rabbit anti-idiotype-F(ab) blocks reformation of ADA-drug complexes by competing with patient ADA for adalimumab binding. Released ADA are measured by an antigen binding test (ABT). The PIA enabled detection of ADA in the presence of large excess of adalimumab and was used to measure ADA in 30 adalimumab treated rheumatoid arthritis (RA) patients during the first 28 weeks of treatment. It revealed ADA in 21 out of 30 tested patients, while the ABT detected ADA in only 5 patients. Indicating that an immunogenic reaction towards adalimumab is present in the majority of adalimumab treated patients.

  20. Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies

    PubMed Central

    2013-01-01

    Background Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. Methods Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. Results Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not

  1. [Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents].

    PubMed

    Polat, Ceylan; Karataş, Ahmet; Sözen, Mustafa; Matur, Ferhat; Abacıoğlu, Hakan; Öktem, Mehmet Ali

    2016-04-01

    Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1

  2. A Facile Method to Prepare Double-Layer Isoporous Hollow Fiber Membrane by In Situ Hydrogen Bond Formation in the Spinning Line.

    PubMed

    Noor, Nazia; Koll, Joachim; Radjabian, Maryam; Abetz, Clarissa; Abetz, Volker

    2016-03-01

    A double-layer hollow fiber is fabricated where an isoporous surface of polystyrene-block-poly(4-vinylpyridine) is fixed on a support layer by co-extrusion. Due to the sulfonation of the support layer material, delamination of the two layers is suppressed without increasing the number of subsequent processing steps for isoporous composite membrane formation. Electron microscope-energy-dispersive X-ray spectroscopy images unveil the existence of a high sulfur concentration in the interfacial region by which in-process H-bond formation between the layers is evidenced. For the very first time, our study reports a facile method to fabricate a sturdy isoporous double-layer hollow fiber.

  3. Ixekizumab, an interleukin-17A specific monoclonal antibody, for the treatment of biologic-naive patients with active psoriatic arthritis: results from the 24-week randomised, double-blind, placebo-controlled and active (adalimumab)-controlled period of the phase III trial SPIRIT-P1

    PubMed Central

    Mease, Philip J; Ritchlin, Christopher T; Okada, Masato; Cuchacovich, Raquel S; Shuler, Catherine L; Lin, Chen-Yen; Braun, Daniel K; Lee, Chin H; Gladman, Dafna D

    2017-01-01

    Objective To assess the safety and efficacy of ixekizumab, a monoclonal antibody that inhibits interleukin-17A, in a double-blind phase III trial enrolling patients with active psoriatic arthritis (PsA). Methods Patients naive to biologic therapy with active PsA were randomised to subcutaneous injections of placebo (N=106), adalimumab 40 mg once every 2 weeks (active reference; N=101), ixekizumab 80 mg once every 2 weeks (IXEQ2W) (N=103), or ixekizumab 80 mg once every 4 weeks (IXEQ4W) (N=107). Both ixekizumab regimens included a 160-mg starting dose. The primary objective was to assess the superiority of IXEQ2W or IXEQ4W versus placebo as measured by the proportion of patients achieving an American College of Rheumatology 20 (ACR20) response at week 24. Results Significantly more patients treated with ixekizumab achieved an ACR20 response with IXEQ2W (62.1%) or IXEQ4W (57.9%) than placebo (30.2%) (p≤0.001; non-responder imputation method). Disease activity and functional disability were significantly improved with both ixekizumab doses versus placebo at weeks 12 and 24, and there was significantly less progression of structural damage at week 24 (p≤0.01). Clearance of plaque psoriasis was greater with ixekizumab than placebo (p≤0.001). Efficacy results with adalimumab, the active reference arm, showed significant improvements versus placebo. Treatment-emergent adverse events were more frequent with ixekizumab (65.7–66.4%) and adalimumab (64.4%) than placebo (47.2%) (p<0.05). Conclusions In biologic-naive patients with active PsA, ixekizumab treatment resulted in improvements in disease activity and physical function, as well as in the inhibition of structural damage progression. Overall, adverse events were more frequent in all active groups compared with placebo. Trial registration number NCT01695239; EudraCT2011-002326-49; Results. PMID:27553214

  4. Antibody Engineering and Therapeutics

    PubMed Central

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  5. Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein.

    PubMed Central

    Bölin, I; Lönroth, H; Svennerholm, A M

    1995-01-01

    Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished. PMID:7714196

  6. Study of Method for Designing the Power and the Capacitance of Fuel Cells and Electric Double-Layer Capacitors of Hybrid Railway Vehicle

    NASA Astrophysics Data System (ADS)

    Takizawa, Kenji; Kondo, Keiichiro

    A hybrid railway traction system with fuel cells (FCs) and electric double layer-capacitors (EDLCs) is discussed in this paper. This system can save FC costs and absorb the regenerative energy. A method for designing FCs and EDLCs on the basis of the output power and capacitance, respectively, has not been reported, even though their design is one of the most important technical issues encountered in the design of hybrid railway vehicles. Such design method is presented along with a train load profile and an energy management strategy. The design results obtained using the proposed method are verified by performing numerical simulations of a running train. These results reveal that the proposed method for designing the EDLCs and FCs on the basis of the capacitance and power, respectively, and by using a method for controlling the EDLC voltage is sufficiently effective in designing efficient EDLCs and FCs of hybrid railway traction systems.

  7. Development and evaluation of an antibody ELISA for sarcoptic mange in sheep and a comparison with the skin-scraping method.

    PubMed

    Rodríguez-Cadenas, F; Carbajal-González, M T; Fregeneda-Grandes, J M; Aller-Gancedo, J M; Huntley, J F; Rojo-Vázquez, F A

    2010-08-01

    In this work an indirect ELISA for detecting serum-specific IgG antibodies in sheep was developed using a crude saline extract from Sarcoptes scabiei var. ovis mites and then the repeatability of the ELISA outcomes was estimated. Subsequently, its diagnostic accuracy was evaluated by Receiver Operating Characteristics (ROC) analysis using a sample collected from the entire sheep population of western Castile and Leon region in Spain, and then compared with that of the skin-scraping method. The reference method used was a combination of clinical examination, skin-scraping analysis and epidemiological surveys, but it introduced selection and probably information biases. Furthermore, we attempted to identify biological factors useful to predict the sensitivity or specificity of the ELISA as determined by comparison with the reference method. Additionally, conventional latent-class analysis [Hui, S.L., Walter, S.D., 1980. Estimating the error rates of diagnostic tests. Biometrics 36, 167-171] was also used to estimate accuracy parameters. The between-run coefficient of variation (CV) for a standard serum was 8.8% and the within-run CV 4.3%. No significant deviation between the OD% means and strength positive correlation between the OD% values (r=0.98) were found for the results from two different batches of antigen. When compared to the reference method, the Area Under the ROC curve (AUC) for the reference population was 0.967 (95% CI: 0.949-0.985) for the ELISA and 0.915 (95% CI: 0.863-0.968) for the skin-scraping method. By logistic regression analysis, one explanatory biological factor-result to the skin-scraping method-and four explanatory biological factors-Tyroglyphidae individual status, Trichophyton verrucosum individual status, Oestrus ovis status of the flock and presence of adjacent animals with a clinical disease neighbour to S. scabiei infection-were found for diagnostic sensitivity and specificity of the ELISA, respectively, although this depended on the

  8. Investigating the quantitative structure-activity relationships for antibody recognition of two immunoassays for polycyclic aromatic hydrocarbons by multiple regression methods.

    PubMed

    Zhang, Yan-Feng; Zhang, Li; Gao, Zhi-Xian; Dai, Shu-Gui

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants found in the environment. Immunoassays represent useful analytical methods to complement traditional analytical procedures for PAHs. Cross-reactivity (CR) is a very useful character to evaluate the extent of cross-reaction of a cross-reactant in immunoreactions and immunoassays. The quantitative relationships between the molecular properties and the CR of PAHs were established by stepwise multiple linear regression, principal component regression and partial least square regression, using the data of two commercial enzyme-linked immunosorbent assay (ELISA) kits. The objective is to find the most important molecular properties that affect the CR, and predict the CR by multiple regression methods. The results show that the physicochemical, electronic and topological properties of the PAH molecules have an integrated effect on the CR properties for the two ELISAs, among which molar solubility (S(m)) and valence molecular connectivity index ((3)χ(v)) are the most important factors. The obtained regression equations for Ris(C) kit are all statistically significant (p < 0.005) and show satisfactory ability for predicting CR values, while equations for RaPID kit are all not significant (p > 0.05) and not suitable for predicting. It is probably because that the Ris(C) immunoassay employs a monoclonal antibody, while the RaPID kit is based on polyclonal antibody. Considering the important effect of solubility on the CR values, cross-reaction potential (CRP) is calculated and used as a complement of CR for evaluation of cross-reactions in immunoassays. Only the compounds with both high CR and high CRP can cause intense cross-reactions in immunoassays.

