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Sample records for double antibody method

  1. 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.

  2. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

    PubMed

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Ling, Shigan; Zhang, Heqiu

    2011-01-01

    A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies. PMID:21029749

  3. 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.

  4. 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.

  5. 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.

  6. The Double Absorbing Boundary method

    NASA Astrophysics Data System (ADS)

    Hagstrom, Thomas; Givoli, Dan; Rabinovich, Daniel; Bielak, Jacobo

    2014-02-01

    A new approach is devised for solving wave problems in unbounded domains. It has common features to each of two types of existing techniques: local high-order Absorbing Boundary Conditions (ABC) and Perfectly Matched Layers (PML). However, it is different from both and enjoys relative advantages with respect to both. The new method, called the Double Absorbing Boundary (DAB) method, is based on truncating the unbounded domain to produce a finite computational domain Ω, and on applying a local high-order ABC on two parallel artificial boundaries, which are a small distance apart, and thus form a thin non-reflecting layer. Auxiliary variables are defined on the two boundaries and inside the layer bounded by them, and participate in the numerical scheme. The DAB method is first introduced in general terms, using the 2D scalar time-dependent wave equation as a model. Then it is applied to the 1D Klein-Gordon equation, using finite difference discretization in space and time, and to the 2D wave equation in a wave guide, using finite element discretization in space and dissipative time stepping. The computational aspects of the method are discussed, and numerical experiments demonstrate its performance.

  7. 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

    2016-01-01

    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 × 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. PMID:26976753

  8. 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

  9. 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.

  10. 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. PMID:26403093

  11. 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.

  12. Hidden anti-double stranded DNA antibodies in autoimmune mice.

    PubMed Central

    Fish, F; Ziff, M

    1982-01-01

    When MRL/l mouse spleen cell culture supernatants were incubated with normal mouse spleen cells, a two-50-fold increase in anti-dsDNA activity was noted. A smaller increase in anti-ssDNA and no change in anti-TNP antibody activity were observed. This 'hidden' antibody in the MRL/l supernatants could not be revealed by DNAse digestion and could not be absorbed by a DNA cellulose column. Hidden antibody was removed from supernatants by sepharose-anti Ig. After DNAase digestion of the MRL/l supernatants, hidden anti-dsDNA could not be revealed by incubation with spleen cells. All the hidden activity was excluded by gel filtration on Sephacryl S-300 (mol. wt greater than 300,000) but was banded in the low density protein area of caesium chloride equilibrium density gradients. It was also noted that MRL/l mouse sera had hidden anti-dsDNA antibodies. Hidden antibodies were present in both the IgG and IgM classes. The revealed antibodies demonstrated impaired ability to bind Fc specific anti-Ig reagents suggesting that they were partially degraded during the incubation with mouse spleen cells. The hidden anti-dsDNA thus appears to represent a DNA-anti-dsDNA complex, perhaps of very high affinity. It may explain why anti-dsDNA but not anti-ssDNA antibodies are of pathological importance in SLE. PMID:6756722

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. A new double views motion deblurring method

    NASA Astrophysics Data System (ADS)

    Hong, Hanyu; Hua, Xia; Zhang, Wenmo; Shi, Yu

    2015-12-01

    With improving of intelligent and automation in modern industrial production area, the detection and reconstruction of the 3D surface of the product has become an important technology, but the image which acquire on the actual production line has motion blur and this problem will affect the later reconstruction work. In order to solve this problem, a deblurring method which based on double view moving target image is proposed in this paper. We can deduce the relationship of the point spread function(PSF) path between the double view image through the epipolar geometry and the camera model. The experimental results show that deblurring with the PSF path solved by the geometric relationship achieves good results.

  18. Use of solid-phase double-antibody radioimmunoassay to identify species from small skeletal fragments.

    PubMed

    Ubelaker, Douglas H; Lowenstein, Jerold M; Hood, Darden G

    2004-09-01

    Protein radioimmunoassay (pRIA) offers the potential to identify species in small skeletal fragments submitted as forensic evidence. The technique consists of protein extraction followed by a solid-phase double-antibody radioimmunoassay using controls of antisera (raised in rabbits) and radioactive (iodine-125) antibody of rabbit gamma globulin (produced in donkeys). Species determination results from evaluation of radioactivity uptake. To demonstrate the potential of this technique, six known bone samples (three human and three nonhuman, including one from a deer [Odocoileus virginianus]) were submitted for blind analysis. pRIA correctly distinguished the human from the nonhuman samples. Using 200 mg or less of each sample, species of the deer specimen was identified correctly, given the choices of cow, deer, dog, goat, and pig. PMID:15461091

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. SPRi-based adenovirus detection using a surrogate antibody method.

    PubMed

    Abadian, Pegah N; Yildirim, Nimet; Gu, April Z; Goluch, Edgar D

    2015-12-15

    Adenovirus infection, which is a waterborne viral disease, is one of the most prevelant causes of human morbidity in the world. Thus, methods for rapid detection of this infectious virus in the environment are urgently needed for public health protection. In this study, we developed a rapid, real-time, sensitive, and label-free SPRi-based biosensor for rapid, sensitive and highly selective detection of adenoviruses. The sensing protocol consists of mixing the sample containing adenovirus with a predetermined concentration of adenovirus antibody. The mixture was filtered to remove the free antibodies from the sample. A secondary antibody, which was specific to the adenovirus antibody, was immobilized onto the SPRi chip surface covalently and the filtrate was flowed over the sensor surface. When the free adenovirus antibodies bound to the surface-immobilized secondary antibodies, we observed this binding via changes in reflectivity. In this approach, a higher amount of adenoviruses resulted in fewer free adenovirus antibodies and thus smaller reflectivity changes. A dose-response curve was generated, and the linear detection range was determined to be from 10 PFU/mL to 5000 PFU/mL with an R(2) value greater than 0.9. The results also showed that the developed biosensing system had a high specificity towards adenovirus (less than 20% signal change when tested in a sample matrix containing rotavirus and lentivirus). PMID:26232675

  4. 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.

  5. 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 ...

  6. 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.

  7. Development and qualification of an antibody rapid deglycosylation method.

    PubMed

    Cook, K Steven; Bullock, Kevin; Sullivan, Timothy

    2012-03-01

    N-linked glycosylation can influence the biological activity and safety of an antibody as well as be a measure of the consistency of the production process. The released N-glycans is an important part of the development of a therapeutic antibody. The traditional method for N-glycan analysis requires complex and laborious sample preparation and lengthy analysis time. Two preparation steps with limited control are removal of the antibody backbone by ice-cold ethanol precipitation and water removal before 2-AB fluorescent dye labeling. Simplification of the sample preparation and better control of key steps that allows for the characterization/quantitation of glycans during all stages of development of a therapeutic antibody is desired. Recently Prozyme introduced a rapid deglycosylation kit and a rapid tagging kit that address some of these issues. The deglycosylation kit immobilizes the antibody on a membrane, thereby eliminating the precipitation step. An instant fluorescent tag kit eliminates the water removal before the 2-AB labeling step. In addition use of a new chromatography column can improve the glycan resolution and shorten the analysis time. The evaluation and qualification of the Rapid Deglycosylation Kit (RDK) and instant 2-AB tagging with the improved chromatography are highlighted. PMID:22257749

  8. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    PubMed Central

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases. PMID:22815145

  9. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement.

    PubMed

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan

    2008-08-01

    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future. PMID:18619538

  10. Kinetic of antigent-antibody reactions with scattering method

    NASA Astrophysics Data System (ADS)

    Bilyi, Olexander I.; Kiselyov, Yevgen M.; Novikov, Volodymyr P.

    2001-07-01

    The immune reactions of interaction antigen-antibody represent specific effect of an antigene with an antibody, which outcome are the complex immune aggregates forming precipitate in case of a soluble antigene, or agglutinate in case of a corpuscular antigene. Immunological methods which uses in the quality of carrier protein latex's polymeric microspheresis, gained name and method latex agglutination. Polymeric microspheresis have the array of advantages before biological carries, which consist in the opportunity of the variation of attributes surface and size microspheresis in the broad band of meanings with the preservation of narrow distribution particles behind measurements, the putting of functional groups, necessary for bunch with ligand on stage their synthesis, in ragidity at storage. The quantitative evaluation of parameters of a response of interaction antigen-antibody in immunology is possible by optical methods on a measurement of a modification of intensity of a light stream of a solution in an outcome of a course of a reaction. Concentration of immune complexes determine both on slacking a taking place stream of light, and on a modification of intensity of a stream of light scattering suspended particles in a solution. The process light scattering by colloidal aggregates are formed from suspension microspheresis with adsorbed on their surface protein is described. In report the physics principle of registration immune reaction by light scattering methods is concerned. The results of the effectiveness latex's preparation created on basis of the polymeric carries is described.

  11. Method for limiting heat flux in double-wall tubes

    DOEpatents

    Hwang, Jaw-Yeu

    1982-01-01

    A method of limiting the heat flux in a portion of double-wall tubes including heat treating the tubes so that the walls separate when subjected to high heat flux and supplying an inert gas mixture to the gap at the interface of the double-wall tubes.

  12. Photoluminescence method of testing double heterostructure wafers

    SciTech Connect

    Besomi, P.R.; Wilt, D.P.

    1984-04-10

    Under photoluminescence (PL) excitation, the lateral spreading of photo-excited carriers can suppress the photoluminescence signal from double heterostructure (DH) wafers containing a p-n junction. In any DH with a p-n junction in the active layer, PL is suppressed if the power of the excitation source does not exceed a threshold value. This effect can be advantageously used for a nondestructive optical determination of the top cladding layer sheet conductance as well as p-n junction misplacement, important parameters for injection lasers and LEDs.

  13. 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...

  14. Occurrence of Newcastle Disease and Infectious Bursal Disease Virus Antibodies in Double-Spurred Francolins in Nigeria

    PubMed Central

    Oluwayelu, Daniel Oladimeji; Adebiyi, Adebowale Idris; Olaniyan, Ibukunoluwa; Ezewele, Phyllis; Aina, Oluwasanmi

    2014-01-01

    The double-spurred francolin Francolinus bicalcaratus has been identified as a good candidate for future domestication due to the universal acceptability of its meat and its adaptability to anthropogenically altered environments. Therefore, in investigating the diseases to which they are susceptible, serum samples from 56 francolins in a major live-bird market (LBM) in Ibadan, southwestern Nigeria, were screened for antibodies against Newcastle disease (ND) and infectious bursal disease (IBD) viruses. Haemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) revealed 25.0% and 35.7% prevalence of ND virus (NDV) antibodies, respectively, while 5.4% and 57.1% prevalence of IBD virus (IBDV) antibodies was detected by agar gel precipitation test (AGPT) and ELISA, respectively. This first report on the occurrence of NDV and IBDV antibodies in apparently healthy, unvaccinated double-spurred francolins from a LBM suggests that they were subclinically infected with either field or vaccine viruses and could thus serve as possible reservoirs of these viruses to domestic poultry. Furthermore, if they are to be domesticated for intensive rearing, a vaccination plan including ND and IBD should be developed and implemented. PMID:26464918

  15. Occurrence of Newcastle Disease and Infectious Bursal Disease Virus Antibodies in Double-Spurred Francolins in Nigeria.

    PubMed

    Oluwayelu, Daniel Oladimeji; Adebiyi, Adebowale Idris; Olaniyan, Ibukunoluwa; Ezewele, Phyllis; Aina, Oluwasanmi

    2014-01-01

    The double-spurred francolin Francolinus bicalcaratus has been identified as a good candidate for future domestication due to the universal acceptability of its meat and its adaptability to anthropogenically altered environments. Therefore, in investigating the diseases to which they are susceptible, serum samples from 56 francolins in a major live-bird market (LBM) in Ibadan, southwestern Nigeria, were screened for antibodies against Newcastle disease (ND) and infectious bursal disease (IBD) viruses. Haemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) revealed 25.0% and 35.7% prevalence of ND virus (NDV) antibodies, respectively, while 5.4% and 57.1% prevalence of IBD virus (IBDV) antibodies was detected by agar gel precipitation test (AGPT) and ELISA, respectively. This first report on the occurrence of NDV and IBDV antibodies in apparently healthy, unvaccinated double-spurred francolins from a LBM suggests that they were subclinically infected with either field or vaccine viruses and could thus serve as possible reservoirs of these viruses to domestic poultry. Furthermore, if they are to be domesticated for intensive rearing, a vaccination plan including ND and IBD should be developed and implemented. PMID:26464918

  16. A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

    PubMed

    Kantor, Aaron B; Moore, Wayne A; Meehan, Stephen; Parks, David R

    2016-01-01

    We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc. PMID:27367287

  17. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

    PubMed

    Baumann, D; Gottstein, B

    1987-06-01

    A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system. PMID:2888183

  18. Measuring Double Stars with the Modified Video Drift Method

    NASA Astrophysics Data System (ADS)

    Iverson, Ernest W.; Nugent, Richard L.

    2015-05-01

    The usefulness of a common CCTV video camera for measuring the position angle and separation of a double star is limited by the camera sensitivity and telescope aperture. The video drift method is enhanced by using an integrating camera but frame integrations longer than 0.132 seconds (4 frames) are impractical. This is due to the target stars elongating (streaking) and moving in incremental steps. A simple modification to the Video Drift Method and corresponding VidPro analysis program significantly increases the magnitude at which double stars can be measured. Double stars down to magnitude +16 have been measured with a 14-inch (35.6-cm) telescope using this method compared to magnitude +12 using the original video drift method under comparable seeing conditions.

  19. On 2D bisection method for double eigenvalue problems

    SciTech Connect

    Ji, X.