  9. Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody.

    PubMed

    Viski, Kornél; Gengeliczki, Zsolt; Lenkey, Krisztián; Baranyáné Ganzler, Katalin

    2016-10-01

    Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.

  10. Drug delivery system for an anticancer agent, chlorogenate-Zn/Al-layered double hydroxide nanohybrid synthesised using direct co-precipitation and ion exchange methods

    SciTech Connect

    Barahuie, Farahnaz; Hussein, Mohd Zobir; Arulselvan, Palanisamy; Fakurazi, Sharida; Zainal, Zulkarnain

    2014-09-15

    A nano-structured drug-inorganic clay hybrid involving an active anticancer compound, which is chlorogenic acid (CA) intercalated into Zn/Al-layered double hydroxide, has been assembled via ion-exchange and co-precipitation methods to form a nanohybrid CZAE (a chlorogenic acid-Zn/Al nanohybrid synthesised using an ion-exchange method) and CZAC (a chlorogenic acid-Zn/Al nanohybrid synthesised using a direct method), respectively. The X-ray diffraction (XRD) results confirmed that the CA-LDH had a hybrid structure in which the anionic chlorogenate is arranged between the interlayers as a horizontal monolayer at 90 and 20° angles from the x axis for CZAE and CZAC, respectively. Both nanohybrids have the properties of mesoporous materials. The high loading percentage of chlorogenic acid (approximately 43.2% for CZAE and 45.3% for CZAC) with basal spacings of 11.7 and 12.6 Å for CZAE and CZAC, respectively, corroborates the successful intercalation of chlorogenic acid into the interlayer gallery of layered double hydroxides. Free chlorogenic acid and the synthesised nanocomposites (CZAE, CZAC) were assessed for their cytotoxicity against various cancer cells. The Fourier transform infrared data supported the formation of both nanohybrids, and a thermal analysis showed that the nanohybrids are more thermally stable than their counterparts. The chlorogenate shows a sustained release, and the release rate of chlorogenate from CZAE and CZAC nanohybrids at pH 7.4 is remarkably lower than that at pH 4.8 due to their different release mechanisms. The release rate of chlorogenate from both nanohybrids can be described as pseudo-second order. The present investigation revealed the potential of the nanohybrids to enhance the in vitro anti-tumour effect of chlorogenic acid in liver and lung cancer cells in vitro. - Highlights: • We intercalated chlorogenic into Zn/Al-layered double hydroxide by ion-exchange and coprecipitation methods. • The two methods gave nanocomposites

  11. Antibody Engineering for Pursuing a Healthier Future

    PubMed Central

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

  12. Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

    PubMed

    Sequeira, Patrícia Carvalho de; Fonseca, Leila de Souza; Silva, Marlei Gomes da; Saad, Maria Helena Féres

    2005-11-01

    Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

  13. Emerging double helical nanostructures.

    PubMed

    Zhao, Meng-Qiang; Zhang, Qiang; Tian, Gui-Li; Wei, Fei

    2014-08-21

    As one of the most important and land-mark structures found in nature, a double helix consists of two congruent single helices with the same axis or a translation along the axis. This double helical structure renders the deoxyribonucleic acid (DNA) the crucial biomolecule in evolution and metabolism. DNA-like double helical nanostructures are probably the most fantastic yet ubiquitous geometry at the nanoscale level, which are expected to exhibit exceptional and even rather different properties due to the unique organization of the two single helices and their synergistic effect. The organization of nanomaterials into double helical structures is an emerging hot topic for nanomaterials science due to their promising exceptional unique properties and applications. This review focuses on the state-of-the-art research progress for the fabrication of double-helical nanostructures based on 'bottom-up' and 'top-down' strategies. The relevant nanoscale, mesoscale, and macroscopic scale fabrication methods, as well as the properties of the double helical nanostructures are included. Critical perspectives are devoted to the synthesis principles and potential applications in this emerging research area. A multidisciplinary approach from the scope of nanoscience, physics, chemistry, materials, engineering, and other application areas is still required to the well-controlled and large-scale synthesis, mechanism, property, and application exploration of double helical nanostructures.

  14. HANFORD DOUBLE SHELL TANK THERMAL AND SEISMIC PROJECT BUCKLING EVALUATION METHODS AND RESULTS FOR THE PRIMARY TANKS

    SciTech Connect

    MACKEY TC; JOHNSON KI; DEIBLER JE; PILLI SP; RINKER MW; KARRI NK

    2009-01-14

    This report documents a detailed buckling evaluation of the primary tanks in the Hanford double-shell waste tanks (DSTs), which is part of a comprehensive structural review for the Double-Shell Tank Integrity Project. This work also provides information on tank integrity that specifically responds to concerns raised by the Office of Environment, Safety, and Health (ES&H) Oversight (EH-22) during a review of work performed on the double-shell tank farms and the operation of the aging waste facility (AWF) primary tank ventilation system. The current buckling review focuses on the following tasks: (1) Evaluate the potential for progressive anchor bolt failure and the appropriateness of the safety factors that were used for evaluating local and global buckling. The analysis will specifically answer the following questions: (a) Can the EH-22 scenario develop if the vacuum is limited to -6.6-inch water gage (w.g.) by a relief valve? (b) What is the appropriate factor of safety required to protect against buckling if the EH-22 scenario can develop? (c) What is the appropriate factor of safety required to protect against buckling if the EH-22 scenario cannot develop? (2) Develop influence functions to estimate the axial stresses in the primary tanks for all reasonable combinations of tank loads based on detailed finite element analysis. The analysis must account for the variation in design details and operating conditions between the different DSTs. The analysis must also address the imperfection sensitivity of the primary tank to buckling. (3) Perform a detailed buckling analysis to determine the maximum allowable differential pressure for each of the DST primary tanks at the current specified limits on waste temperature, height, and specific gravity. Based on the concrete anchor bolt loads analysis and the small deformations that are predicted at the unfactored limits on vacuum and axial loads, it is very unlikely that the EH-22 scenario (i.e., progressive anchor bolt

  15. Reactions of organoaluminum compounds with acetylene as a method for the synthesis of aliphatic derivatives with a z-disubstituted double bond

    SciTech Connect

    Andreeva, N.I.; Kuchin, A.V.; Tolstikov, G.A.

    1985-11-01

    This paper develops a method for the synthesis of aliphatic compounds with a Z-disubstituted double bond, which are important synthons for the preparation of such natural products as insect pheromones, aromatic principles, etc. In the carbalumination reaction of acetylene Z-alkenyldialkylaluminums are formed selectively. A-Alkenyldialkylaluminums are highly reactive and can readily be converted into Z-allyl alcohols and their ethers, and into Z-iodovinyl derivatives. By the reactions of vinyl organoaluminum compounds with the complex CH/sub 3/COClhaAlCl/sub 3/ E-conjugated ketones were obtained.

  16. Encephalitis and GABAB receptor antibodies

    PubMed Central

    Höftberger, Romana; Titulaer, Maarten J.; Sabater, Lidia; Dome, Balazs; Rózsás, Anita; Hegedus, Balazs; Hoda, Mir Alireza; Laszlo, Viktoria; Ankersmit, Hendrik Jan; Harms, Lutz; Boyero, Sabas; de Felipe, Alicia; Saiz, Albert; Dalmau, Josep

    2013-01-01

    Objective: To report the clinical features of 20 newly diagnosed patients with GABAB receptor (GABABR) antibodies and determine the frequency of associated tumors and concurrent neuronal autoantibodies. Methods: Clinical data were retrospectively obtained and evaluated. Serum and CSF samples were examined for additional antibodies using methods previously reported. Results: Seventeen patients presented with seizures, memory loss, and confusion, compatible with limbic encephalitis (LE), one patient presented with ataxia, one patient presented with status epilepticus, and one patient presented with opsoclonus-myoclonus syndrome (OMS). Nineteen (95%) patients eventually developed LE during the course of the disease. Small-cell lung cancer (SCLC) was identified in 10 (50%) patients, all with LE. Treatment and outcome was available from 19 patients: 15 showed complete (n = 7) or partial (n = 8) neurologic improvement after steroids, IV immunoglobulins, or plasma exchange and oncologic treatment when indicated; 1 patient died of tumor progression shortly after the first cycle of immunotherapy, and 3 were not treated. Five patients with SCLC had additional onconeuronal antibodies (Ri, amphiphysin, or SOX1), and 2 without tumor had GAD65 and NMDAR antibodies, respectively. GABABR antibodies were not detected in serum of 116 patients with SCLC without neurologic symptoms. Conclusion: Our study confirms GABABR as an autoantigen of paraneoplastic and nonparaneoplastic LE and expands the phenotype of GABABR antibodies to ataxia, OMS, and status epilepticus. The long-term prognosis is dictated by the presence of a tumor. Recognition of syndromes associated with GABABR antibodies is important because they usually respond to treatment. PMID:24068784

  17. The detection and discovery of glycan motifs in biological samples using lectins and antibodies: new methods and opportunities

    PubMed Central

    Tang, Huiyuan; Hsueh, Peter; Kletter, Doron; Bern, Marshall; Haab, Brian

    2015-01-01

    Recent research is uncovering unexpected ways that glycans contribute to biology, as well as new strategies for combatting disease using approaches involving glycans. To make full use of glycans for clinical applications, we need more detailed information on the location, nature, and dynamics of glycan expression in vivo. Such studies require the use of specimens acquired directly from patients. Effective studies of clinical specimens require low-volume assays, high precision measurements, and the ability to process many samples. Assays using affinity reagents—lectins and glycan-binding antibodies—can meet these requirements, but further developments are needed to make the methods routine and effective. Recent advances in the use of glycan-binding proteins could meet that need. The advances involve improved determination of specificity using glycan arrays; the availability of databases for mining and analyzing glycan array data; lectin engineering methods; and the ability to quantitatively interpret lectin measurements. Here we describe many of the challenges and opportunities involved in the application of these new approaches to the study of biological samples. The new tools hold promise for developing methods to improve the outcomes of patients afflicted with diseases characterized by aberrant glycan expression. PMID:25727148

  18. Rapid calculation of diffuse reflectance from a multilayered model by combination of the white Monte Carlo and adding-doubling methods

    PubMed Central

    Yoshida, Kenichiro; Nishidate, Izumi

    2014-01-01

    To rapidly derive a result for diffuse reflectance from a multilayered model that is equivalent to that of a Monte-Carlo simulation (MCS), we propose a combination of a layered white MCS and the adding-doubling method. For slabs with various scattering coefficients assuming a certain anisotropy factor and without absorption, we calculate the transition matrices for light flow with respect to the incident and exit angles. From this series of precalculated transition matrices, we can calculate the transition matrices for the multilayered model with the specific anisotropy factor. The relative errors of the results of this method compared to a conventional MCS were less than 1%. We successfully used this method to estimate the chromophore concentration from the reflectance spectrum of a numerical model of skin and in vivo human skin tissue. PMID:25426319

  19. A double-blind evaluation of the activity of an anti-cellulite product containing retinol, caffeine, and ruscogenine by a combination of several non-invasive methods.

    PubMed

    Bertin, C; Zunino, H; Pittet, J C; Beau, P; Pineau, P; Massonneau, M; Robert, C; Hopkins, J

    2001-01-01

    A double-blind, randomized, placebo-controlled study was conducted with 46 healthy female volunteers in order to test an anti-cellulite product containing retinol, caffeine and ruscogenine. An evaluation of different parameters related to cellulite appearance, i.e., the skin macrorelief, the dermal and hypodermal structures, the skin mechanical characteristics, and the cutaneous flowmetry was assessed using several non-invasive methods. This combination of different evaluation methods resulted in the demonstration of significant activity of the anti-cellulite product versus baseline and showed its superiority versus the placebo in skin macrorelief (decrease of the "orange peel" effect) and an increase in cutaneous microcirculation. By using a combination of methods, it was possible to detail the activity of an anti-cellulite product and to show superiority of the product in comparison with the placebo.

  20. NEACRP standard problem exercise on criticality codes for dissolving fissile oxides in acids: A reference method for treating the fuel double heterogeneity

    SciTech Connect

    Santamarina, A.; Smith, H.J. . Div. d'Etudes et de Developpement des Reacteurs); Whitesides, G.E. )

    1990-01-01

    The value of international comparison studies by the OECD-NEA Criticality Working Group has again been demonstrated by this study. Computational methods that had been commonly used for criticality safety calculations and which were shown to be valid for systems for which experimental data existed, were demonstrated to be inadequate when extrapolated to some simulated actual situations. The major source of dispersion in the results in international criticality benchmark calculations on problems treating a fuel double heterogeneity is shown to be the incorrect evaluation of effective resonance cross sections for 238U. A reference calculational method is proposed and used to evaluate theoretically the errors created by various standard methods of calculating the effects of self-shielding of resonance cross sections. 7 figs., 3 tabs.

  1. Serum herpes simplex antibodies

    MedlinePlus

    ... gov/ency/article/003352.htm Serum herpes simplex antibodies To use the sharing features on this page, please enable JavaScript. Serum herpes simplex antibodies is a blood test that looks for antibodies ...

  2. Antibodies specific for nucleic acids and applications in genomic detection and clinical diagnostics.

    PubMed

    Hu, Zonglin; Leppla, Stephen H; Li, Baoguang; Elkins, Christopher A

    2014-09-01

    Detection of nucleic acids using antibodies is uncommon. This is in part because nucleic acids are poor immunogens and it is difficult to elicit antibodies having high affinity to each type of nucleic acid while lacking cross-reactivity to others. We describe the origins and applications of a variety of anti-nucleic acid antibodies, including ones reacting with modified nucleosides and nucleotides, single-stranded DNA, double-stranded DNA, RNA, DNA:RNA hybrids, locked-nucleic acids or peptide nucleic acid:nucleic acid hybrids. Carefully selected antibodies can be excellent reagents for detecting bacteria, viruses, small RNAs, microRNAs, R-loops, cancer cells, stem cells, apoptotic cells and so on. The detection may be sensitive, simple, rapid, specific, reproducible, quantitative and cost-effective. Current microarray and diagnostic methods that depend on cDNA or cRNA can be replaced by using antibody detection of nucleic acids. Therefore, development should be encouraged to explore new utilities and create a robust arsenal of new anti-nucleic acid antibodies.

  3. [Double responses].

    PubMed

    Motté, G; Dinanian, S; Sebag, C; Drieu, L; Slama, M

    1995-12-01

    Double response is a rare electrocardiographic phenomenon requiring two atrioventricular conduction pathways with very different electrophysiological properties. Double ventricular responses are the usual manifestation: an atrial depolarisation (spontaneous or provoked, anticipated or not) is followed by a first ventricular response dependent on an accessory pathway or a rapid nodal pathway and then a second response resulting from sufficiently delayed transmission through a nodal pathway for the ventricles to have recovered their excitability when the second wave of activation reaches them. A simple curiosity when isolated and occurring under unusual conditions, particularly during electrophysiological investigation of the Wolff-Parkinson-White syndrome, the double response may initiate symptomatic non-reentrant junctional tachycardia when associated with nodal duality and repeating from atria in sinus rhythm. The functional incapacity and resistance to antiarrhythmic therapy may require referral for ablation of the slow pathway.

  4. Preparation of Nickel-Aluminum-Containing Layered Double Hydroxide Films by Secondary (Seeded) Growth Method and Their Electrochemical Properties.

    PubMed

    Zhang, Fazhi; Guo, Li; Xu, Sailong; Zhang, Rong

    2015-06-23

    Thin films of nickel-aluminum-containing layered double hydroxide (NiAl-LDH) have been prepared on nickel foil and nickel foam substrates by secondary (seeded) growth of NiAl-LDH seed layer. The preparation procedure consists of deposition of LDH seeds from a colloidal suspension on the substrate by dip coating, followed by hydrothermal treatment of the nanocrystals to form the LDH film. The secondary grown film is found to provide a higher crystallinity and more uniform composition of metal cations in the film layer than the in situ grown film on seed-free substrate under identical hydrothermal conditions. A systematic investigation of the film evolution process reveals that the crystallite growth rate is relatively fast for the secondary grown film because of the presence of LDH nanocrystal seeds. Electrochemical performance of the resulting NiAl-LDH films as positive electrode material was further assessed as an example of their practical applications. The secondary grown film electrode delivers improved recharge-discharge capacity and cycling stability compared with that of the in situ grown film, which can be explained by the existence of a unique microstructure of the former. Our findings show an example for the effective fabrication of LDH film with controllable microstructure and enhanced application performance through a secondary (seeded) growth procedure.

  5. Shock sensitivity of a double-base propellant as a function of age, processing method, and size

    NASA Astrophysics Data System (ADS)

    Sandusky, H. W.