    1996-06-01

    The two-dimensional bisection method presented in (SIAM J. Matrix Anal. Appl. 13(4), 1085 (1992)) is efficient for solving a class of double eigenvalue problems. This paper further extends the 2D bisection method of full matrix cases and analyses its stability. As in a single parameter case, the 2D bisection method is very stable for the tridiagonal matrix triples satisfying the symmetric-definite condition. Since the double eigenvalue problems arise from two-parameter boundary value problems, an estimate of the discretization error in eigenpairs is also given. Some numerical examples are included. 42 refs., 1 tab.

  20. Proprietary arabinogalactan extract increases antibody response to the pneumonia vaccine: a randomized, double-blind, placebo-controlled, pilot study in healthy volunteers

    PubMed Central

    2010-01-01

    Background Arabinogalactan from Larch tree (Larix spp.) bark has previously demonstrated immunostimulatory activity. The purpose of this study was to test the hypothesis that ingestion of a proprietary arabinogalactan extract, ResistAid™, would selectively enhance the antibody response to the pneumococcal (pneumonia) vaccine in healthy adults. Methods This randomized, double-blind, placebo-controlled, parallel group pilot study included 45 healthy adults who had not previously been vaccinated against Streptococcus pneumoniae. The volunteers began taking the study product or placebo (daily dosage 4.5 g) at the screening visit (V1-Day 0) and continued over the entire 72 day study period. After 30 days the subjects received the 23-valent pneumococcal vaccine (V2). They were monitored the following day (V3-Day 31), as well as 21 days (V4-Day 51) and 42 days (V5-Day 72) after vaccination. Responses by the adaptive immune system (antigen specific) were measured via pneumococcal IgG antibodies (subtypes 4, 6B, 9V, 14, 18C, 19F, and 23F) and salivary IgA levels. Responses by the innate immune system (non-specific) were measured via white blood cell counts, inflammatory cytokines and the complement system. Results Vaccination significantly increased pneumococcal IgG levels as expected. The arabinogalactan group demonstrated a statistically significant greater IgG antibody response than the placebo group in two antibodies subtypes (18C and 23F) at both Day 51 (p = 0.006 and p = 0.002) and at Day 72 (p = 0.008 and p = 0.041). These same subtypes (18C and 23F) also demonstrated change scores from baseline which were significant, in favor of the arabinogalactan group, at Day 51 (p = 0.033 and 0.001) and at Day 72 (p = 0.012 and p = 0.003). Change scores from baseline and mean values were greater in the arabinogalactan group than placebo for most time points in antibody subtypes 4, 6B, 9V, and 19F, but these differences did not reach statistical significance. There was no

  1. 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. PMID:25982722

  2. 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.

  3. Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh; Jaliani, Hossein Zarei

    2014-02-01

    Because they are monovalent for antigen, single chain antibodies display a different antibody-antigen interaction pattern from that of full-length antibodies. Using the law of mass action and considering the antibody-antigen binding pattern at OD-100% and OD-50% points, we introduced a formula for estimating single chain antibody affinity. Sigmoid curves of optical density values versus antibody concentrations were drawn and used to determine antibody concentrations at OD-50% points using statistical software SigmaPlot. The OD-50% points were then used to calculate the affinity via the mathematical formula. A software-adapted format of the equation is also presented for further facilitation of the calculation process. The accuracy of this method for affinity calculation was proved by surface plasma resonance. This method offers a precise evaluation of antibody affinity without requiring special material or apparatus, making it possible to be performed in any biological laboratory with minimum facilities. PMID:24555931

  4. [Detection of antigen-antibody interaction of human adenovirus by the method of surface plasmon resonance].

    PubMed

    Nosach, L M; Boltovets', P M; Povnytsia, O Iu; Zhovnovata, V L; Zakharenko, O M; Snopok, B A; Shyrshov, Iu M; Diachenko, N S

    2005-01-01

    A possibility to detect adenoviral protein--hexon, using specific antibodies by surface plasmon resonance (SPR) was demonstrated. The hexon of the human adenovirus 2 (Ad2) binds to antibodies immobilized on the sensor surface treated by KNCS and protein A Staphylococcus aureus. The specificity of antihexon antibodies was demonstrated by indirect method of fluorescent antibodies (MFA) and cellular variant of the immunoassay (cELISA). PMID:16250237

  5. Comparison of double-ended transition state search methods

    NASA Astrophysics Data System (ADS)

    Koslover, Elena F.; Wales, David J.

    2007-10-01

    While a variety of double-ended transition state search methods have been developed, their relative performance in characterizing complex multistep pathways between structurally disparate molecular conformations remains unclear. Three such methods (doubly-nudged elastic band, a string method, and a growing string method) are compared for a series of benchmarks ranging from permutational isomerizations of the seven-atom Lennard-Jones cluster (LJ7) to highly cooperative LJ38 and LJ75 rearrangements, and the folding pathways of two peptides. A database of short paths between LJ13 local minima is used to explore the effects of parameters and suggest reasonable default values. Each double-ended method was employed within the framework of a missing connection network flow algorithm to construct more complicated multistep pathways. We find that in our implementation none of the three methods definitively outperforms the others, and that their relative effectiveness is strongly system and parameter dependent.

  6. Comparison of double-ended transition state search methods.

    PubMed

    Koslover, Elena F; Wales, David J

    2007-10-01

    While a variety of double-ended transition state search methods have been developed, their relative performance in characterizing complex multistep pathways between structurally disparate molecular conformations remains unclear. Three such methods (doubly-nudged elastic band, a string method, and a growing string method) are compared for a series of benchmarks ranging from permutational isomerizations of the seven-atom Lennard-Jones cluster (LJ(7)) to highly cooperative LJ(38) and LJ(75) rearrangements, and the folding pathways of two peptides. A database of short paths between LJ(13) local minima is used to explore the effects of parameters and suggest reasonable default values. Each double-ended method was employed within the framework of a missing connection network flow algorithm to construct more complicated multistep pathways. We find that in our implementation none of the three methods definitively outperforms the others, and that their relative effectiveness is strongly system and parameter dependent. PMID:17919006

  7. 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

  8. 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.

  9. Double micropipettes configuration method of scanning ion conductance microscopy

    NASA Astrophysics Data System (ADS)

    Zhuang, Jian; Li, Zeqing; Jiao, Yangbohan

    2016-07-01

    In this paper, a new double micropipettes configuration mode of scanning ion conductance microscopy (SICM) is presented to better overcome ionic current drift and further improve the performance of SICM, which is based on a balance bridge circuit. The article verifies the feasibility of this new configuration mode from theoretical and experimental analyses, respectively, and compares the quality of scanning images in the conventional single micropipette configuration mode and the new double micropipettes configuration mode. The experimental results show that the double micropipettes configuration mode of SICM has better effect on restraining ionic current drift and better performance of imaging. Therefore, this article not only proposes a new direction of overcoming the ionic current drift but also develops a new method of SICM stable imaging.

  10. Double micropipettes configuration method of scanning ion conductance microscopy.

    PubMed

    Zhuang, Jian; Li, Zeqing; Jiao, Yangbohan

    2016-07-01

    In this paper, a new double micropipettes configuration mode of scanning ion conductance microscopy (SICM) is presented to better overcome ionic current drift and further improve the performance of SICM, which is based on a balance bridge circuit. The article verifies the feasibility of this new configuration mode from theoretical and experimental analyses, respectively, and compares the quality of scanning images in the conventional single micropipette configuration mode and the new double micropipettes configuration mode. The experimental results show that the double micropipettes configuration mode of SICM has better effect on restraining ionic current drift and better performance of imaging. Therefore, this article not only proposes a new direction of overcoming the ionic current drift but also develops a new method of SICM stable imaging. PMID:27475561

  11. Evaluating method for the double image phenomenon of LED lighting

    NASA Astrophysics Data System (ADS)

    Wu, Wen-Hong; Kuo, Chao-Hui; Hung, Min-Wei; Huang, Kuo-Cheng

    In recent years, the overriding advantages long life, high efficiency, small size and short reaction time have made LED become a viable alternative to conventional light sources. LED lighting sources are usually composed of several individual LED cells which must be mounted on a panel as a lighting module. Being composed of several individual LED cells, the LED sources will cause the double image phenomenon. The double image phenomenon is more obvious when the LED sources are more closer, such as LED table lamp, and limits the applications of LED sources. By using a proper secondary optical lens, the double image phenomenon can be reduced. In this research, an evaluating method based on image processing is developed for the double image phenomenon of a LED sources. By analyzing the gray-scale of the grabbed image which is obtained by putting a rob under a LED source, an index of double image can be established and be a criterion to judge different LED sources. Furthermore, a series of LED lighting simulations are shown in this paper and several type of secondary optical lens are compared and discussed in this paper as well.

  12. 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.

  13. 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. PMID:27127340

  14. Concerning the Video Drift Method to Measure Double Stars

    NASA Astrophysics Data System (ADS)

    Nugent, Richard L.; Iverson, Ernest W.

    2015-05-01

    Classical methods to measure position angles and separations of double stars rely on just a few measurements either from visual observations or photographic means. Visual and photographic CCD observations are subject to errors from the following sources: misalignments from eyepiece/camera/barlow lens/micrometer/focal reducers, systematic errors from uncorrected optical distortions, aberrations from the telescope system, camera tilt, magnitude and color effects. Conventional video methods rely on calibration doubles and graphically calculating the east-west direction plus careful choice of select video frames stacked for measurement. Atmospheric motion is one of the larger sources of error in any exposure/measurement method which is on the order of 0.5-1.5. Ideally, if a data set from a short video can be used to derive position angle and separation, with each data set self-calibrating independent of any calibration doubles or star catalogues, this would provide measurements of high systematic accuracy. These aims are achieved by the video drift method first proposed by the authors in 2011. This self calibrating video method automatically analyzes 1,000's of measurements from a short video clip.

  15. Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab')2 Fragment of a Polyclonal Antibody by Two Different Methods.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Goli, Leila Balaei; Babaei, Mahdokht; Ghods, Roya

    2010-04-01

    R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity. PMID:23407609

  16. Differentiation between Human Coronaviruses NL63 and 229E Using a Novel Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay Based on Specific Monoclonal Antibodies

    PubMed Central

    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. PMID:21084464

  17. 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. PMID:21084464

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

    DOEpatents

    Lindenmeyer, C.W.

    1988-04-14

    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. 4 figs.

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

    DOEpatents

    Lindenmeyer, Carl W.

    1989-01-01

    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.

  20. 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.

  1. Heart antibodies in cardiomyopathies.

    PubMed Central

    Trueman, T; Thompson, R A; Cummins, P; Littler, W A

    1981-01-01

    The reported frequency of circulating heart reactive antibodies in cardiomyopathies has varied and their significance is unknown. In this study such antibodies were sought in patients with primary congestive and hypertrophic cardiomyopathies and other heart diseases. Standard "single sandwich" and the more sensitive "double sandwich" indirect immunofluorescence techniques failed to disclose a significant difference between any cardiomyopathic group and controls in repeated experiments. With both techniques results were subject to considerable method-specific artefacts and observer variation. No published work associating heart antibodies detected by immunofluorescence methods with cariomyopathies adequately takes these into account. PMID:7028058

  2. 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.

  3. 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).

  4. Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera ▿

    PubMed Central

    Hu, Wei Ping; Lu, Yang; Precioso, Nestor Amadeo; Chen, Hsiao Ying; Howard, Teresa; Anderson, David; Guan, Ming

    2008-01-01

    A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n = 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications. PMID:18495846

  5. A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles.

    PubMed

    Filbrun, Seth L; Driskell, Jeremy D

    2016-06-21

    The ability to evaluate antibody immobilization onto gold nanoparticles is critical for assessing coupling chemistry and optimizing the sensitivity of nanoparticle-enabled biosensors. Herein, we developed a fluorescence-based method for directly quantifying antibodies bound onto gold nanoparticles. Antibody-modified gold nanoparticles were treated with KI/I2 etchant to dissolve the gold nanoparticles. A desalting spin column was used to recover the antibody released from the nanoparticles, and NanoOrange, a fluorescent dye, was used to quantify the antibody. We determined 309 ± 93 antibodies adsorb onto a 60 nm gold nanoparticles (2.6 × 10(10) NP mL(-1)), which is consistent with a fully adsorbed monolayer based on the footprint of an IgG molecule. Moreover, the increase in hydrodynamic diameter of the conjugated nanoparticle (76 nm) compared to that of the unconjugated nanoparticle (62 nm) confirmed that multilayers did not form. A more conventional method of indirectly quantifying the adsorbed antibody by analysis of the supernatant overestimated the antibody surface coverage (660 ± 87 antibodies per nanoparticle); thus we propose the method described herein as a more accurate alternative to the conventional approach. PMID:27113720

  6. 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. PMID:25874406

  7. A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.

    PubMed

    Yoo, S K; Yoon, M; Park, U J; Han, H S; Kim, J H; Hwang, H J

    1999-09-30

    High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA. PMID:10551259

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

    PubMed Central

    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. PMID:24278299

  14. Biotin radioligand assay with an /sup 125/I-labeled biotin derivative, avidin, and avidin double-antibody reagents

    SciTech Connect

    Livaniou, E.; Evangelatos, G.P.; Ithakissios, D.S.

    1987-11-01

    We describe a new radioligand assay for determining biotin in biological fluids by using a mixture of N-(beta-(4-OH-3-125I-phenyl)ethyl)- and N-(beta-(4-OH-3,5-di-125I-phenyl)ethyl)biotinamides as radiotracer, avidin as a binding protein, and an avidin double-antibody as a separation reagent. The radiotracer is synthesized by coupling (at pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to radioiodinated tyramine. The assay curve is linear and the assay itself is sensitive (less than 10 ng/L), reproducible (intra- and interassay CVs 4.1% and 7.0%, respectively), and allows the simultaneous handling of more than 100 samples in less than 4 h. Serum samples from apparently normal subjects contained 100-840 ng of biotin per liter (mean 340 ng/L). Pregnant women had low concentrations of biotin (100-300 ng/L) in their serum. Patients undergoing chronic hemodialysis treatment showed high concentrations (0.5-3.0 micrograms/L), which may be ascribable to the inability of avidin, which was used as the assay binding protein, to distinguish biotin from biotinyl derivatives with an intact ureido ring.