    2017-01-01

    Shock sensitivity of a double-base propellant (nitroglycerin and nitrocellulose with smaller quantities of other ingredients) was compared for fresh and aged samples. Because the available amount of propellant was less than that needed for extrusion, most samples were pressed. The large-scale gap test (LSGT) was consistent for 240-g samples of fresh propellant whether extruded as one piece or 6-mm pieces pressed together directly into the confinement tube. The latter had the same density but with microscopic gaps between pieces and nitrocellulose fibers randomly oriented instead of being aligned with the extrusion axis. In a further reduction of the amount of required propellant, 11-g samples pressed from 3-mm pieces had the same critical shock pressure in the Insensitive High Explosive gap test (IHEGT), which has a LSGT donor and replicates its confinement. There was just enough propellant aged for 13 and 16 years to press several IHEGT samples each. The critical shock pressure was somewhat lower for aged propellant. These results demonstrate how smaller tests can mimic results in larger ones upon proper consideration of shock, detonation, and material science.

  6. Quantum dots-based double imaging combined with organic dye imaging to establish an automatic computerized method for cancer Ki67 measurement

    NASA Astrophysics Data System (ADS)

    Wang, Lin-Wei; Qu, Ai-Ping; Liu, Wen-Lou; Chen, Jia-Mei; Yuan, Jing-Ping; Wu, Han; Li, Yan; Liu, Juan

    2016-02-01

    As a widely used proliferative marker, Ki67 has important impacts on cancer prognosis, especially for breast cancer (BC). However, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study is to establish quantum dots (QDs)-based double imaging of nuclear Ki67 as red signal by QDs-655, cytoplasmic cytokeratin (CK) as yellow signal by QDs-585, and organic dye imaging of cell nucleus as blue signal by 4‧,6-diamidino-2-phenylindole (DAPI), and to develop a computer-aided automatic method for Ki67 index measurement. The newly developed automatic computerized Ki67 measurement could efficiently recognize and count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 index, visual Ki67 index, and marked Ki67 index for 30 patients of 90 images with Ki67 ≤ 10% (low grade), 10% < Ki67 < 50% (moderate grade), and Ki67 ≥ 50% (high grade) showed computerized Ki67 counting is better than visual Ki67 counting, especially for Ki67 low and moderate grades. Based on QDs-based double imaging and organic dye imaging on BC tissues, this study successfully developed an automatic computerized Ki67 counting method to measure Ki67 index.

  7. The effective encapsulation of a hydrophobic lipid-insoluble drug in solid lipid nanoparticles using a modified double emulsion solvent evaporation method.

    PubMed

    Nabi-Meibodi, Mohsen; Vatanara, Alireza; Najafabadi, Abdolhossein Rouholamini; Rouini, Mohammad Reza; Ramezani, Vahid; Gilani, Kambiz; Etemadzadeh, Seyed Mohammad Hossein; Azadmanesh, Kayhan

    2013-12-01

    Raloxifene HCl (RH), a selective estrogen receptor modulator (SERM), is indicated for the prophylaxis or treatment of postmenopausal osteoporosis. RH shows extremely poor bioavailability due to limited solubility and an extensive intestinal/hepatic first-pass metabolism. Solid lipid nanoparticles (SLNs) are valuable carriers that can enhance drug bioavailability. However, in the case of RH, the encapsulation of the drug in SLNs remains a challenge because of its poor solubility in both water and lipids. In this study, a series of RH-containing SLNs (RH-SLNs) were generated using a modified double emulsion solvent evaporation (DESE) method. Briefly, RH with various drug/lipid ratios was solubilized in the inner core of a double emulsion using different water/organic solvent mixtures. Our best formulation was achieved with the formation of negatively charged nanoparticles, 180nm in diameter, with an encapsulation and loading efficiency of 85% and 4.5%, respectively. It also showed a Fickian mechanism of the drug release in the basic dissolution media. Thermal analysis revealed a distinct decrease in the crystallinity of lipids and RH in comparison with the unprocessed materials. The results of a cell viability assay also showed a better antiproliferative effect of the drug-loaded SLNs versus the free drug solution. Thus, these results indicated that the modified DESE method could be proposed for the effective encapsulation of RH in SLNs with appropriate physicochemical and biological properties.

  8. Distinction between MOG antibody-positive and AQP4 antibody-positive NMO spectrum disorders

    PubMed Central

    Sato, Douglas Kazutoshi; Callegaro, Dagoberto; Lana-Peixoto, Marco Aurelio; Waters, Patrick J.; Jorge, Frederico M. de Haidar; Takahashi, Toshiyuki; Nakashima, Ichiro; Apostolos-Pereira, Samira Luisa; Talim, Natalia; Simm, Renata Faria; Lino, Angelina Maria Martins; Misu, Tatsuro; Leite, Maria Isabel; Aoki, Masashi

    2014-01-01

    Objective: To evaluate clinical features among patients with neuromyelitis optica spectrum disorders (NMOSD) who have myelin oligodendrocyte glycoprotein (MOG) antibodies, aquaporin-4 (AQP4) antibodies, or seronegativity for both antibodies. Methods: Sera from patients diagnosed with NMOSD in 1 of 3 centers (2 sites in Brazil and 1 site in Japan) were tested for MOG and AQP4 antibodies using cell-based assays with live transfected cells. Results: Among the 215 patients with NMOSD, 7.4% (16/215) were positive for MOG antibodies and 64.7% (139/215) were positive for AQP4 antibodies. No patients were positive for both antibodies. Patients with MOG antibodies represented 21.1% (16/76) of the patients negative for AQP4 antibodies. Compared with patients with AQP4 antibodies or patients who were seronegative, patients with MOG antibodies were more frequently male, had a more restricted phenotype (optic nerve more than spinal cord), more frequently had bilateral simultaneous optic neuritis, more often had a single attack, had spinal cord lesions distributed in the lower portion of the spinal cord, and usually demonstrated better functional recovery after an attack. Conclusions: Patients with NMOSD with MOG antibodies have distinct clinical features, fewer attacks, and better recovery than patients with AQP4 antibodies or patients seronegative for both antibodies. PMID:24415568

  9. Recombinant antibodies and their use in biosensors.

    PubMed

    Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

    2012-04-01

    Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

  10. [Antinuclear antibodies].

    PubMed

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  11. Engineered antibodies take center stage.

    PubMed

    Huston, J S; George, A J

    2001-01-01

    implications across many disciplines. The growth in antibody engineering was highlighted by the attendance of some 600 participants at the meeting, doubling that of the 1999 meeting. Dramatic clinical acceptance of monoclonal antibodies during the past two years has fostered this growth, with sales in 2000 of 1.8 billion dollars and projections for 2001 of 3 billion dollars. However, economic measures cannot begin to convey the medical revolution that is being effected by these first humanized and chimerized monoclonal antibodies. At this juncture, the 10 monoclonal antibody therapeutics in clinical use are of murine origin, of which 3 are entirely murine (OKT3, Mylotarg, 90Y-labeled Bexxar), 4 have been chimerized (human constant domains replacing murine) (ReoPro, Rituxan and its 131I-labeled analogue (Zevalin), Simulect, Remicade) and 3 were chimerized and humanized (human residues being substituted for at least some mouse-specific framework residues in VH and VL) (Zenapax, Herceptin, Synagis). Fully humanized anti-CD52 (CAMPATH-1H) has also been approved by the FDA for the treatment of B-cell chronic lymphocytic leukemia and should become available in late 2001. Humanization was initially developed by Dr. Greg Winter at the MRC Laboratory of Molecular Biology (Cambridge, UK), who presented the meeting's keynote address, "Antibodies as a Paradigm for Molecular Evolution". His pioneering work in antibody phage display libraries has been reformulated into a daring approach to develop truly novel proteins with genetically paired structural elements. He described studies in combinatorial protein engineering with enormous implications for both industrial and therapeutic applications of macromolecules.

  12. Growth of(Fe xMg 1 - x) 2SiO 4 single crystals by the double pass floating zone method

    NASA Astrophysics Data System (ADS)

    Tsai, Tse-Lun; Markgraf, Steven A.; Higuchi, Mikio; Dieckmann, Ru¨diger

    1996-12-01

    Good quality olivine single crystals,(Fe xMg 1 - x) 2SiO 4, have been successfully grown by the floating zone method using an image furnace. The double pass floating zone method was utilized to grow crystals with uniform composition along the growth axis. Chemical analyses by electron microprobe confirmed that the crystals were homogeneous in composition along the growth direction over the entire length of the boule. The primary crystal defects observed were bubbles. Precipitates, such as iron oxide and silica-rich phases were only occasionally found in iron-rich olivine crystals. An after-heater, either active or passive, was placed just below the freezing interface to control the interface shape and reduce the thermal gradient, enabling the growth of crack-free single crystals.

  13. Precision treatment of single and double multiphoton ionization of He atoms by strong laser fields: Time-dependent generalized pseudospectral method in internal coordinates

    NASA Astrophysics Data System (ADS)

    Telnov, Dmitry A.; Heslar, John; Chu, Shih-I.