  15. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited

    PubMed Central

    Kumar, Yashwant; Bhatia, Alka; Minz, Ranjana Walker

    2009-01-01

    It has been more than 50 years since antinuclear antibodies were first discovered and found to be associated with connective tissue diseases. Since then different methods have been described and used for their detection or confirmation. For many decades immunofluorescent antinuclear antibody test has been the "gold standard" in the diagnosis of these disorders. However to increase the sensitivity and specificity of antinuclear antibody detection further approaches were explored. Today a battery of newer techniques are available some of which are now considered better and are competing with the older methods. This article provides an overview on advancement in antinuclear antibody detection methods, their future prospects, advantages, disadvantages and guidelines for use of these tests. PMID:19121207

  16. 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.

  17. 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.

  18. A simple method for the production of anti-C3d monoclonal antibody.

    PubMed

    Cruz, Carlos; León, Graciela

    2007-12-01

    Production of monoclonal antibodies to C3d usually involves the purification of protein. Our method does not require C3 purification; it relies on attachment of C3b to mouse erythrocytes by activation of alternative pathways and further conversion in C3d. We prepared human complement-coated mouse red cells and sensitized mice of the same strain with our own schedule of immunization and applied the classical methods to obtain a mouse monoclonal antibody. We obtained a clone called BMS-11 which produces a monoclonal antibody of IgM class, to C3d with a title of 1:500000. The monoclonal antibody obtained has shown that it is suitable for use as an antiglobulin reagent. PMID:18158789

  19. Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs.

    PubMed

    van Wyngaardt, Wouter; Mashau, Cordelia; Wright, Isabel; Fehrsen, Jeanni

    2013-01-01

    There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping. PMID:23388433

  20. Upscaling of Hydraulic Conductivity using the Double Constraint Method

    NASA Astrophysics Data System (ADS)

    El-Rawy, Mustafa; Zijl, Wouter; Batelaan, Okke

    2013-04-01

    The mathematics and modeling of flow through porous media is playing an increasingly important role for the groundwater supply, subsurface contaminant remediation and petroleum reservoir engineering. In hydrogeology hydraulic conductivity data are often collected at a scale that is smaller than the grid block dimensions of a groundwater model (e.g. MODFLOW). For instance, hydraulic conductivities determined from the field using slug and packer tests are measured in the order of centimeters to meters, whereas numerical groundwater models require conductivities representative of tens to hundreds of meters of grid cell length. Therefore, there is a need for upscaling to decrease the number of grid blocks in a groundwater flow model. Moreover, models with relatively few grid blocks are simpler to apply, especially when the model has to run many times, as is the case when it is used to assimilate time-dependent data. Since the 1960s different methods have been used to transform a detailed description of the spatial variability of hydraulic conductivity to a coarser description. In this work we will investigate a relatively simple, but instructive approach: the Double Constraint Method (DCM) to identify the coarse-scale conductivities to decrease the number of grid blocks. Its main advantages are robustness and easy implementation, enabling to base computations on any standard flow code with some post processing added. The inversion step of the double constraint method is based on a first forward run with all known fluxes on the boundary and in the wells, followed by a second forward run based on the heads measured on the phreatic surface (i.e. measured in shallow observation wells) and in deeper observation wells. Upscaling, in turn is inverse modeling (DCM) to determine conductivities in coarse-scale grid blocks from conductivities in fine-scale grid blocks. In such a way that the head and flux boundary conditions applied to the fine-scale model are also honored at the

  1. Production of monoclonal antibodies specific for Haemophilus ducreyi: a screening method to discriminate specific and cross-reacting antibodies.

    PubMed

    Odumeru, J A; Alfa, M J; Martin, C F; Ronald, A R; Jay, F T

    1989-06-01

    Haemophilus ducreyi is the etiological agent of chancroid. The organism shares extensive immunological cross-reactivity with other Haemophilus species. This presents substantial difficulties for the production of specific monoclonal antibodies (MAbs). A competition ELISA was devised for hybridoma screening which allowed the detection of H. ducreyi-specific antibody-producing hybridoma cultures during the initial screening process. With this screening method, seven MAbs specific for H. ducreyi were obtained in a single cell fusion exercise. The specificities of the 7 MAbs were demonstrated by direct ELISA and dot immunobinding assays against several strains each of H. influenzae, H. parainfluenzae and Neisseria gonorrhoeae. Five of the MAbs reacted against all ten strains of H. ducreyi. These MAbs may permit the development of rapid and efficient immunodiagnostics for chancroid. The principle of the competition ELISA for hybridoma screening should be widely applicable to the development of specific MAbs to other organisms in which immunological cross-reactivity is an impediment to hybridoma screening by conventional methods. PMID:2787274

  2. Antineutrophil cytoplasmic antibodies (ANCA) testing: detection methods and clinical application.

    PubMed

    Sinico, Renato Alberto; Radice, Antonella

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are considered the diagnostic biomarker of some necrotising vasculitis such as granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and, to a lesser extent, eosinophilic granulomatosis with polyangiitis (EGPA). According to the current recommendations, combining indirect immunofluorescence and proteinase 3 (PR3) and myeloperoxidase (MPO) antigen specific immunometric assays, in the proper clinical setting, assures the best diagnostic specificity. When such conditions are satisfied, ANCA are detected in up to 90% of patients with active generalised GPA and MPA and in about 40% of patients with EGPA. Cytoplasmic ANCA (C-ANCA) with specificity for PR3 are usually found in patients with GPA whereas perinuclear ANCA (P-ANCA) in patients with MPA and EGPA. However, ANCA antigen specificity is more closely associated with disease phenotype and prognosis than clinical diagnosis. The clinical value of serial ANCA testing in monitoring disease activity is still debated. Recently, new promising developments in methodology and techniques (computer-based image analysis of immunofluorescence patterns, novel generation of PR3-/MPO-ANCA immunometric assays and multiplex technology) have been proposed but studies comparing the performances of the different assays are scarce. PMID:24854381

  3. Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins.

    PubMed

    Ranz, A I; Miguet, J G; Anaya, C; Venteo, A; Cortés, E; Vela, C; Sanz, A

    1992-11-01

    A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-specific MAbs allowed visualization by immunofluorescence of tubule-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported. PMID:1481354

  4. A homogeneous fluorescence-based method to measure antibody internalization in tumor cells.

    PubMed

    Gong, Haibiao; Urlacher, Teresa

    2015-01-15

    We have developed a simple fluorescence-based method to monitor antibody internalization. Panitumumab was dual-labeled with the fluorophore IRDye 800CW and quencher IRDye QC-1 to yield the biomolecular probe Pan800QC. The fluorescence of IRDye 800CW is quenched by IRDye QC-1 on the same intact antibody. After incubation with epidermal growth factor receptor (EGFR)-expressing cells, internalization of Pan800QC was detected by an increase in fluorescence signal due to enzymatic digestion of the antibody and separation of IRDye 800CW and IRDye QC-1. By optimizing reaction conditions, a signal-to-background ratio of 8.5 was obtained. This homogeneous assay can be applied in the characterization and screening of internalizing antibodies. PMID:25245185

  5. [The development of methods for obtaining monoclonal antibody-producing cells].

    PubMed

    Skowicki, Michał; Lipiński, Tomasz

    2016-01-01

    Monoclonal antibodies (mAbs) are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given. PMID:27117113

  6. 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.

  7. 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

  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. One-Step Conjugation Method for Site-Specific Antibody-Drug Conjugates through Reactive Cysteine-Engineered Antibodies.

    PubMed

    Shinmi, Daisuke; Taguchi, Eri; Iwano, Junko; Yamaguchi, Tsuyoshi; Masuda, Kazuhiro; Enokizono, Junichi; Shiraishi, Yasuhisa

    2016-05-18

    Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC. PMID:27074832

  10. Development of Double Eastern Blotting for Major Licorice Components, Glycyrrhizin and Liquiritin for Chemical Quality Control of Licorice Using anti-Glycyrrhizin and anti-Liquiritin Monoclonal Antibodies.

    PubMed

    Fujii, Shunsuke; Morinaga, Osamu; Uto, Takuhiro; Nomura, Shuichi; Shoyama, Yukihiro

    2016-02-10

    Licorice is utilized in various food industries around the world for seasoning agents, confectioneries, drinks, and functional foods. Glycyrrhizin (GL) and liquiritin (Liq) are major quality control chemical markers of licorice that have multifunctional bioactivities. Chemical quality control of licorice is important because its component profiles change depending environmental factors (climate, soil condition, and water deficit) and differences between species. Double eastern blotting using anti-GL and anti-Liq monoclonal antibodies was developed for more convenient, rapid, and specific quality control analysis of GL and Liq, respectively. Moreover, double eastern blotting was applied to investigate the immunohistochemical distributions of GL and Liq in the root of fresh licorice; the localization of both components was then clarified visually. This double eastern blotting technique for GL and Liq may serve as a powerful approach for visually determining the chemical quality of licorice. PMID:26765784

  11. A comparative evaluation of six principal IgY antibody extraction methods.

    PubMed

    Ren, Hao; Yang, Wenjing; Thirumalai, Diraviyam; Zhang, Xiaoying; Schade, Rüdiger

    2016-03-01

    Egg yolk has been considered a promising source of antibodies. Our study was designed to compare six principal IgY extraction methods (water dilution, polyethylene glycol [PEG] precipitation, caprylic acid extraction, chloroform extraction, phenol extraction, and carrageenan extraction), and to assess their relative extraction efficiencies and the purity of the resulting antibodies. The results showed that the organic solvents (chloroform or phenol) minimised the lipid ratio in the egg yolk. The water dilution, PEG precipitation and caprylic acid extraction methods resulted in high yields, and antibodies purified with PEG and carrageenan exhibited high purity. Our results indicate that phenol extraction would be more suitable for preparing high concentrations of IgY for non-therapeutic usage, while the water dilution and carrageenan extraction methods would be more appropriate for use in the preparation of IgY for oral administration. PMID:27031600

  12. 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...

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

    PubMed

    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

  14. 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

  15. 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. PMID:25843170

  16. [Determination of anti-rabies antibodies by means of the indirect immunofluorescence method].

    PubMed

    Jiran, E; Závora, M

    1975-06-01

    The paper presents a description of the method of indirect immunofluorescence (IFRA test) for the demonstration of post-vaccination anti-rabies antibodies. The model of immune hamster serum was used to study the reproducibility of the test. The dynamics of antibody formation was quantitatively examined in rabbits immunized by the Flury-LEP virus. For brief information, the test was also used for the demonstration of post-vaccination antibodes against rabies in dogs. The method can be recommended as an additional and orientation test for the evaluation of the immunogenic properties of vaccines against rabies. PMID:810938

  17. 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

  18. Phase 1 Randomized, Double-Blind, Placebo-Controlled Study of RG7667, an Anticytomegalovirus Combination Monoclonal Antibody Therapy, in Healthy Adults

    PubMed Central

    Ishida, Julie H.; Burgess, Tracy; Derby, Michael A.; Brown, Pearline A.; Maia, Mauricio; Deng, Rong; Emu, Brinda; Feierbach, Becket; Fouts, Ashley E.; Liao, X. Charlene

    2015-01-01

    Cytomegalovirus can cause debilitating and life-threatening disease in newborns infected in utero and immunocompromised individuals, including transplant recipients. RG7667 is a unique combination of two monoclonal antibodies that binds glycoprotein complexes on the surface of cytomegalovirus and inhibits its entry into host cells. A phase 1 first-in-human, randomized, double-blind, placebo-controlled, dose-escalation study of RG7667 given intravenously was conducted in 181 healthy adults. The study involved a single ascending dose stage (1, 3, 5, and 10 mg/kg each antibody; n = 21), a multiple ascending dose stage (5 and 10 mg/kg each antibody monthly for 3 doses; n = 10), and a multiple dose expansion stage (10 mg/kg each antibody monthly for 3 doses; n = 150). Subjects were followed for 85 to 141 days to evaluate safety, tolerability, pharmacokinetics, and immunogenicity. Most adverse events were mild, and the incidence of adverse events was similar among the RG7667 and placebo groups. RG7667 had dose-proportional pharmacokinetics in all three dosing stages, a mean terminal half-life of 20 to 30 days, and an overall pharmacokinetic profile consistent with that of a human monoclonal antibody that lacks endogenous host targets. The proportion of subjects developing an antitherapeutic antibody response was not higher in the RG7667 group than in the placebo group. In summary, single and multiple doses of RG7667 were found to be safe and well-tolerated in healthy adults and had a favorable pharmacokinetic and immunogenicity profile. This study supports further development of RG7667 as a therapy for the prevention and treatment of cytomegalovirus infection in susceptible populations. (This study has been registered at ClinicalTrials.gov under registration no. NCT01496755.) PMID:26055360

  19. 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.

  20. 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. PMID:27432793

  1. 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…

  2. Histochemistry of nerve fibres double labelled with anti-TRPV2 antibodies and sensory nerve marker AM1-43 in the dental pulp of rat molars.

    PubMed

    Nishikawa, Sumio

    2008-09-01

    AM1-43 can label sensory nerve fibres and sensory neurons. Permeation of non-selective cation channels of the nerve cell membrane is suggested to be the mechanism responsible for labelling. To identify these channels, two candidates, TRPV1 and TRPV2 were examined by immunocytochemistry in the dental pulp and trigeminal ganglion of rats injected with AM1-43. A part of AM1-43-labelled nerve fibres was also positive for anti-TRPV2 antibody but negative for anti-TRPV1 antibody in the dental pulp. In the trigeminal ganglion, a part of the neuron showed both bright AM1-43 labelling and anti-TRPV2 immunolabelling, but neurons double labelled with AM1-43 and TRPV1 were rare. These results suggest that TRPV2 channels, but not TRPV1 channels, contribute to the fluorescent labelling of AM1-43 in the dental pulp. PMID:18405879

  3. 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.

  4. 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.

  5. An Improved Method for Estimating Antibody Titers in Microneutralization Assay Using Green Fluorescent Protein

    PubMed Central

    Yang, Hongmei; Baker, Steven F.; González, Mario E.; Topham, David J.; Martínez-Sobrido, Luis; Zand, Martin; Holden-Wiltse, Jeanne; Wu, Hulin

    2015-01-01

    Viruses that express reporter genes upon infection have been recently used to evaluate neutralizing antibody responses, where a lack of reporter expression indicates specific virus inhibition. The traditional model-based methods using standard outcome of percent neutralization could be applied to the data from the assays to estimate antibody titers. However, the data produced is sometimes irregular, which can yield meaningless outcomes of percent neutralization that do not fit the typical curves for immunoassays, making automated or semi-high throughput antibody titer estimation unreliable. We developed a type of new outcomes model, which is biologically meaningful and fits typical immunoassay curves well. Our simulation study indicates that the new response approach outperforms the traditional response approach regardless of the data variability. The proposed new response approach can be used in similar assays for other disease models. PMID:26010892

  6. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

    PubMed

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-11-15

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use. PMID:25076184

  7. Comparative of three methods (ELIZA, MAIPA and flow cytometry) to determine anti-platelet antibody in children with ITP.

    PubMed

    Hamidpour, Mohsen; Khalili, Ghader; Tajic, Nader; Shamsian, Bi Bi Shahin; Hamidpour, Rafie

    2014-01-01

    Immune (idiopathic) thrombocytopenic purpurea (ITP) is an autoimmune disease characterized by the increased anti-platelet antibodies in the patient's sera and decreased platelets in the blood circulation. This study has determined and characterized the antiplatelet glycoproteins in children with ITP. Thirty eight children, who were hospitalized with clinical signs of ITP in Mofid Children Hospital (Tehran, Iran) during 18 months, went under our clinical studies in a research project. ELISA, Flow cytometry and MAIPA (Monoclonal Antibody Immobilization of Platelet Antigens) methods were employed to determine serum anti-platelet antibodies level. The anti-platelet antibodies level above mean + 3SD of control group was assumed as positive. The platelet counts ranged between 2 × 10(9)/L and 100 × 10(9)/L. Among the patients 63.5% of them were anti-platelet antibodies positive with ELISA method. Results of platelet lysate method showed that 51.7% of patients had antibodies against platelet antigens. Antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 48%, 54% and 25% respectively. In flow cytometry 62% of patients showed anti-platelet antibodies. The comparison of three methods shows that since MAIPA is the specific method for the detection of very small amount of antibody against glycoprotein antigens, it has the advantage of differentiating between immune and non-immune thrombocytopenia. PMID:25755908

  8. Comparative of three methods (ELIZA, MAIPA and flow cytometry) to determine anti-platelet antibody in children with ITP

    PubMed Central

    Hamidpour, Mohsen; Khalili, Ghader; Tajic, Nader; Shamsian, Bi Bi Shahin; Hamidpour, Rafie

    2014-01-01

    Immune (idiopathic) thrombocytopenic purpurea (ITP) is an autoimmune disease characterized by the increased anti-platelet antibodies in the patient’s sera and decreased platelets in the blood circulation. This study has determined and characterized the antiplatelet glycoproteins in children with ITP. Thirty eight children, who were hospitalized with clinical signs of ITP in Mofid Children Hospital (Tehran, Iran) during 18 months, went under our clinical studies in a research project. ELISA, Flow cytometry and MAIPA (Monoclonal Antibody Immobilization of Platelet Antigens) methods were employed to determine serum anti-platelet antibodies level. The anti-platelet antibodies level above mean + 3SD of control group was assumed as positive. The platelet counts ranged between 2 × 109/L and 100 × 109/L. Among the patients 63.5% of them were anti-platelet antibodies positive with ELISA method. Results of platelet lysate method showed that 51.7% of patients had antibodies against platelet antigens. Antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 48%, 54% and 25% respectively. In flow cytometry 62% of patients showed anti-platelet antibodies. The comparison of three methods shows that since MAIPA is the specific method for the detection of very small amount of antibody against glycoprotein antigens, it has the advantage of differentiating between immune and non-immune thrombocytopenia. PMID:25755908

  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. 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)

  11. 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

  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. [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. PMID:79979

  14. An improved method for covalently conjugating morpholino oligomers to antitumor antibodies.

    PubMed

    He, Jiang; Liu, Guozheng; Dou, Shuping; Gupta, Suresh; Rusckowski, Mary; Hnatowich, Donald

    2007-01-01

    Whether for conventional pretargeting, amplification pretargeting, or affinity enhancement pretargeting, it will be necessary to conjugate an antitumor antibody as the first injectate. This laboratory is investigating phosphorodiamidate morpholinos (MORFs) for pretargeting, and accordingly we are examining methods of attaching MORFs to antitumor antibodies that provide at least one group per molecule (gpm) without adversely influencing antibody properties. The aim of this investigation was to evaluate the commercial Hydralink for the conjugation of the anti-CEA MN14 antibody with an 18 mer amine-derivatized MORF. The conjugation was approached in both directions by first reacting MN14 with the NHS derivatives of 4-hydrozinonicotinate acetone hydrazone (SANH) or 4-formylbenzoate (SFB) and then combining with MORF that was previously reacted with SFB or SANH to yield MN14(SANH)-MORF and MN14(SFB)-MORF respectively. The storage stability, immunoreactive fraction, and the biodistribution in normal mice were compared for both conjugates. Thereafter, MN14(SANH)-MORF was used in a pretargeting study in tumored nude mice, and the results were compared to that obtained historically with MN14-MORF prepared by carbodiimide (EDC) coupling. Both new methods of conjugation provided between 1 and 2 gpm compared to 0.2 achieved previously by EDC. Furthermore, by repeat SE HPLC with and without CEA, both showed an unimpaired immunoreactive fraction. MN14(SANH)-MORF tolerated long-term storage best. More importantly, when labeled by hybridization with 99mTc-labeled complementary MORF (99mTc-cMORF), the biodistribution of MN14(SANH)-MORF was more favorable than that of MN14(SFB)-MORF in normal mice with lower liver (5.7 vs 9.4 %ID/g at 18 h) and spleen (3.5 vs 8.4 %ID/g) accumulations and higher blood levels (4.8 vs 3.4 %ID/g). Accordingly, only MN14(SANH)-MORF was used in a pretargeting study in tumored mice. When targeted with 99mTc-cMORF and at 2 days postinjection of antibody

  15. 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.

  16. MTF measurement of IRFPA based on double-knife edge scanning method

    NASA Astrophysics Data System (ADS)

    Ying, Cheng-ping; Wu, Bin; Wang, Heng-fei; Shi, Xue-shun; Liu, Hong-yuan

    2013-09-01

    Modulation transfer function (MTF) is one of the most important parameters of infrared focal plane array (IRFPA). A double-knife edge scanning method is proposed for MTF measurement of IRFPA. In this method, a double-knife edge was used as a target, and the IRFPA under test was positioned in the focal plane of the imaging optical system by a 3-axis translation stage. With an IRFPA data acquisition system, the image of the double-knife edge was restored. By scanning in the direction orthogonal to the double-knife edge image, edge spread function (ESF) curve of each pixel swept across the knife-edge image was obtained. MTF could be calculated from the subsequent fitting, differential and Fourier transformation procedures. With double-knife edge scanning, two ESF curves of double-knife edge were obtained simultaneously, and symmetry of the two ESF curves could be used to evaluate the verticality between photosensitive surface of IRFPA and optical axis of the double-knife edge imaging system. In addition, this method can be used to judge the existing of interference from outside such as vibration, stray light and electrical noise. A measurement facility for IRFPA's MTF based on double-knife edge scanning method was also established in this study. The facility is composed of double-knife edge imaging optical system, 3-axis translation stage and data acquisition system, et al. As the kernel of the facility, the double-knife edge imaging optical system mainly comprises two symmetrical parabolic mirrors coating with reflective material, and the magnification of the optical system is 1 with an operation wavelength range of (1˜14) μm.

  17. 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. PMID:25634077

  18. MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics.

    PubMed

    Razavi, Morteza; Pope, Matthew E; Soste, Martin V; Eyford, Brett A; Jackson, Angela M; Anderson, N Leigh; Pearson, Terry W

    2011-02-01

    A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma. PMID:21078325

  19. Conjugation behaviours of CdTe quantum dots and antibody by a novel immunochromatographic method.

    PubMed

    Wang, Y; Bai, Y; Wei, X

    2011-03-01

    Three water-soluble CdTe quantum dots (QDs) (green-emitting, yellow-emitting and red-emitting) were synthesised for different refluxing time with 3-mercaptopropionic acid (MPA) as stabiliser. Then the red-emitting CdTe QDs and mouse immunoglobulin G (IgG) were taken as the representative to study the conjugation behaviour of QDs and antibody by a novel immunochromatographic method. After comparing with several methods, that is, direct conjugation, 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC)-mediated conjugation, N-hydroxysuccinimide (NHS)-mediated conjugation, EDC/NHS-mediated conjugation by immunochromatographic strips, EDC and NHS were selected together as coupling agents to conjugate QDs with antibody efficiently. Finally, the K562 leukaemia cells were incubated with the EDC/NHS-mediated conjugates to evaluate the performance in practical application, and the result from fluorescence images showed that it was successfully applied to label cells. The immunochromatographic strip was a superior method to study the conjugation of the fluorophore and antibody. PMID:21241157

  20. Production of anti-SAG1 IgY antibody against Toxoplasma gondii parasites and evaluation of antibody activity by ELISA method.

    PubMed

    Cakir-Koc, Rabia

    2016-08-01

    Chicken egg yolk antibody, also known as immunoglobulin Y (IgY), is the predominant class of serum immunoglobulin in birds. IgY has many advantages over mammalian antibodies, such as enhanced immunogenicity conserved mammalian proteins exhibited in birds due to their phylogenetic distance, non-invasive rapid, and economical collection system. However, there are limited studies about IgY production against Toxoplasma, which is a worldwide veterinary and public health problem. In this study, the production of specific IgY antibodies against the surface antigen 1 (SAG1) protein of Toxoplasma gondii and the determination of antibody activity via the enzyme-linked immunosorbent assay (ELISA) method were conducted. According to ELISA, Western blot, and NanoDrop results, specific and higher amounts of IgY antibody against SAG1 were obtained with this study. Considering the advantages of IgY and importance of SAG1 for the diagnosis of toxoplasmosis, it is expected that anti-SAG1 IgY will play an increasing role and gain commercial value in research, diagnostics, and immunotherapy against toxoplasmosis in the future. PMID:27079459

  1. The double exponential sinc collocation method for singular Sturm-Liouville problems

    NASA Astrophysics Data System (ADS)

    Gaudreau, P.; Slevinsky, R.; Safouhi, H.

    2016-04-01

    Sturm-Liouville problems are abundant in the numerical treatment of scientific and engineering problems. In the present contribution, we present an efficient and highly accurate method for computing eigenvalues of singular Sturm-Liouville boundary value problems. The proposed method uses the double exponential formula coupled with sinc collocation method. This method produces a symmetric positive-definite generalized eigenvalue system and has exponential convergence rate. Numerical examples are presented and comparisons with single exponential sinc collocation method clearly illustrate the advantage of using the double exponential formula.

  2. Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei.

    PubMed Central

    Bühler, R

    1991-01-01

    A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted. PMID:1781689

  3. Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence

    PubMed Central

    Nikoloudis, Dimitris; Pitts, Jim E.

    2014-01-01

    The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak. PMID:25071985

  4. Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence.

    PubMed

    Nikoloudis, Dimitris; Pitts, Jim E; Saldanha, José W

    2014-01-01

    The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak. PMID:25071985

  5. 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

  6. Single versus double testing of meat-juice samples for Salmonella antibodies, in the Danish pig-herd surveillance programme.

    PubMed

    Ekeroth, Lars; Alban, Lis; Feld, Niels

    2003-08-01

    In Denmark, a national serological surveillance-and-control programme for Salmonella in pigs has been in operation since 1995. The programme is based on the Danish mix-ELISA and uses double testing (two ELISA-wells used per sample) of meat-juice samples taken in relation to slaughter. All herds are classified monthly into one of the three levels; the classification is based on the percentage of positive serological results in the previous 3 months. In connection with evaluation of the programme in 2001, we investigated whether single testing (testing in one well only) could be expected to be sufficiently precise compared to double testing. Data from the year 2000 were used, and mathematical modelling. Single testing was simulated by randomised selection of one of the two results in the double testing. A slight increase in the prevalence of Salmonella-positive samples (1.02-1.09 times more through the four quarters of the year 2000) was found in the simulated single testing, as compared to the double testing. Around 0.5% of the herds would be allocated to another herd level in single testing-almost equal numbers one level up and one level down. No herd being seronegative in double testing would be allocated to levels 2 or 3 (herds with >40 or >70%, respectively, serological reactors) in single testing. The prevalence of "false-positive" diagnoses (positive in single testing and negative in double testing) and inversely defined "false-negative" diagnoses varied from 4.2 to 8.7% and from 3.2 to 4.5%, respectively, through the four quarters of the year 2000. The probability of allocating a herd to a wrong level due to sampling error was on the average 6.2 (varying from 1.66 to over 100) times higher than the probability of allocating a herd to a wrong level due to the test inaccuracy introduced by going from double to single testing. This is, however, an average; a herd with a true prevalence close to one of the level border cut-offs (40 and 70% weighted seroprevalence

  7. 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.