    2012-06-01

    We have developed a new computational method for accurate and efficient numerical solution of the time-dependent Schr"odinger equation for two-electron atoms. Our approach is full-dimensional and makes use of the internal coordinates of the electrons in the plane defined by the electrons and the nucleus (r1, r2, and θ12) as well as Euler angles which determine the orientation of the plane in space. The internal coordinates can be optimally discretized by means of the generalized pseudospectral method while the Euler angles appear through the basis set functions with the definite total angular momentum and its projections. The results of the single and double ionization of the helium atom by strong 800 nm laser fields will be presented. The accurate time-dependent electron density obtained can be used for testing and improvement of various approximate exchange-correlation functionals of the time-dependent density functional theory.

  14. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  15. Double fiber probe with a single fiber Bragg grating based on the capillary-driven self-assembly fabrication method for dimensional measurement of micro parts.

    PubMed

    Cui, Jiwen; Feng, Kunpeng; Hu, Yang; Li, Junying; Dang, Hong; Tan, Jiubin

    2015-12-28

    Focusing on the ultra-precision dimensional measurement of parts with micro-scale dimensions and high aspect ratios, a two-dimensional double fiber probe with a single fiber Bragg grating (DS-FBG probe) is investigated in detail in this paper. The theoretical analysis of the sensing principle is verified by spectrum simulations of the DS-FBG probe with a modified transfer matrix method using the strain distribution within the DS-FBG probe. The fabrication process and physical principle of the capillary-driven self-assembly of double fibers in the UV adhesive with a low viscosity are demonstrated. Experimental results indicate that resolutions of 30 nm in radial direction and 15 nm in axial direction can be achieved, and the short-term displacement drifts within 90 seconds are 28.0 nm in radial direction and 7.9 nm in axial direction, and the long-term displacement drifts within 1 hour are 61.3 nm in radial direction and 17.3 nm in axial direction. The repeatability of the probing system can reach 60 nm and the measurement result of a standard nozzle is 300.49 μm with a standard deviation of 20 nm.

  16. Optical properties of double layer thin films zinc oxide doping aluminum (ZnO/Al) were deposited on glass substrates by sol gel method spray coating technique

    NASA Astrophysics Data System (ADS)

    Permatasari, Anes; Sutanto, Heri; Marito Siagian, Sinta

    2017-01-01

    Thin films of double layer of ZnO/Al has succeeded in deposition on a glass substrate using sol-gel method and spray coating techniques. Variations of doping Al as much as 2%, 4%, 6% and 8%. ZnO precursor synthesized using zinc acetate dehydrate (Zn(COOCH3)2.2H2O), isopropanol ((CH3)2CHOH) and monoethanolamine (MEA) were stirred using a magnetic stirrer for 45 minutes. ZnO precursor get homogeneous and then added of aluminum nitrate nonahydrate predetermined doping concentration and stirred again for 15 minutes. Deposition solution is done by the spray on a glass substrate and then heated at a temperature of 450°C. A layer of ZnO/Al deposited over the ZnO to produce a thin layer of a double layer. Optical properties layer of ZnO/Al characterized using UV-Vis spectrophotometer. Based on data from UV-Vis absorbance was determined the value of the energy band gap. Pure and dopped layers has different energy due the Al dopping. For pure ZnO layer has energy band gap of 3.347 eV and decreased to 3.09 eV for ZnO layer with Al dopant.

  17. Visualisation of Microglia with the use of Immunohistochemical Double Staining Method for CD-68 and Iba-1 of Cerebral Tissue Samples in Cases of Brain Contusions.

    PubMed

    Stankov, Aleksandar; Belakaposka-Srpanova, Viktorija; Bitoljanu, Natasa; Cakar, Ljupco; Cakar, Zdravko; Rosoklija, Gorazd

    2015-01-01

    In the recent years it has been confirmed that the main component of the immune response in an injury of the nerve cell comes from microglia and macrophages. The main challenge in the field of microglia research is to detect the different stages of cellular activation by visualization of the cell morphology. The existing visualization techniques are based on surface molecules expression in resting and activated microglia cells. For visualization of the microglial cells and their functional state we used double labeling method for cd-68 and iba1 in brain contusions with different survival time. Microglia are stained brown with Iba-1, whereas microglia impregnated with black, grainy color, represents activated microglia stained with CD 68. We had significantly positive results, and we were able to observe changes in the morphology of the microglia that correlated with the survival time. Using double labeling with Iba-1 and cd68 we were able to determine their physiological state based on the morphology and immunoreactivity.

  18. New treatment method for boron in aqueous solutions using Mg-Al layered double hydroxide: Kinetics and equilibrium studies.

    PubMed

    Kameda, Tomohito; Oba, Jumpei; Yoshioka, Toshiaki

    2015-08-15

    Mg-Al layered double hydroxides (LDHs) intercalated with NO3(-) (NO3 · Mg - Al LDHs) and with Cl(-) (Cl · Mg - Al LDHs) were found to take up boron from aqueous solutions. Boron was removed by anion exchange of B(OH)4(-) in solution with NO3(-) and Cl(-) intercalated in the interlayer of the LDH. Using three times the stoichiometric quantity of NO3 · Mg-Al LDH, the residual concentration of B decreased from 100 to 1.9 mg L(-1) in 120 min. Using five times the stoichiometric quantity of Cl · Mg - Al LDH, the residual concentration of B decreased from 100 to 5.6 mg L(-1) in 120 min. It must be emphasized that, in both cases, the residual concentration of B was less than the effluent standards in Japan (10 mg L(-1)). The rate-determining step of B removal by the NO3 · Mg - Al and Cl · Mg - Al LDHs was found to be chemical adsorption involving anion exchange of B(OH)4(-) with intercalated NO3(-) and Cl(-). The removal of B was well described by a pseudo second-order kinetic equation. The adsorption of B by NO3 · Mg - Al LDH and Cl · Mg - Al LDH followed a Langmuir-type adsorption. The values of the maximum adsorption and the equilibrium adsorption constant were 3.6 mmol g(-1) and 1.7, respectively, for NO3 · Mg - Al LDH, and 3.8 mmol g(-1) and 0.7, respectively, for Cl · Mg-Al LDH. The B(OH)4(-) in B(OH)4 · Mg - Al LDH produced by removal of B was found to undergo anion exchange with NO3(-) and Cl(-) in solution. The NO3 · Mg - Al and Cl · Mg - Al LDHs obtained after this regeneration treatment were able to remove B from aqueous solutions, indicating the possibility of recycling NO3 · Mg - Al and Cl · Mg - Al LDHs for B removal.

  19. A method for identification of the peptides that bind to a clone of thyroid-stimulating antibodies in the serum of Graves' disease patients.

    PubMed

    Na, Chan Hyun; Lee, Mi Hwa; Cho, Bo Youn; Chae, Chi-Bom

    2003-04-01

    A method was developed for identification of the peptide sequences that bind to thyroid-stimulating antibody (TSAb) clones from phage-displayed peptide library. Immunoglobulin G (IgG) was purified from the serum of a Graves' disease patient that stimulates the synthesis of cAMP in the cells that express TSH receptor (TSHR). The IgG that binds to TSHR was purified by an affinity column packed with the resin cross-linked with the extracellular domain of human TSHR. The receptor-binding IgG was then mixed with phages that display linear or cyclic peptides at the end of tail protein pIII. The bound phages were eluted with acidic glycine after extensive washing. From sequencing of the pIII gene of the bound phages, one can deduce the sequences of the peptides that bind to the receptor-binding IgG. Each peptide sequence was then tested for inhibition of the synthesis of cAMP from thyroid cells induced by the serum of a Graves' patient. In this way, one can obtain the peptides that bind to a clone of TSAb. We obtained a peptide sequence that inhibits the action of TSAb at an extremely low concentration (<10(-14) M). Such a peptide will be useful for various studies on TSAb.

  20. Introducing a new method for evaluation of the interaction between an antigen and an antibody: single frequency impedance analysis for biosensing systems.

    PubMed

    Özcan, Burcu; Demirbakan, Burçak; Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2014-07-01

    This paper illustrates the application of an antibody, anti-parathyroid hormone (anti-PTH), as a bioreceptor in a biosensor system for the first time, and demonstrates how this biosensor can be used in parathyroid hormone (PTH) determination. The interaction between the biosensor and parathyroid hormone was firstly investigated by a novel electrochemical method, single frequency impedance analysis. The biosensor was based on the gold electrode modified by cysteine self-assembled monolayers. Anti-PTH was covalently immobilized onto cysteine layer by using an EDC/NHS couple. The immobilization of anti-PTH was monitored by cyclic voltammetry and electrochemical impedance spectroscopy techniques. The performance of the biosensor was evaluated in terms of linearity, sensitivity, repeatability and reproducibility, after a few important optimization studies were carried out. In particular, parathyroid hormone was detected within a linear range of 10-60 fg/mL. Kramers-Kronig transform was also performed on the impedance data. The specificity of the biosensor was also evaluated. The biosensor was validated by using a complementary reference technique. Lastly the developed biosensor was used to monitor PTH levels in artificial serum samples.