  8. Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness

    PubMed Central

    Bub, Carolina Bonet; Martinelli, Beatriz Moraes; Avelino, Thayná Mendonça; Gonçalez, Ana Cláudia; Barjas-Castro, Maria de Lourdes; Castro, Vagner

    2013-01-01

    Background Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. Objective The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. Methods A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. Results Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. Conclusion This study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness. PMID:24106442

  9. 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.

  10. Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies

    PubMed Central

    Eakin, Catherine M.; Miller, Amanda; Kerr, Jennifer; Kung, James; Wallace, Alison

    2014-01-01

    A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab’2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab’2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method. PMID:24808864

  11. 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

  12. Extracorporeal adsorption therapy: A Method to improve targeted radiation delivered by radiometal-labeled monoclonal antibodies.

    SciTech Connect

    Nemecek, Eneida R.; Green, Damian J.; Fisher, Darrell R.; Pagal, John M.; Lin, Yukang; Gopal, A. K.; Durack, Lawrence D.; Rajendran, Joseph G.; Wilbur, D. S.; Nilsson, Rune; Sandberg, Bengt; Press, Oliver W.

    2008-04-01

    Many investigators have demonstrated the ability to treat hematologic malignancies with radiolabeled monoclonal antibodies targeting hematopoietic antigens such as anti-CD20 and anti-CD45. [1-5] Although the remission rates achieved with radioimmunotherapy (RIT) are relatively high, many patients subsequently relapse presumably due to suboptimal delivery of enough radiation to eradicate the malignancy. The dose-response of leukemia and lymphoma to radiation has been proven. Substantial amounts of radiation can be delivered by RIT if followed by hematopoietic cell transplantation to rescue the bone marrow from myeloablation.[ref] However, the maximum dose of RIT that can be used is still limited by toxicity to normal tissues affected by nonspecific delivery of radiation. Efforts to improve RIT focus on improving the therapeutic ratios of radiation in target versus non-target tissues by removing the fraction of radioisotope that fails to bind to target tissues and circulates freely in the bloodstream perfusing non-target tissues. Our group and others have explored several alternatives for removal of unbound circulating antibody. [refs] One such method, extracorporeal adsorption therapy (ECAT) consists of removing unbound antibody by a method similar to plasmapheresis after critical circulation time and distribution of antibody into target tissues have been achieved. Preclinical studies of ECAT in murine xenograft models demonstrated significant improvement in therapeutic ratios of radioactivity. Chen and colleagues demonstrated that a 2-hour ECAT procedure could remove 40 to 70% of the radioactivity from liver, lung and spleen. [ref] Although isotope concentration in the tumor was initially unaffected, a 50% decrease was noted approximately 36 hours after the procedure. This approach was also evaluated in a limited phase I pilot study of patients with refractory B-cell lymphoma. [ref] After radiographic confirmation of tumor localization of a test dose of anti-CD20

  13. 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. PMID:26930597

  14. 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 Central

    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. PMID:26930597

  15. Double Force Compensation Method to Enhance the Performance of a Null Balance Force Sensor

    NASA Astrophysics Data System (ADS)

    Choi, In-Mook; Choi, Dong-June; Kim, Soo Hyun

    2002-06-01

    Microforce measurement is becoming more essential as precision industries such as biomedicine, precision chemistry, semiconductor manufacturing, and so forth develop. A null balance method has been introduced in order to improve on force measurement performances involving a loadcell. The null-balance type force sensor is analyzed and designed for the improvement of measurement performances. The measurement range and the resolution are dependent on the force generation capacity and the various error sources. These characteristics are estimated and verified according to the mechanical sensitivity and the force compensation sensitivity. Two different coil systems are designed and tested experimentally. Double force compensation is proposed in order to obtain a large range and high resolution. The measurement range of the large coil system and the resolution of the small one are fully realized by the double compensation method. After manufacturing, a range over 300 gf and resolution under ± 0.1 mgf were obtained by the double compensation method.

  16. 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. PMID:26025812

  17. 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.

  18. 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.

  19. Double-layered target and identification method of individual target correlated with evaporation residues

    NASA Astrophysics Data System (ADS)

    Kaji, D.; Morimoto, K.

    2015-08-01

    A double-layered target system and an identification method (target ID) for individual targets mounted on a rotating wheel using correlation with evaporation residues were newly developed for the study of superheavy elements (SHE). The target system can be used in three modes: conventional single-layered mode, double-layered mode, and energy-degrader mode. The target ID method can be utilized for masking a target, measuring an excitation function without changing the beam energy from the accelerator, and searching for SHE nuclides using multiple targets during a single irradiation.

  20. 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.

  1. Novel EGFR mutation specific antibodies for NSCLC: Immunohistochemistry as a possible screening method for EGFR mutations

    PubMed Central

    Kato, Yasufumi; Peled, Nir; Wynes, Murry W.; Yoshida, Koichi; Pardo, Marta; Mascaux, Celine; Ohira, Tatsuo; Tsuboi, Masahiro; Matsubayashi, Jun; Nagao, Toshitaka; Ikeda, Norihiko; Hirsch, Fred R.

    2010-01-01

    Background Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict better outcome to EGFR tyrosine kinase inhibitors (TKIs). The most common mutations are exon 19 deletions (most frequently E746-A750) and L858R point mutation in exon 21. Here, we evaluated the accuracy of novel EGFR mutation specific antibodies in a Japanese cohort with NSCLC and compared to direct DNA sequencing and clinical outcome. Materials and methods Immunohistochemistry (IHC) using antibodies specific for the E746-A750 and L858R mutations in EGFR was performed on tissue microarrays of tumors from 70 gefitinib treated NSCLC patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21. Results DNA sequencing showed EGFR mutations in 41 patients (58.6%), and exon 19 deletions in 18 patients (25.7%), 61% (11/18) had a deletion in the range of E746-A750) and 12 (17.1%) had exon 21 mutations (L858R). IHC showed, for the E746-A750 and L858R mutations, sensitivity (81.8% and 75%), specificity (100%, 96.6%), PPV (100%, 81.8%) and NPV (96.7%, 94.9%). Analysis for objective response rates (ORR) and survival were not correlated to IHC staining, although the combined staining showed non-significant trends towards better overall survival for patients with EGFR mutations. Conclusions The mutation specific IHC antibodies have high sensitivity and specificity for pre-defined EFGR mutations and may be suitable for screening for these pre-defined mutations. However, negative IHC results require further mutation analyses prior to excluding EGFR-targeted therapy. PMID:20697298

  2. 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.

  3. 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.

  4. A method to confer Protein L binding ability to any antibody fragment

    PubMed Central

    Lakhrif, Zineb; Pugnière, Martine; Henriquet, Corinne; di Tommaso, Anne; Dimier-Poisson, Isabelle; Billiald, Philippe; Juste, Matthieu O.; Aubrey, Nicolas

    2016-01-01

    abstract Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10-11 M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection. PMID:26683650

  5. A FORTRAN Program for Computing Refractive Index Using the Double Variation Method.

    ERIC Educational Resources Information Center

    Blanchard, Frank N.

    1984-01-01

    Describes a computer program which calculates a best estimate of refractive index and dispersion from a large number of observations using the double variation method of measuring refractive index along with Sellmeier constants of the immersion oils. Program listing with examples will be provided on written request to the author. (Author/JM)

  6. Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates.

    PubMed

    Beck, Alain; Terral, Guillaume; Debaene, François; Wagner-Rousset, Elsa; Marcoux, Julien; Janin-Bussat, Marie-Claire; Colas, Olivier; Dorsselaer, Alain Van; Cianférani, Sarah

    2016-02-01

    Antibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual drug-linker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiple-level structural assessment protocols. PMID:26653789

  7. The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

    PubMed Central

    Stemshorn, B; Nielsen, K

    1981-01-01

    A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests. Images Fig. 1. Fig. 2. PMID:6790144

  8. Humanization of predicted T-cell epitopes reduces the immunogenicity of chimeric antibodies: new evidence supporting a simple method.

    PubMed

    Roque-Navarro, Lourdes; Mateo, Cristina; Lombardero, Josefa; Mustelier, Geraudis; Fernández, Alicia; Sosa, Katya; Morrison, Sherrie L; Pérez, Rolando

    2003-08-01

    Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between re-shaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential. PMID:14511570

  9. 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. PMID:26298680

  10. 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.

  11. Double Hypernuclei Experiment with Hybrid Emulsion Method at J-PARC

    NASA Astrophysics Data System (ADS)

    Ekawa, Hiroyuki

    Double hypernuclei are important probes to study the system with strangeness S = -2. Several emulsion experiments had been performed to search for them. We are planning a new experiment to search for double hypernuclei at the K1.8 beam line in the Hadron Experimental Facility (J-PARC E07 experiment). Ξ- tracks in the emulsion plates and SSD will be automatically connected by a hybrid method. The estimated Ξ- stopped statistics is 10 times as high as that of the KEK E373 experiment. Discoveries of 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 theemulsion plates by tagging Ξ- stopped events. This will be the first measurement to give information on the Ξ- potential at the nuclear surface region.

  12. An analytical method for analyzing symmetry-breaking bifurcation and period-doubling bifurcation

    NASA Astrophysics Data System (ADS)

    Zou, Keguan; Nagarajaiah, Satish

    2015-05-01

    A new modification of homotopy analysis method (HAM) is proposed in this paper. The auxiliary differential operator is specifically chosen so that more than one secular term must be eliminated. The proposed method can capture asymmetric and period-2 solutions with satisfactory accuracy and hence can be used to predict symmetry-breaking and period-doubling bifurcation points. The variation of accuracy is investigated when different number of frequencies are considered.

  13. Applying a kinetic method to an indirect ELISA measuring Ostertagia ostertagi antibodies in milk

    PubMed Central

    Vanderstichel, Raphaël; Dohoo, Ian; Markham, Fred

    2015-01-01

    Indirect enzyme-linked immunosorbent assays (ELISAs) are frequently run as endpoint ELISAs (e-ELISAs). However, kinetic ELISAs (k-ELISAs) have certain advantages over e-ELISAs. The objective of this study was to understand the relationship between e-ELISA and k-ELISA results. Specifically, to determine whether it was possible to run both k-ELISA and e-ELISA on the same plate and establish an appropriate time interval for k-ELISA measurements. A normalization method for k-ELISA slopes (slope ratio) is proposed. Using an indirect e-ELISA test measuring antibodies against Ostertagia ostertagi in milk from dairy cattle, we found that running a k-ELISA had no effect on optical density ratio results of an e-ELISA on the same plate, and that agreement was very strong at 10, 15, and 28 min, allowing for a reduction in the total processing time for ELISA tests. PMID:26130849

  14. 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.

  15. 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.

  16. 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

  17. Investigation of an innovative method for DC flow suppression of double-inlet pulse tube coolers

    NASA Astrophysics Data System (ADS)

    Hu, J. Y.; Luo, E. C.; Wu, Z. H.; Dai, W.; Zhu, S. L.

    2007-05-01

    The use of double-inlet mode in the pulse tube cooler opens up a possibility of DC flow circulating around the regenerator and the pulse tube. The DC flow sometimes deteriorates the performance of the cryocooler because such a steady flow adds an unwanted thermal load to the cold heat exchanger. It seems that this problem is still not well solved although a lot of effort has been made. Here we introduce a membrane-barrier method for DC flow suppression in double-inlet pulse tube coolers. An elastic membrane is installed between the pulse tube cooler inlet and the double-inlet valve to break the closed-loop flow path of DC flow. The membrane is acoustically transparent, but would block the DC flow completely. Thus the DC flow is thoroughly suppressed and the merit of double-inlet mode is remained. With this method, a temperature reduction of tens of Kelvin was obtained in our single-stage pulse tube cooler and the lowest temperature reached 29.8 K.

  18. 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.

  19. 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.

  20. A double filtering method for measuring the translational velocity of fluorescently stained cells

    SciTech Connect

    Yasokawa, Toshiki; Ishimaru, Ichirou; Kuriyama, Shigeki; Masaki, Tsutomu; Takegawa, Kaoru; Tanaka, Naotaka

    2007-09-24

    The authors propose a double filtering method to measure translational velocity for tracking fluorescently stained cells. This method employs two diffraction gratings installed in the infinity space through which the parallel pencil beam of the fluorescence passes. With this method, the change in light intensity whose period is proportional to the translational velocity of the sample can be obtained at the imaging surface. By using a sample that has a random distribution of fluorescence intensity, the authors verified that translational velocity measurements could be achieved using the proposed method.

  1. Chromatography-based methods for determining molar extinction coefficients of cytotoxic payload drugs and drug antibody ratios of antibody drug conjugates.

    PubMed

    Wang, Chunlei; Chen, Sike; Caceres-Cortes, Janet; Huang, Richard Y-C; Tymiak, Adrienne A; Zhang, Yingru

    2016-07-15

    UV spectrophotometry is widely used to determine the molar extinction coefficients (MECs) of cytotoxic drugs as well as the drug antibody ratios (DARs) of antibody drug conjugates (ADCs). However, the unknown purity of a drug due to interfering impurities can lead to erroneous MECs and DARs. Hence, reliable methods to accurately determine purity and the MECs of drugs with limited quantity is urgently needed in Drug Discovery. Such a method has been developed. It achieves absolute purity and accurate MEC determination by a single automated HPLC analysis that uses less than 5μg of material. Specifically, analytical HPLC separation with online UV detection was used to resolve impurities and measure absorbance from only the compound of interest. Simultaneously, an online chemiluminescence nitrogen detector (CLND) was used to determine the concentration of the analyte. The MECs were then calculated from the absorbance and concentration results. The accuracy of the method was demonstrated using caffeine and a commercial cytotoxic drug, DM1. This approach is particularly suited to analyzing mixtures or samples with low purities. Excellent reproducibility was demonstrated by analyzing a proprietary drug with linker synthesized from different batches with very different levels of purity. In addition, the MECs of drug with linker, along with ADC peak areas measured from size exclusion chromatography (SEC), were used to calculate DARs for 21 in-house ADCs. The DAR results were consistent with those obtained by MS analysis. PMID:27286648

  2. A pathologist-in-the-loop IHC antibody test selection using the entropy-based probabilistic method

    PubMed Central

    Shin, Dmitriy; Arthur, Gerald; Caldwell, Charles; Popescu, Mihail; Petruc, Marius; Diaz-Arias, Alberto; Shyu, Chi-Ren

    2012-01-01

    Background: Immunohistochemistry (IHC) is an important tool to identify and quantify expression of certain proteins (antigens) to gain insights into the molecular processes in a diseased tissue. However, it is a challenge for pathologists to remember the discriminative characteristics of the growing number of such antigens across multiple diseases. The complexity of their expression patterns, fueled by continuous discoveries in molecular pathology, gives rise to a combinatorial explosion that places an unprecedented burden on a practicing pathologist and therefore increases cost and variability of IHC studies. Materials and Methods: To tackle these issues, we have developed antibody test optimized selection method, a novel informatics tool to help pathologists in improving the IHC antibody selection process. The method uses extensions of Shannon's information entropies and Bayesian probabilities to dynamically build an efficient diagnostic tree. Results: A comparative analysis of our method with the expert and World Health Organization classification guidelines showed that the proposed method brings threefold reduction in number of antibody tests required to reach a diagnostic conclusion. Conclusion: The developed method can significantly streamline the antibody test selection process, decrease associated costs and reduce inter- and intrapathologist variability in IHC decision-making. PMID:22439121

  3. Description of a double centrifugation tube method for concentrating canine platelets

    PubMed Central

    2013-01-01

    Background To evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was processed by a double centrifugation tube method to obtain platelet concentrates, which were then analyzed by a flow cytometry haematology system for haemogram. Platelet concentration and white blood cell count were determined in all samples. Results Platelet concentration at least 4 times higher than the baseline value and a white blood cell count not exceeding twice the reference range were obtained respectively in 10 cases out of 12 (83.3%) and 11 cases out of 12 (91.6%). Conclusions This double centrifugation tube method is a relatively simple and inexpensive method for obtaining platelet-rich canine plasma, potentially available for therapeutic use to improve the healing process. PMID:23876182

  4. Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

    PubMed Central

    Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Gabriel; González-Sapienza, Gualberto

    2015-01-01

    BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. PMID:25819371

  5. Immunoscintigraphy with indium-111 labeled monoclonal antibodies: The importance of a good display method

    SciTech Connect

    Liehn, J.C.; Hannequin, P.; Nasca, S.; Lebrun, D.; Fernandez-Valoni, A.; Valeyre, J. )

    1989-03-01

    A major drawback of In-111-labeled monoclonal antibodies (MoAb) is the presence of intense liver, renal, and bone marrow nonspecific activity. This makes the display of the images hardly optimal and their visual interpretation difficult. In this study, the intrinsic color scale (which consists of selecting the limits of the color scale as the highest and the lowest pixel value of the image) was compared to a new, simple algorithm for the determination of the limits of the color scale. This algorithm was based on the count density in the iliac crest areas. OC-125 or anti-CEA In-111 MoAb F(ab')2 fragments were used in 32 patients with suspected recurrence of ovarian (19 patients) or colorectal cancer (13 patients). Final diagnosis was assessed by surgery (21 patients), biopsy (five patients), or followup (six patients). A 10-minute abdomino-pelvic anterior view was recorded two days after injection. These views are displayed using the two methods and interpreted by two observers. Using their responses in each quadrant of the pelvis, the authors calculated two ROC curves. The comparison of the ROC curves showed better performances for the new method. For example, for the same specificity (73%), the sensitivity of the new method was significantly better (78% versus 68%). This result confirmed the importance of a good methodology for displaying immunoscintigraphic images.

  6. 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. PMID:26216971

  7. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26216971

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

    PubMed Central

    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. PMID:27195009

  10. Research on text encryption and hiding method with double-random phase-encoding

    NASA Astrophysics Data System (ADS)

    Xu, Hongsheng; Sang, Nong

    2013-10-01

    By using optical image processing techniques, a novel text encryption and hiding method applied by double-random phase-encoding technique is proposed in the paper. The first step is that the secret message is transformed into a 2- dimension array. The higher bits of the elements in the array are used to fill with the bit stream of the secret text, while the lower bits are stored specific values. Then, the transformed array is encoded by double random phase encoding technique. Last, the encoded array is embedded on a public host image to obtain the image embedded with hidden text. The performance of the proposed technique is tested via analytical modeling and test data stream. Experimental results show that the secret text can be recovered either accurately or almost accurately, while maintaining the quality of the host image embedded with hidden data by properly selecting the method of transforming the secret text into an array and the superimposition coefficient.

  11. Key management of the double random-phase-encoding method using public-key encryption

    NASA Astrophysics Data System (ADS)

    Saini, Nirmala; Sinha, Aloka

    2010-03-01

    Public-key encryption has been used to encode the key of the encryption process. In the proposed technique, an input image has been encrypted by using the double random-phase-encoding method using extended fractional Fourier transform. The key of the encryption process have been encoded by using the Rivest-Shamir-Adelman (RSA) public-key encryption algorithm. The encoded key has then been transmitted to the receiver side along with the encrypted image. In the decryption process, first the encoded key has been decrypted using the secret key and then the encrypted image has been decrypted by using the retrieved key parameters. The proposed technique has advantage over double random-phase-encoding method because the problem associated with the transmission of the key has been eliminated by using public-key encryption. Computer simulation has been carried out to validate the proposed technique.

  12. Temperature measurement of wood flame based on the double line method of atomic emission spectra

    NASA Astrophysics Data System (ADS)

    Hao, Xiaojian; Liu, Zhenhua; Sang, Tao

    2016-01-01

    Aimed at the testing requirement of the transient high temperature in explosion field and the bore of barrel weapon, the temperature measurement system of double line of atomic emission spectrum was designed, the method of flame spectrum testing system were used for experimental analysis. The experimental study of wood burning spectra was done with flame spectrum testing system. The measured spectra contained atomic emission spectra of the elements K, Na, and the excitation ease of two kinds atomic emission spectra was analyzed. The temperature was calculated with two spectral lines of K I 766.5nm and 769.9nm. The results show that, compared with Na, the excitation temperature of K atomic emission spectra is lower. By double line method, the temperature of wood burning is 1040K, and error is 3.7%.

  13. Chosen-plaintext attack on double-random-phase-encoding-based image hiding method

    NASA Astrophysics Data System (ADS)

    Xu, Hongsheng; Li, Guirong; Zhu, Xianchen

    2015-12-01

    By using optical image processing techniques, a novel text encryption and hiding method applied by double-random phase-encoding technique is proposed in the paper. The first step is that the secret message is transformed into a 2- dimension array. The higher bits of the elements in the array are used to fill with the bit stream of the secret text, while the lower bits are stored specific values. Then, the transformed array is encoded by double random phase encoding technique. Last, the encoded array is embedded on a public host image to obtain the image embedded with hidden text. The performance of the proposed technique is tested via analytical modeling and test data stream. Experimental results show that the secret text can be recovered either accurately or almost accurately, while maintaining the quality of the host image embedded with hidden data by properly selecting the method of transforming the secret text into an array and the superimposition coefficient.

  14. 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.

  15. 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.

  16. 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

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

    PubMed

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

    2015-01-01

    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. PMID:25827484

  18. 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.

  19. Cell proliferation method: click chemistry based on BrdU coupling for multiplex antibody staining.

    PubMed

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

    2008-07-01

    Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle (see UNIT 7.7). 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 an alkenyl deoxyuridine (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 preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging. PMID:18770651

  20. 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)

  1. A Simple Method for the Immunocytochemical Detection of Proteins Inside Nuclear Structures That Are Inaccessible to Specific Antibodies

    PubMed Central

    Svistunova, Darya M.; Musinova, Yana R.; Polyakov, Vladimir Yu.

    2012-01-01

    It has been demonstrated elsewhere that a high concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies. In this study, the authors found that a short proteinase treatment allowed for the detection of antigens in the nucleoli. The described approach is compatible with the simultaneous observation of proteins fused to fluorescent tags and with preembedding electron microscopy. It appears that the described method can be useful in situations when the proper recognition of antigens by specific antibodies is disturbed by a high density of cellular structures or a high concentration of antigens inside these structures. PMID:22114257

  2. 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. PMID:26527062

  3. Targeting Aberrant DNA double strand break repair in triple negative breast cancer with alpha particle emitter radiolabeled anti-EGFR antibody

    PubMed Central

    Song, Hong; Hedayati, Mohammad; Hobbs, Robert F.; Shao, Chunbo; Bruchertseifer, Frank; Morgenstern, Alfred; DeWeese, Theodore L.; Sgouros, George

    2013-01-01

    The higher potential efficacy of alpha-particle radiopharmaceutical therapy lies in the 3 to 8-fold greater biological effectiveness (RBE) of alpha particles relative to photon or beta-particle radiation. This greater RBE, however, also applies to normal tissue, thereby reducing the potential advantage of high RBE. Since alpha particles typically cause DNA double strand breaks (DSBs), targeting tumors that are defective in DSB repair effectively increases the RBE, yielding a secondary, RBE-based differentiation between tumor and normal tissue that is complementary to conventional, receptor-mediated tumor targeting. In some triple negative breast cancers (TNBC, ER−/PR−/HER-2−), germline mutation in BRCA-1, a key gene in homologous recombination (HR) DSB repair, predisposes patients to early onset of breast cancer. These patients have few treatment options once the cancer has metastasized. In this study, we investigated the efficacy of alpha particle emitter, 213Bi labeled anti-EGFR antibody, Cetuximab, in BRCA-1 defective TNBC. 213Bi-Cetuximab was found to be significantly more effective in the BRCA-1 mutated TNBC cell line HCC1937 than BRCA-1 competent TNBC cell MDA-MB-231. siRNA knockdown of BRCA-1 or DNA-PKcs, a key gene in non-homologous end joining (NHEJ) DSB repair pathway, also sensitized TNBC cells to 213Bi-Cetuximab. Furthermore, the small molecule inhibitor of DNA-PKcs, NU7441, sensitized BRCA-1 competent TNBC cells to alpha particle radiation. Immunofluorescent staining of γH2AX foci and comet assay confirmed that enhanced RBE is caused by impaired DSB repair. These data offer a novel strategy for enhancing conventional receptor-mediated targeting with an additional, potentially synergistic radiobiological targeting that could be applied to TNBC. PMID:23873849

  4. Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure.

    PubMed

    Chen, Kang; Long, Dianna S; Lute, Scott C; Levy, Michaella J; Brorson, Kurt A; Keire, David A

    2016-09-01

    Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures (e.g., oligomerized mAb). Here, several commonly available analytical methods, i.e., size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR. PMID:27344629

  5. 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'.

  6. 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.

  7. 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.

  8. Improved Methods to Detect Low Levels of HIV Using Antibody-Based Technologies.

    PubMed

    Eugenin, Eliseo A; Berman, Joan W

    2016-01-01

    Persistence of latent virus represents a major barrier to eradicating HIV even in the current antiretroviral therapy era. A critical limitation to eliminating these viral reservoirs is the lack of reliable methods to detect, quantify, and characterize cells harboring low levels of virus. However, recent work of several laboratories indicates that PCR and viral amplification based technologies underestimate or overestimate the size of the reservoirs. Thus, new technologies and methodologies to detect, quantify, and characterize these viral reservoirs are necessary to monitor and eradicate HIV. Recent developments in imaging technologies have enabled the development or improvement of detection protocols and have facilitated the identification and quantification of several markers with exquisite resolution. In the context of HIV, we developed new protocols for the detection of low amounts of viral proteins. In this chapter, we describe several antibody-based technologies for signal amplification to improve and detect low amounts of HIV proteins in cells, tissues, and other biological samples. The improvement in these techniques is essential to detect viral reservoirs and to design strategies to eliminate them. PMID:26714718

  9. Hypersingular meshless method using double-layer potentials for three-dimensional exterior acoustic problems.

    PubMed

    Young, D L; Chen, K H; Liu, T Y; Wu, C S

    2016-01-01

    Three-dimensional exterior acoustic problems with irregular domains are solved using a hypersingular meshless method. In particular, the method of fundamental solutions (MFS) is used to formulate and analyze such acoustic problems. It is well known that source points for MFS cannot be located on the real boundary due to the singularity of the kernel functions. Thus, the diagonal terms of the influence matrices are unobtainable when source points are located on the boundary. An efficient approach is proposed to overcome such difficulties, when the MFS is used for three-dimensional exterior acoustic problems. This work is an extension of previous research on two-dimensional problems. The solution of the problem is expressed in terms of a double-layer potential representation on the physical boundary. Three examples are presented in which the proposed method is compared to the MFS and boundary element method. Good numerical performance is demonstrated by the proposed hypersingular meshless method. PMID:26827046

  10. Low-noise multiple watermarks technology based on complex double random phase encoding method

    NASA Astrophysics Data System (ADS)

    Zheng, Jihong; Lu, Rongwen; Sun, Liujie; Zhuang, Songlin

    2010-11-01

    Based on double random phase encoding method (DRPE), watermarking technology may provide a stable and robust method to protect the copyright of the printing. However, due to its linear character, DRPE exist the serious safety risk when it is attacked. In this paper, a complex coding method, which means adding the chaotic encryption based on logistic mapping before the DRPE coding, is provided and simulated. The results testify the complex method will provide better security protection for the watermarking. Furthermore, a low-noise multiple watermarking is studied, which means embedding multiple watermarks into one host printing and decrypt them with corresponding phase keys individually. The Digital simulation and mathematic analysis show that with the same total embedding weight factor, multiply watermarking will improve signal noise ratio (SNR) of the output printing image significantly. The complex multiply watermark method may provide a robust, stability, reliability copyright protection with higher quality printing image.