  1. Nonlinear transient response analysis for double walls with a porous material supported by nonlinear springs using FEM and MSKE method

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Takao; Hozumi, Hiroaki; Hirano, Yuta; Tobita, Kazuhiro; Kurosawa, Yoshio

    2014-01-01

    In this paper, we newly propose a fast computation method for the nonlinear transient responses including coupling between nonlinear springs and sound proof structures having porous materials using FEM. In this method, we extend our numerical method named as Modal Strain and Kinetic Method (i.e. MSKE method proposed previously by Yamaguchi who is one of the authors) from linear damping analysis to nonlinear dynamic analysis. We assume that the restoring force of the spring has cubic nonlinearity and linear hysteresis damping. To calculate damping properties for soundproof structures including elastic body, viscoelastic body and porous body, displacement vectors as common unknown variable are solved under coupled condition. The damped sound fields in the porous materials are defined by complex effective density and complex bulk modulus. The discrete equations in physical coordinate for this system are transformed into nonlinear ordinary coupled differential equations using normal coordinates corresponding to linear natural modes. Further, using MSKE method, modal damping can be derived approximately under coupled conditions between hysteresis damping of viscoelastic materials, damping of the springs and damping due to flow resistance in porous materials. The modal damping is used for the nonlinear differential equation to compute nonlinear transient responses.

  2. Double Layers in Astrophysics

    NASA Technical Reports Server (NTRS)

    Williams, Alton C. (Editor); Moorehead, Tauna W. (Editor)

    1987-01-01

    Topics addressed include: laboratory double layers; ion-acoustic double layers; pumping potential wells; ion phase-space vortices; weak double layers; electric fields and double layers in plasmas; auroral double layers; double layer formation in a plasma; beamed emission from gamma-ray burst source; double layers and extragalactic jets; and electric potential between plasma sheet clouds.

  3. Drug delivery system for an anticancer agent, chlorogenate-Zn/Al-layered double hydroxide nanohybrid synthesised using direct co-precipitation and ion exchange methods

    NASA Astrophysics Data System (ADS)

    Barahuie, Farahnaz; Hussein, Mohd Zobir; Arulselvan, Palanisamy; Fakurazi, Sharida; Zainal, Zulkarnain

    2014-09-01

    A nano-structured drug-inorganic clay hybrid involving an active anticancer compound, which is chlorogenic acid (CA) intercalated into Zn/Al-layered double hydroxide, has been assembled via ion-exchange and co-precipitation methods to form a nanohybrid CZAE (a chlorogenic acid-Zn/Al nanohybrid synthesised using an ion-exchange method) and CZAC (a chlorogenic acid-Zn/Al nanohybrid synthesised using a direct method), respectively. The X-ray diffraction (XRD) results confirmed that the CA-LDH had a hybrid structure in which the anionic chlorogenate is arranged between the interlayers as a horizontal monolayer at 90 and 20° angles from the x axis for CZAE and CZAC, respectively. Both nanohybrids have the properties of mesoporous materials. The high loading percentage of chlorogenic acid (approximately 43.2% for CZAE and 45.3% for CZAC) with basal spacings of 11.7 and 12.6 Å for CZAE and CZAC, respectively, corroborates the successful intercalation of chlorogenic acid into the interlayer gallery of layered double hydroxides. Free chlorogenic acid and the synthesised nanocomposites (CZAE, CZAC) were assessed for their cytotoxicity against various cancer cells. The Fourier transform infrared data supported the formation of both nanohybrids, and a thermal analysis showed that the nanohybrids are more thermally stable than their counterparts. The chlorogenate shows a sustained release, and the release rate of chlorogenate from CZAE and CZAC nanohybrids at pH 7.4 is remarkably lower than that at pH 4.8 due to their different release mechanisms. The release rate of chlorogenate from both nanohybrids can be described as pseudo-second order. The present investigation revealed the potential of the nanohybrids to enhance the in vitro anti-tumour effect of chlorogenic acid in liver and lung cancer cells in vitro.

  4. Precise quantitation of antinuclear antibodies on HEp-2 cells without the need for serial dilution.

    PubMed

    Hollingsworth, P N; Dawkins, R I; Peter, J B

    1996-07-01

    Using HEp-2 cells as a substrate, we developed a method to quantitate antinuclear antibodies (ANA) by comparing the green fluorescence intensity of unknown samples with that of calibrated standards. Intensity was then converted to international units per milliliter by reference to a standard curve. This method is accurate and precise around the cutoff for positively (5 to 10 IU/ml) and therefore provides a reliable screening test for active, untreated systemic lupus erythematosus. Furthermore, the method can identify sera likely to contain autoantibodies commonly detected in ANA-positive sera (SS-A, SS-B, Sm, small nuclear ribonucleoprotein, Scl-70, and double-stranded DNA).

  5. Double screening

    SciTech Connect

    Gratia, Pierre; Hu, Wayne; Joyce, Austin; Ribeiro, Raquel H.

    2016-06-15

    Attempts to modify gravity in the infrared typically require a screening mechanism to ensure consistency with local tests of gravity. These screening mechanisms fit into three broad classes; we investigate theories which are capable of exhibiting more than one type of screening. Specifically, we focus on a simple model which exhibits both Vainshtein and kinetic screening. We point out that due to the two characteristic length scales in the problem, the type of screening that dominates depends on the mass of the sourcing object, allowing for different phenomenology at different scales. We consider embedding this double screening phenomenology in a broader cosmological scenario and show that the simplest examples that exhibit double screening are radiatively stable.

  6. A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining.

    PubMed

    Cappella, Paolo; Gasparri, Fabio; Pulici, Maurizio; Moll, Jürgen

    2008-07-01

    Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments.

  7. General principles and methods for routine automated microRNA in situ hybridization and double labeling with immunohistochemistry.

    PubMed

    Singh, U; Keirstead, N; Wolujczyk, A; Odin, M; Albassam, M; Garrido, R

    2014-05-01

    MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression by binding to complementary sequences on target messenger RNA transcripts. Changes in the expression levels of specific miRNAs have been associated with a variety of disease conditions. We developed a reliable and high throughput in situ hybridization (ISH) method and optimized tissue fixation conditions for formalin fixed, paraffin embedded (FFPE) tissues. ISH methods were automated to visualize four miRNAs: miRNA-145 (smooth muscle cells), miRNA-126 (endothelial cells), miRNA-21 (neoplastic cells) and U6 small nuclear RNA (nuclear marker) using locked nucleic acid (LNA) probes and the Discovery Ultra Ventana(™) platform. The FFPE tissue sections were pretreated with protease 3, hybridized with probe concentrations of ≤ 25 nM; signal was detected using an enhanced, polymer-based detection method. The ISH signal was stronger and more uniform for tissue samples fixed for ≥ 48 h. To investigate the specificity of the method, we developed an automated dual ISH for miRNA-145 coupled with immunohistochemistry for smooth muscle actin, which confirmed the specific distribution of miRNA-145 to smooth muscle cells. These methods may be used routinely for exploratory studies of biomarker development, sample screening and understanding the role of miRNA in the pathophysiology of specific diseases.

  8. A Non-Destructive Culturing and Cell Sorting Method for Cardiomyocytes and Neurons Using a Double Alginate Layer

    PubMed Central

    Terazono, Hideyuki; Kim, Hyonchol; Hayashi, Masahito; Hattori, Akihiro; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-01-01

    A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture. PMID:22870332

  9. A simplified method to attach antibodies on liposomes by biotin-streptavidin affinity for rapid and economical screening of targeted liposomes.

    PubMed

    Papadia, Konstantina; Markoutsa, Eleni; Antimisiaris, Sophia G

    2014-05-01

    The biotin-Streptavidin (STREP) technique for attachment of monoclonal antibodies (mAbs) (or other ligand types) on liposome surface offers high attachment yield, however it is time consuming and expensive due to the number of steps used and the consumption of large quantities of STREP. Herein, a simplified, fast and economic technique, by incubating pre-mixed biotin-mAb/STREP with biotin-liposomes, at a 3:1:1 biotin-mAb/STREP/biotin-LIP ratio (mol/mol/mol) was evaluated. The physichochemical properties, final mAb attachment yield and targeting potential of liposomes decorated with an anti-transferrin receptor mAb (TfR-mAb), prepared by the simple method (SM) and the conventional method (CM), were compared. The vesicle uptake by hCMEC/D3 cells (known to overexpress TfR) were considered as a measure of liposome targeting capability. Results show that both targeted liposome types (SM and CM) have small size (mean diameters around 150 nm), low poly-dispersity (approx. 0.20) and similar mAb attachment yield (between 64-88%). However, the uptake of the SM-liposomes is slightly lower compared to CM-LIP (24-30% decrease), suggesting that the modulated conformation of mAbs on the liposome surface (triplets attached to one single STREP molecule) results in decreased targeting capability. Nevertheless, the simpler and faster one-step preparation procedure which has very high lipid recovery (> 95%) compared to the CM (50-60%) and 15-30 times lower consumption of STREP, may be a good alternative for initial screening of various mAbs as ligands for targeted liposomal or other nanotechnologies, during pre-clinical development.

  10. NDE evaluation of the intergranular corrosion susceptibility of a 2205 duplex stainless steel using thermoelectric power and double loop electrochemical potentiokinetic reactivation methods

    NASA Astrophysics Data System (ADS)

    Ortiz, N.; Carreón, H.; Ruiz, A.

    2013-01-01

    There is a need for a nondestructive technique to assess rapidly and with confidence the degree of sensitization (DOS) in duplex stainless steel (DSS). In this investigation, we present the use of thermoelectric power (TEP) measurements as nondestructive method for the determination of DOS in isothermally aged 2205 DSS at 700°C for different aging times. The DOS of the aged samples was first established by performing the double loop electrochemical potentiokinetic reactivation (DL-EPR) test. The microstructural evolution was evaluated by scanning electron microscopy (SEM). Experimental results indicate that TEP coefficient is sensitive to gradual microstructural changes produced by thermal aging and can be used to monitor IGC sensitization of 2205 duplex stainless steel.

  11. An efficient formulation and implementation of the analytic energy gradient method to the single and double excitation coupled-cluster wave function - Application to Cl2O2

    NASA Technical Reports Server (NTRS)

    Rendell, Alistair P.; Lee, Timothy J.

    1991-01-01

    The analytic energy gradient for the single and double excitation coupled-cluster (CCSD) wave function has been reformulated and implemented in a new set of programs. The reformulated set of gradient equations have a smaller computational cost than any previously published. The iterative solution of the linear equations and the construction of the effective density matrices are fully vectorized, being based on matrix multiplications. The new method has been used to investigate the Cl2O2 molecule, which has recently been postulated as an important intermediate in the destruction of ozone in the stratosphere. In addition to reporting computational timings, the CCSD equilibrium geometries, harmonic vibrational frequencies, infrared intensities, and relative energetics of three isomers of Cl2O2 are presented.

  12. Direct visualization method of the atomic structure of light and heavy atoms with double-detector C{sub s}-corrected scanning transmission electron microscopy

    SciTech Connect

    Kotaka, Yasutoshi

    2012-09-24

    The advent of C{sub s}-corrected scanning transmission electron microscopy (STEM) has advanced the observation of atomic structures in materials and nanotechnology devices. High-angle annular dark-field (HAADF)-STEM using an annular detector visualizes heavy elements as bright spots at atomic resolution that can be observed with the Z-contrast technique. In this study, the atomic column of light elements is directly observed as bright spots by middle-angle bright-field (MABF)-STEM imaging. Therefore, a double-detector STEM imaging method was developed, exploiting the advantage of both MABF-STEM and HAADF-STEM to maximum, which consists of multiple exposures of simultaneously observed MABF- and HAADF-STEM images in red-green-blue color.

  13. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

    PubMed

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C

    2014-06-01

    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  14. Teaching the Double Layer.

    ERIC Educational Resources Information Center

    Bockris, J. O'M.

    1983-01-01

    Suggests various methods for teaching the double layer in electrochemistry courses. Topics addressed include measuring change in absolute potential difference (PD) at interphase, conventional electrode potential scale, analyzing absolute PD, metal-metal and overlap electron PDs, accumulation of material at interphase, thermodynamics of electrified…

  15. Double Helix Revisited.

    ERIC Educational Resources Information Center

    Glickstein, Neil M.

    1995-01-01

    Describes the use of James Watson's book, "The Double Helix," as a multidisciplinary way of introducing students to actual science; the scientific method; dilemmas encountered in the world of research; and the rich setting of personalities, politics, and history in post-World War II Europe. (MKR)

  16. [Method of double feedback from EEG oscillators of the patient for correction of stress-induced functional disorders].

    PubMed

    Fedotchev, A I; Bondar', A T

    2008-01-01

    Method of correction of human stress-induced functional disorders is proposed which is based on two feedback contours from narrow-band EEG oscillators of the patient. The first one is a usual biofeedback contour in which the feedback signals from the narrow-band EEG oscillator are consciously perceived by the patient and serve him as a guide for voluntary reduction/activation of these EEG components. The second one is an additional contour of resonance stimulation which helps a subject to overcome the difficulties of conscious control of feedback signals. In this contour the parameters of audio-visual stimulation are automatically tuned to the frequency of the dominant narrow-band EEG oscillator of the patient (from the same or other EEG frequency range) to reach its resonance activation. Scientific basis of the method is provided, the results of its experimental testing are presented.

  17. Double arch mirror study

    NASA Technical Reports Server (NTRS)

    Vukobratovich, D.; Hillman, D.

    1983-01-01

    The development of a method of mounting light weight glass mirrors for astronomical telescopes compatible with the goals of the Shuttle Infrared Telescope Facility (SIRTF) was investigated. A 20 in. diameter double arch lightweight mirror previously fabricated was modified to use a new mount configuration. This mount concept was developed and fabricated. The mounting concept of the double mounting mirror is outlined. The modifications made to the mirror, fabrication of the mirror mount, and room temperature testing of the mirror and mount and the extension of the mirror and mount concept to a full size (40 in. diameter) primary mirror for SIRTF are discussed.

  18. Synthesis of layered double hydroxides containing Mg2+, Zn2+, Ca2+ and Al3+ layer cations by co-precipitation methods-A review

    NASA Astrophysics Data System (ADS)

    Theiss, Frederick L.; Ayoko, Godwin A.; Frost, Ray L.

    2016-10-01

    Co-precipitation is a common method for the preparation of layered double hydroxides (LDHs) and related materials. This review article is aimed at providing newcomers to the field with some examples of the types of co-precipitation reactions that have been reported previously and to briefly investigate some of the properties of the products of these reactions. Due to the sheer volume of literature on the subject, the authors have had to limit this article to the synthesis of Mg/Al, Zn/Al and Ca/Al LDHs by co-precipitation and directly related methods. LDHs have been synthesised from various reagents including metal salts, oxides and hydroxides. Co-precipitation is also useful for the direct synthesis of LDHs with a wide range of interlayer anions and various bases have been successfully employed to prepare LDHs. Examples of other synthesis techniques including the urea method, hydrothermal synthesis and various mechanochemical methods that are undoubtedly related to co-precipitation have also been included in this review. The effect of post synthesis hydrothermal has also been summarised.

  19. A double-spike method for K-Ar measurement: A technique for high precision in situ dating on Mars and other planetary surfaces

    NASA Astrophysics Data System (ADS)

    Farley, K. A.; Hurowitz, J. A.; Asimow, P. D.; Jacobson, N. S.; Cartwright, J. A.

    2013-06-01

    A new method for K-Ar dating using a double isotope dilution technique is proposed and demonstrated. The method is designed to eliminate known difficulties facing in situ dating on planetary surfaces, especially instrument complexity and power availability. It may also have applicability in some terrestrial dating applications. Key to the method is the use of a solid tracer spike enriched in both 39Ar and 41K. When mixed with lithium borate flux in a Knudsen effusion cell, this tracer spike and a sample to be dated can be successfully fused and degassed of Ar at <1000 °C. The evolved 40Ar∗/39Ar ratio can be measured to high precision using noble gas mass spectrometry. After argon measurement the sample melt is heated to a slightly higher temperature (˜1030 °C) to volatilize potassium, and the evolved 39K/41K ratio measured by Knudsen effusion mass spectrometry. Combined with the known composition of the tracer spike, these two ratios define the K-Ar age using a single sample aliquot and without the need for extreme temperature or a mass determination. In principle the method can be implemented using a single mass spectrometer. Experiments indicate that quantitative extraction of argon from a basalt sample occurs at a sufficiently low temperature that potassium loss in this step is unimportant. Similarly, potassium isotope ratios measured in the Knudsen apparatus indicate good sample-spike equilibration and acceptably small isotopic fractionation. When applied to a flood basalt from the Viluy Traps, Siberia, a K-Ar age of 351 ± 19 Ma was obtained, a result within 1% of the independently known age. For practical reasons this measurement was made on two separate mass spectrometers, but a scheme for combining the measurements in a single analytical instrument is described. Because both parent and daughter are determined by isotope dilution, the precision on K-Ar ages obtained by the double isotope dilution method should routinely approach that of a pair of