  11. A time-resolved Langmuir double-probe method for the investigation of pulsed magnetron discharges

    SciTech Connect

    Welzel, Th.; Dunger, Th.; Kupfer, H.; Richter, F.

    2004-12-15

    Langmuir probes are important means for the characterization of plasma discharges. For measurements in plasmas used for the deposition of thin films, the Langmuir double probe is especially suited. With the increasing popularity of pulsed deposition discharges, there is also an increasing need for time-resolved characterization methods. For Langmuir probes, several single-probe approaches to time-resolved measurements are reported but very few for the double probe. We present a time-resolved Langmuir double-probe technique, which is applied to a pulsed magnetron discharge at several 100 kHz used for MgO deposition. The investigations show that a proper treatment of the current measurement is necessary to obtain reliable results. In doing so, a characteristic time dependence of the charge-carrier density during the ''pulse on'' time containing maximum values of almost 2{center_dot}10{sup 11} cm{sup -3} was found. This characteristic time dependence varies with the pulse frequency and the duty cycle. A similar time dependence of the electron temperature is only observed when the probe is placed near the magnesium target.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. 349 Detection of Anti-nucclear Antibodies (ana) Used for Diagnostic Approach of Systemic Autoimmune Diseases. Correlation with Double Stranded DNA (DSDNA) and Extractable Nuclear Antigen (ENA) Antibodies

    PubMed Central

    Anastasiou, Ekarerini; Vakaloudi, Anastasia; Papadopoulos, Georgios; Mavridou, Styliani; Koteli, Asimoula

    2012-01-01

    Background To determine the correlation between the titer of ANA and anti-dsDNA and anti-ENA antibodies and the contribution of ANA detection to the diagnosis of connective tissue diseases (CTD). Methods Our samples consisted of 516 specimens, from Rheumatology Department, collected during January 2010 – July 2010. The detection of ANA was performed using indirect immunofluorescence (IFA) and the detection of anti-dsDNA and anti-ENA using ELISA. Results Of the 364 (70.54%) samples with negative ANA 4 (1%) had positive anti-ENA and 2 (0.5%) had positive anti-dsDNA while positive anti-ENA and anti-dsDNA were detected in the 44.73% (n = 68) and 21% (n = 32) of the specimens with positive ANA respectively. The probability of detecting positive anti-ENA and anti-dsDNA rises proportionately to the titer of ANA. Specifically, the correlation between the probability of detecting positive anti-ENA and the titer of ANA is 0.577 (P < 0.001) while the correlation between the probability of detecting positive anti-dsDNA and the titer of ANA is 0.18 (P = 0.003). Probability calculations on the basis of the ANA titer showed that samples with low titer ANAs (1:160 or less) had low probabilities for positive anti-ENA. The receiver operating (ROC) curves of the ANA titer for anti-ENA had a larger under the curve area compared to the ROC curve for anti-dsDNA, indicating that ANA titer is better for predicting anti-ENA than anti-dsDNA. The sensitivity of positive ANA in the prediction of the anti-ENA and anti-dsDNA was 94.40% and 94.10%, the specificity was 81% and 75.10%, the positive prognostic value was 44.70% and 21.10% and negative prognostic value was 98.90 and 99.50%. Conclusions The detection of ANA using indirect IFA has high sensitivity in predicting the presence of specialized antibodies and may be used as a screening method for the diagnosis of CTD. It is cost and time effective too. Our study also shows that the ANA titer is useful in predicting anti-ENA. Samples with

  14. 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. PMID:26990372

  15. Sensitivity of a radioimmunoassay method for detection of certain viral antibodies in sera and cerebrospinal fluids.

    PubMed Central

    Forghani, B; Schmidt, N J; Lennette, E H

    1976-01-01

    An indirect solid-phase radioimmunoassay (RIA) was applied to titration of serum and cerebrospinal fluid (CSF) antibodies against a variety of viruses including rubella, mumps, measles, herpes simplex, varicella-zoster, and vaccinia. The test used fixed, virus-infected cells as a source of antigen, and conditions for optimal production of viral antigen were determined for each virus-host cell system. In acute, uncomplicated viral infections, sera taken 2 to 5 days after onset generally had low homotypic RIA titers ranging from less than 1:100 to 1:500, whereas convalescent-phase titers ranged from 1:128,000 to 1:512,000. Rubella and measles antibody titers as high as 1:256,000 were demonstrated by RIA in CSF from patients with chronic panencephalitis, whereas homologous antibody titers of 1:4,000 were detected in CSF from acute mumps, herpes simplex, and varicella-zoster virus infections with central nervous system involvement. Some heterotypic antibody was demonstrable by RIA in CSF, but, with the exception of herpes simplex antibody in a mumps virus infection, titers were markedly lower than those to the infecting virus type. RIA generally demonstrated titers at least 1,000 times higher than those obtained by conventional assays such as complement fixation, hemagglutination inhibition, neutralization, and immunofluorescent staining. PMID:187618

  16. Estimates of Mumps Seroprevalence May Be Influenced by Antibody Specificity and Serologic Method

    PubMed Central

    McGrew, Marcia; Williams, Nobia J.; Sowers, Sun B.; Bellini, William J.; Hickman, Carole J.

    2014-01-01

    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. PMID:24371258

  17. Methods for quantitative detection of antibody-induced complement activation on red blood cells.

    PubMed

    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 - fatal(1). 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 lysis(1-4). 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

  18. Investigation of lasing in lead vapor by the double pulse method

    SciTech Connect

    Kazakov, V.V.; Markova, S.V.; Molchanova, L.V.; Petrash, G.G.

    1983-05-01

    The double pulse method was to study the 722.9 nm line emitted by a lead vapor laser. The parameters of the second excitation pulse were measured simultaneously with the lasing characteristics. It was found that the main factor determining a decrease in the laser output power in the second pulse was the residual population of the lower active level. Variations in the excitation pulse had a relatively weak influence on the form of this dependence and were essentially manifested when there was a relatively long delay between the excitation pulses.

  19. Double-filtering method based on two acousto-optic tunable filters for hyperspectral imaging application.

    PubMed

    Wang, Pengchong; Zhang, Zhonghua

    2016-05-01

    A hyperspectral imaging system was demonstrated based on two acousto-optic tunable filters (AOTFs). Efficient regulation of the incoherent beam was executed by means of the wide-angular regime of Bragg diffraction in the birefringent materials. A double-filtering process was achieved when these two AOTFs operated with a central wavelength difference. In comparison with the single-filtering method, the spectral bandwidth was greatly compressed, giving an increment of 42.02% in spectral resolution at the wavelength of 651.62 nm. Experimental results and theoretical calculations are basically identical. Furthermore, the sidelobe was found to be suppressed by the double-filtering process with the first order maximum decreased from -9.25 dB to -22.35 dB. The results indicated high spectral resolution and high spectral purity were obtained simultaneously from this method. The basic spectral resolution performance was examined with a didymium glass by this configuration. We present our experimental methods and the detailed results obtained. PMID:27137600

  20. Anti-vascular endothelial cell antibodies (AECA): comparison of two assay methods and clinical applications.

    PubMed

    Meyer, O; Kaiser, P; Haim, T; Edgell, C J; Pasquier, C; de Bandt, M; Bridey, F; Sellak, H; Lansaman, J; Kahn, M F

    1995-12-01

    Vascular endothelial cells may be a target for autoantibodies (AECAs) against membrane antigens that are constitutively expressed, induced or bound to their surface. To test this hypothesis, we used an enzyme-linked immunosorbent assay (ELISA) with two types of human endothelial cells as the substrate, i.e., human umbilical cord vein endothelial cells (HUVECs) or the hybrid cell line EAhy-926 obtained by fusion of HUVECs with the bronchial carcinoma cell line A549. A comparative functional study of these two cell types demonstrated that EAhy-926 cells produced only small amounts of VIII von Willebrand factor and tissular factor, did not contain Weibel Palade bodies visible under the electron microscope, and expressed ICAM-1 and selectin E in levels of no more than 15% of those expressed by human umbilical cord vein endothelial cells both after stimulation by bacterial lipopolysaccharide and under basal conditions. However, the two assay methods yielded similar IgG AECA titers when used on sera from patients with rheumatoid vasculitis or antiphospholipid syndrome. These antibodies did not exhibit cytotoxicity for cord vein or EAhy-926 cells. They were not specific for endothelium, since their activity decreased by a mean of 40% after incubation of sera with the epithelial cell line A549. A cross-sectional study of 565 sera demonstrated that anti-vascular IgG and IgM AECAs reactive with EAhy-926 cells occurred mainly in patients with dermatomyositis (IgG, 58%; IgM, 22%), systemic scleroderma (IgG, 48%; IgM, 18%), primary Sjögren's syndrome (IgG, 44%; IgM, 12%) and secondary and primary systemic vasculitides (IgG, 38%; IgM, 18%) including Wegener's granulomatosis. A longitudinal study in patients with Wegener's granulomatosis showed that AECAS were predictive of disease activity. PMID:8869215

  1. 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

  2. A comparison of error detection rates between the reading aloud method and the double data entry method.

    PubMed

    Kawado, Miyuki; Hinotsu, Shiro; Matsuyama, Yutaka; Yamaguchi, Takuhiro; Hashimoto, Shuji; Ohashi, Yasuo

    2003-10-01

    Data entry and its verification are important steps in the process of data management in clinical studies. In Japan, a kind of visual comparison called the reading aloud (RA) method is often used as an alternative to or in addition to the double data entry (DDE) method. In a typical RA method, one operator reads previously keyed data aloud while looking at a printed sheet or computer screen, and another operator compares the voice with the corresponding data recorded on case report forms (CRFs) to confirm whether the data are the same. We compared the efficiency of the RA method with that of the DDE method in the data management system of the Japanese Registry of Renal Transplantation. Efficiency was evaluated in terms of error detection rate and expended time. Five hundred sixty CRFs were randomly allocated to two operators for single data entry. Two types of DDE and RA methods were performed. Single data entry errors were detected in 358 of 104,720 fields (per-field error rate=0.34%). Error detection rates were 88.3% for the DDE method performed by a different operator, 69.0% for the DDE method performed by the same operator, 59.5% for the RA method performed by a different operator, and 39.9% for the RA method performed by the same operator. The differences in these rates were significant (p<0.001) between the two verification methods as well as between the types of operator (same or different). The total expended times were 74.8 hours for the DDE method and 57.9 hours for the RA method. These results suggest that in detecting errors of single data entry, the RA method is inferior to the DDE method, while its time cost is lower. PMID:14500053

  3. 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.

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

    PubMed

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

    2016-08-01

    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. PMID:27387556

  5. 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).

  6. 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

  7. 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. PMID:25716465

  8. Research on absorption test methods of Yb-doped double cladding fiber

    NASA Astrophysics Data System (ADS)

    Wang, Pupu; Li, Rundong; Rong, Liang; Ji, Wei; Gao, Yankun; Jiang, Cong; Gu, Shaoyi

    2016-01-01

    Absorption coefficient is a very useful feature for active fiber. In fiber laser system, the length of active fiber is chosen according to absorption coefficient. And the length of fiber can directly influence the feature of fiber laser. Therefore, how to obtain an accurate absorption coefficient is very important. Because fiber exists re-emission in typical absorption band pumped by power. It is difficult to accurately measure absorption coefficient. The absorption coefficients of Yb-doped double cladding fiber at 975 nm measured by several methods were compared. In conclusion, for the fibers with same length pumped by white light, the absorption coefficient is the highest when cutback only once. Meanwhile, when fibers with different length were measured by the same method, the absorption coefficient is inversely proportional to optical fiber length.

  9. 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.

  10. Method for determining effective nonradiative lifetime and leakage losses in double-heterostructure lasers

    SciTech Connect

    van Opdorp, C.; 't Hooft, G.W.

    1981-06-01

    Carrier losses in double-heterostructure lasers are twofold: (i) nonradiative recombination through killers in the bulk of the active region and at all its boundaries (interfaces and surfaces), and (ii) leakage out of the active region. A simple theory shows the following. In the high-injection regime (papprox. =n) all processes under (i) are directly proportional to n. Consequently their contributions can be lumped together in a single effective nonradiative carrier lifetime tau/sub nr/ ; this tau/sub nr/ is constant (i.e., independent of n) owing to the constant degree of occupation of all killers in the mentioned regime. On the other hand, the leakage losses (ii) are superlinear in n. This provides a well-grounded basis for disentangling the contributions of (i) and (ii) in a given sample. Further, a simple method is presented for accurately determining tau/sub nr/ from data of the external quantum efficiency eta/sub ext/ measured as a function of current I in the spontaneous high-injection regime below the laser threshold. Knowledge of the light-extraction factor (i.e., the ratio of external and internal quantum efficiencies) is essentially unnecessary with this method. However, optionally it can be determined easily from a slight extension of the method. For illustration the method of determining tau/sub nr/, which is also applicable to double-hetero LED's, has been applied to some thirty LPE and metal-organic VPE GaAs-(Ga,Al)As lasers of widely varying qualities. The values found vary between 0.8 and 55 ns. From the measured values of tau/sub nr/ it follows that the upper limit for the interface recombination velocity in the best samples is 270 cm/s. For most samples tau/sub nr/ cannot account for all electrical losses at laser threshold. The superlinear excess losses are ascribable to leakage.