  20. High Prevalence of Antinuclear Antibodies in Children with Thyroid Autoimmunity

    PubMed Central

    Segni, Maria; Pucarelli, Ida; Truglia, Simona; Turriziani, Ilaria; Serafinelli, Chiara; Conti, Fabrizio

    2014-01-01

    Background. Antinuclear antibodies (ANA) are a hallmark of many autoimmune diseases and can be detected many years before disease onset. Autoimmune thyroid diseases (AITD) are frequently associated with other organ- and non-organ-specific autoimmune disorders. Objectives. To assess the prevalence of ANA in pediatric patients with AITD and their clinical correlations. Methods. Ninety-three consecutive pediatric patients with AITD were enrolled (86 children with chronic lymphocytic thyroiditis and 7 with Graves' disease). ANA, anti-double DNA (anti-dsDNA) antibodies, anti-extractable nuclear antigen (anti-ENA), anti-cyclic citrullinated peptide antibodies (anti-CCP), and rheumatoid factor (RF) was obtained. Signs and symptoms potentially related to rheumatic diseases in children were investigated by a questionnaire. Results. ANA positivity was found in 66/93 children (71%), anti-ENA in 4/93 (4.3%), anti-dsDNA in 1/93 (1.1%), RF in 3/93 (3.2%), and anti-CCP in none. No significant differences were found between the ANA-positive and ANA-negative groups with respect to age, sex, L-thyroxine treatment, or prevalence of other autoimmune diseases. Overall, parental autoimmunity was found in 23%. Conclusions. ANA positivity was demonstrated in 71% of children with AITD. ANA positivity was not related to overt immune-rheumatic diseases. However, because the positivity of ANA can occur even many years before the onset of systemic autoimmune diseases, prospective studies are warranted. PMID:24741574

  1. Enantioselective Michael additions of nitromethane by a catalytic double activation method using chiral lewis Acid and achiral amine catalysts.

    PubMed

    Itoh, Kennosuke; Kanemasa, Shuji

    2002-11-13

    Reactions of nitromethane with 1-(2-alkenoyl)-3,5-dimethylpyrazoles can be effectively catalyzed by R,R-DBFOX/Ph.Ni(ClO4)2.3H2O and achiral amine bases, each in a catalytic loading of 10 mol %, to give 1-(3-substituted 4-nitrobutanoyl)-3,5-dimethylpyrazoles in high chemical yields. Excellent enantioselectivities up to 98% ee have been achieved. The nitro moiety can be easily reduced on Raney nickel at atmospheric pressure, followed by concurrent cyclization, to give enantiomers of 4-substituted 2-pyrrolidinone derivatives after usual workup. This method can be successfully applied to a short step synthesis of (R)-(-)-rolipram.

  2. Antibody titers in Group O platelet donors

    PubMed Central

    Tendulkar, Anita Amar; Jain, Puneet Ashok; Velaye, Sanjay

    2017-01-01

    BACKGROUND AND OBJECTIVES: The occurrence of hemolysis due to transfusion of ABO plasma-incompatible platelets (PLTs) is challenging. There has been no consensus for critical antibody titers in the transfusion community. This study was conducted to understand the trends of anti-A and anti-B antibody titer levels in O group donors and to identify any specific patterns of distribution in relation to age and gender. MATERIALS AND METHODS: A total of 1635 Group O PLT donors were randomly selected for this prospective study. Serial 2-fold doubling dilutions were prepared for each sample to calculate the titer of anti-A and anti-B in a standard 96 well micro-plate. Tube technique was used for comparison with the microplate method for 100 samples. RESULTS: Out of 1635 donors, 1430 (87.46%) were males and 205 (12.54%) were females. The median titer for anti-A and anti-B was 128 with range from 4 to 2048. Spearman's correlation coefficient for microplate versus tube technique was estimated to be 0.803 (P < 0.01, two-tailed). 57.12% and 51.19% of all donors had titers ≥128 for anti-A and anti-B, respectively. The geometric mean of anti-A and anti-B was 155.7 and 137.28, respectively. The titers were significantly higher (P < 0.001) in female donors. An inverse relation between titer levels and age was seen. CONCLUSION: Microplate can be used to perform titers in resource-constrained settings. Screening for critical titers in O group donors is essential as they are more implicated in hemolytic transfusion reactions. In the absence of a global consensus on this topic, institutes may need to formulate their own guidelines on handling ABO plasma-incompatible PLT transfusions. PMID:28316436

  3. Uni-dimensional double development HPTLC-densitometry method for simultaneous analysis of mangiferin and lupeol content in mango (Mangifera indica) pulp and peel during storage.

    PubMed

    Jyotshna; Srivastava, Pooja; Killadi, Bharti; Shanker, Karuna

    2015-06-01

    Mango (Mangifera indica) fruit is one of the important commercial fruit crops of India. Similar to other tropical fruits it is also highly perishable in nature. During storage/ripening, changes in its physico-chemical quality parameters viz. firmness, titrable acidity, total soluble solid content (TSSC), carotenoids content, and other biochemicals are inevitable. A uni-dimensional double-development high-performance thin-layer chromatography (UDDD-HPTLC) method was developed for the real-time monitoring of mangiferin and lupeol in mango pulp and peel during storage. The quantitative determination of both compounds of different classes was achieved by densitometric HPTLC method. Silica gel 60F254 HPTLC plates and two solvent systems viz. toluene/EtOAC/MeOH and EtOAC/MeOH, respectively were used for optimum separation and selective evaluation. Densitometric quantitation of mangiferin was performed at 390nm, while lupeol at 610nm after post chromatographic derivatization. Validated method was used to real-time monitoring of mangiferin and lupeol content during storage in four Indian cultivars, e.g. Bombay green (Bgreen), Dashehari, Langra, and Chausa. Significant correlations (p<0.05) between of acidity and TSSC with mangiferin and lupeol in pulp and peel during storage were also observed.

  4. [Titration comparative study of TOPINA Tablets in patients with localization related epilepsy: double-blind comparative study by rapid and slow titration methods].

    PubMed

    Kaneko, Sunao; Inoue, Yushi; Sasagawa, Mutsuo; Kato, Masaaki

    2012-04-01

    To compare the tolerability and efficacy of two titration methods (rapid and slow titration) for TOPINA Tablets with different dosages and periods of escalation, a double-blind comparative study was conducted in patients with localization-related epilepsy. A total of 183 patients were randomized to either rapid titration (initial dosage 100 mg/day increased by 100-200 mg at weekly intervals) or to slow titration (initial dosage 50 mg/day increased in 50 mg/day increments at weekly intervals). TOPINA Tablets were administered for 12 weeks to the maximum dosage of 400 mg/day. The incident of adverse events leading to treatment interruptions or withdrawals was 18.9% in rapid titration and 14.8% in slow titration, with no statistical significance (p = 0.554). The incident of adverse events and adverse reactions of slow titration was slightly lower than that of rapid titration. The common adverse events and adverse reactions reported in the two titration methods were comparable and were well tolerated. On the other hand, the efficacy of slow titration, percent reduction in seizure rate and responder rate, was comparable with that of rapid titration. In conclusion, there were no significant differences of therapeutic response to TOPINA Tablets between the two titration methods.

  5. Synthesis of organo cobalt-aluminum layered double hydroxide via a novel single-step self-assembling method and its use as flame retardant nanofiller in PP.

    PubMed

    Wang, De-Yi; Das, Amit; Costa, Francis Reny; Leuteritz, Andreas; Wang, Yu-Zhong; Wagenknecht, Udo; Heinrich, Gert

    2010-09-07

    Synthesis of polypropylene/organo-layered double hydroxide (PP/OLDH) has been carried out based on self-assembled organocobalt-aluminum LDH (O-CoAl-LDH). The novel method of synthesizing self-assembled CoAl-LDH and its characterization have also been reported in details. This method is proven to be very efficient way of producing OLDH in a single step with homogeneous composition and structure. As flame-retardant nanofiller, O-CoAl-LDH shows significant decrease in heat release rate (HRR), the total heat release (THR) and the heat release capacity (HRC) of the PP composites, though the thermal stability of the compounds decreases slightly compared to the base polymer. Morphological analyses show that the LDH particles are dispersed in PP matrix in a partially exfoliated form. The activation energy calculation based on the Kissinger method reveals that O-CoAl-LDH has a positive effect on the activation energy of thermal decomposition of PP. However, in the presence of this filler, decomposition of the composites starts at an earlier stage than that of pure PP.

  6. Microscale purification of antigen-specific antibodies.

    PubMed

    Brown, Eric P; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E; Chan, Ying N; Lai, Jennifer I; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E

    2015-10-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method acros