  11. A new three-dimensional shape measurement method based on double-frequency fringes

    NASA Astrophysics Data System (ADS)

    Li, Biao; Yang, Jie; Wu, Haitao; Fu, Yanjun

    2015-10-01

    Fringe projection profilometry (FPP) is a rapidly developing technique which is widely used for industrial manufacture, heritage conservation, and medicine etc. because of its high speed, high precision, non-contact operation, full-field acquisition, and easy information processing. Among the various FFP methods, the squared binary defocused projection method (SBM) has been promptly expanding with several advantages: (1) high projection speed because of 1-bit grayscale fringe; (2) eliminating nonlinear gamma of the projector for the defocusing effect. Nevertheless, the method is not trouble-free. When the fringe stripe is wide, it brings down the fringe contrast and is difficult to control the defocused degree, resulting in a low measurement accuracy. In order to further improve high-speed and high-precision three-dimensional shape measurement, this paper presents a new three-dimensional shape measurement method based on double-frequency fringes projection. This new method needs to project two sets of 1-bit grayscale fringe patterns (low-frequency fringe and high-frequency fringe) onto the object surface under slightly defocused projection mode. The method has the following advantages: (1) high projection speed because of 1-bit grayscale fringe; (2) high measurement precision for selectively removing undesired harmonics. Low-frequency fringe is produced by error-diffusion dithering (Dithering) technique and high-frequency fringe is generated by optimal pulse-width modulation (OPWM) technique. The two kinds of fringe patterns have each superiorities and flaws. The low-frequency fringe has a low measurement accuracy, but the continue phase can be easily retrieved. However, the property of high-frequency fringe and low-frequency fringe is the opposite. The general idea of this method proposed is as follows: Because the both fringes test the same object, the height is the same. The low-frequency fringe can be used to assist the high frequency fringe to retrieve

  12. 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

  13. MARCC (Matrix-Assisted Reader Chromatin Capture): an antibody-free method to enrich and analyze combinatorial nucleosome modifications

    PubMed Central

    Su, Zhangli

    2016-01-01

    Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody-free method named MARCC (Matrix-Assisted Reader Chromatin Capture) to enrich combinatorial histone modifications. The combinatorial patterns are enriched on native nucleosomes extracted from cultured mammalian cells and prepared by micrococcal nuclease digestion. Such enrichment is achieved by recombinant chromatin-interacting protein modules, or so-called reader domains, which can bind in a combinatorial modification-dependent manner. The enriched chromatin can be quantified by western blotting or mass spectrometry for the co-existence of histone modifications, while the associated DNA content can be analyzed by qPCR or next-generation sequencing. Altogether, MARCC provides a reproducible, efficient and customizable solution to enrich and analyze combinatorial histone modifications. PMID:26131849

  14. Double isotopic method using dansyl chloride for the determination of GABA in rat C6 astrocytoma cell cultures

    SciTech Connect

    Kohl, R.L.; Quay, W.B.; Perez-Polo, J.R.

    1986-01-01

    Methods are described for the quantitative measurement of GABA in culture. The method can be adapted to any amino acid or dansyl-chloride-reactive species. The sensitivity and selectivity of the procedure result from the double isotopic design in which (/sup 14/C)-labeled internal standard was added to the samples before reaction with (3M)-labeled dansyl chloride. Values obtained by ion-exchange amino acid analysis of cultures agree closely with the values obtained by the double isotopic method. This method is sensitive enough to measure GABA intracellularly and the condition medium.

  15. SEC Based Method for Size Determination of Immune Complexes of Therapeutic Antibodies in Animal Matrix

    PubMed Central

    Boysen, Marta; Dreher, Ingeborg; Loebbert, Ralf

    2016-01-01

    Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC). PMID:27556050

  16. Simulated likelihood methods for complex double-platform line transect surveys.

    PubMed

    Schweder, T; Skaug, H J; Langaas, M; Dimakos, X K

    1999-09-01

    The conventional line transect approach of estimating effective search width from the perpendicular distance distribution is inappropriate in certain types of surveys, e.g., when an unknown fraction of the animals on the track line is detected, the animals can be observed only at discrete points in time, there are errors in positional measurements, and covariate heterogeneity exists in detectability. For such situations a hazard probability framework for independent observer surveys is developed. The likelihood of the data, including observed positions of both initial and subsequent observations of animals, is established under the assumption of no measurement errors. To account for measurement errors and possibly other complexities, this likelihood is modified by a function estimated from extensive simulations. This general method of simulated likelihood is explained and the methodology applied to data from a double-platform survey of minke whales in the northeastern Atlantic in 1995. PMID:11314993

  17. 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.

  18. 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.

  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. PMID:22087899

  20. 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

  1. 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.

  2. Uniscale multi-view registration using double dog-leg method

    NASA Astrophysics Data System (ADS)

    Chen, Chao-I.; Sargent, Dusty; Tsai, Chang-Ming; Wang, Yuan-Fang; Koppel, Dan

    2009-02-01

    3D computer models of body anatomy can have many uses in medical research and clinical practices. This paper describes a robust method that uses videos of body anatomy to construct multiple, partial 3D structures and then fuse them to form a larger, more complete computer model using the structure-from-motion framework. We employ the Double Dog-Leg (DDL) method, a trust-region based nonlinear optimization method, to jointly optimize the camera motion parameters (rotation and translation) and determine a global scale that all partial 3D structures should agree upon. These optimized motion parameters are used for constructing local structures, and the global scale is essential for multi-view registration after all these partial structures are built. In order to provide a good initial guess of the camera movement parameters and outlier free 2D point correspondences for DDL, we also propose a two-stage scheme where multi-RANSAC with a normalized eight-point algorithm is first performed and then a few iterations of an over-determined five-point algorithm is used to polish the results. Our experimental results using colonoscopy video show that the proposed scheme always produces more accurate outputs than the standard RANSAC scheme. Furthermore, since we have obtained many reliable point correspondences, time-consuming and error-prone registration methods like the iterative closest points (ICP) based algorithms can be replaced by a simple rigid-body transformation solver when merging partial structures into a larger model.

  3. Comparing high-throughput methods to measure NK cell-mediated antibody dependent cellular cytotoxicity during HIV-infection.

    PubMed

    Konstantinus, Iyaloo N; Gamieldien, Hoyam; Mkhize, Nonhlanhla N; Kriek, Jean-Mari; Passmore, Jo-Ann S

    2016-07-01

    HIV-specific binding antibody responses, including those mediating antibody-dependent cellular cytotoxicity (ADCC), provided the best functional correlate of lower risk of infection in the RV144 HIV-1 vaccine clinical trial. The aim of this study was to compare two high-throughput flow cytometry based methods to measure HIV-specific ADCC responses, the GranToxilux and PanToxilux assays. Plasma from nine HIV-1 seropositive individuals was screened for binding antibody titres against HIV-1 subtype C gp120 by ELISA and western blot. Plasma from six HIV-negative individuals was included as controls. Both ADCC assays used subtype C gp120-coated CEM.NKRCCR5 cells as targets. The PanToxilux assay (which measured both granzyme B and caspase activity) measured higher levels of direct natural killer (NK) cell killing of K562 tumour cells than the GranToxilux assay (granzyme B alone; p<0.05). In ADCC assays in which NK cell killing was directed against gp120-coated CEM.NKRCCR5 cells in an antibody-dependent manner, plasma from HIV-positive individuals yielded significantly higher levels of ADCC activity than the HIV-negative controls. In contrast to direct killing, the GranToxilux assay measured similar levels of ADCC killing as the PanToxilux assay but had significantly lower background cytotoxicity against target cells coated with HIV negative serum. In conclusion, the PanToxilux assay was more sensitive for detecting direct NK cell killing of K562 cells than the GranToxilux assay, although the GranToxilux assay performed better at detecting HIV-specific ADCC activity, because of lower background cytotoxicity from HIV-negative serum. This is the first study to compare GranToxilux and PanToxilux ability to detect ADCC during HIV infection. PMID:27094485

  4. Validation of an automated method for compounding monoclonal antibody patient doses

    PubMed Central

    Peters, Bas J.M.; Capelle, Martinus A.H.; Arvinte, Tudor; van de Garde, Ewoudt M.W.

    2013-01-01

    Automation robots have recently come to the market as an alternative for manual compounding of drugs for intravenous administration. Our aim was to assess whether robotic compounding can be performed with monoclonal antibodies (mAbs) without influencing the aggregation state of the proteins. Three frequently used mAbs were studied: infliximab (Remicade®, Janssen Biotech) and trastuzumab (Herceptin®, Roche) in lyophilised form, and bevacizumab (Avastin®, Roche) as a liquid formulation stored at 2°C to 8°C. The effects of different procedures to prepare the patient doses on antibody aggregation were evaluated. Remicade® and Herceptin® were reconstituted both manually and by a robotic arm (i.v.STATION®, Health Robotics). Additionally, the influence of vigorous shaking during reconstitution was investigated. The effects of rapid aspiration and dispensing on antibody aggregation were investigated for all three mAbs. Aggregation state was assessed by UV-Vis absorbance, 90° light scatter, fluorescence spectroscopy, Nile red fluorescence microscopy, and field flow fractionation without cross and focus flow. Robotic reconstituted samples showed similar findings compared with manual reconstitution if performed exactly according to the summary of product characteristics (SPC). Vials that were vigorously shaken showed a significant increase in aggregates. Similarly, rapid aspiration/dispense cycles resulted in a strong increase in the number and sizes of aggregates for all three mAbs; this result was observed after just one rapid aspiration/dispense cycle. Our study showed that robotic compounding of mAbs is feasible if the robot is exactly programmed according to the SPC, indicating that robotic compounding can be used to achieve reproducible high-quality compounding for delicate formulations. PMID:23255057

  5. 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 factor…

  6. A hybrid ensemble method based on double disturbance for classifying microarray data.

    PubMed

    Chen, Tao; Xue, Huifeng; Hong, Zenglin; Cui, Man; Zhao, Hui

    2015-01-01

    Microarray data has small samples and high dimension, and it contains a significant amount of irrelevant and redundant genes. This paper proposes a hybrid ensemble method based on double disturbance to improve classification performance. Firstly, original genes are ranked through reliefF algorithm and part of the genes are selected from the original genes set, and then a new training set is generated from the original training set according to the previously selected genes. Secondly, D bootstrap training subsets are produced from the previously generated training set by bootstrap technology. Thirdly, an attribute reduction method based on neighborhood mutual information with a different radius is used to reduce genes on each bootstrap training subset to produce new training subsets. Each new training subset is applied to train a base classifier. Finally, a part of the base classifiers are selected based on the teaching-learning-based optimization to build an ensemble by weighted voting. Experimental results on six benchmark cancer microarray datasets showed proposed method decreased ensemble size and obtained higher classification performance compared with Bagging, AdaBoost, and Random Forest. PMID:26405970

  7. Low-order mathematical modelling of electric double layer supercapacitors using spectral methods

    NASA Astrophysics Data System (ADS)

    Drummond, Ross; Howey, David A.; Duncan, Stephen R.

    2015-03-01

    This work investigates two physics-based models that simulate the non-linear partial differential algebraic equations describing an electric double layer supercapacitor. In one model the linear dependence between electrolyte concentration and conductivity is accounted for, while in the other model it is not. A spectral element method is used to discretise the model equations and it is found that the error convergence rate with respect to the number of elements is faster compared to a finite difference method. The increased accuracy of the spectral element approach means that, for a similar level of solution accuracy, the model simulation computing time is approximately 50% of that of the finite difference method. This suggests that the spectral element model could be used for control and state estimation purposes. For a typical supercapacitor charging profile, the numerical solutions from both models closely match experimental voltage and current data. However, when the electrolyte is dilute or where there is a long charging time, a noticeable difference between the numerical solutions of the two models is observed. Electrical impedance spectroscopy simulations show that the capacitance of the two models rapidly decreases when the frequency of the perturbation current exceeds an upper threshold.

  8. 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. PMID:25399127

  9. 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.

  10. 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.

  11. Development of an antibody-based diagnostic method for the identification of Bemisia tabaci biotype B.

    PubMed

    Baek, Ji Hyeong; Lee, Hye Jung; Kim, Young Ho; Lim, Kook Jin; Lee, Si Hyeock; Kim, Bum Joon

    2016-07-01

    The whitefly Bemisia tabaci is a very destructive pest. B. tabaci is composed of various morphologically undistinguishable biotypes, among which biotypes B and Q, in particular, draw attention because of their wide distribution in Korea and differential potentials for insecticide resistance development. To develop a biotype-specific protein marker that can readily distinguishes biotypes B from other biotypes in the field, we established an ELISA protocol based on carboxylesterase 2 (COE2), which is more abundantly expressed in biotypes B compared with Q. Recombinant COE2 was expressed, purified and used for antibody construction. Polyclonal antibodies specific to B. tabaci COE2 [anti-COE2 pAb and deglycosylated anti-COE2 pAb (DG anti-COE2 pAb)] revealed a 3-9-fold higher reactivity to biotype B COE2 than biotype Q COE2 by Western blot and ELISA analyses. DG anti-COE2 pAb exhibited low non-specific activity, demonstrating its compatibility in diagnosing biotypes. Western blot and ELISA analyses determined that one of the 11 field populations examined was biotype B and the others were biotype Q, suggesting the saturation of biotype Q in Korea. DG anti-COE2 pAb discriminates B. tabaci biotypes B and Q with high specificity and accuracy and could be useful for the development of a B. tabaci biotype diagnosis kit for on-site field applications. PMID:27265822

  12. Multiplex method for initial complex testing of antibodies to blood transmitted diseases agents.

    PubMed

    Poltavchenko, Alexander G; Nechitaylo, Oleg V; Filatov, Pavel V; Ersh, Anna V; Gureyev, Vadim N

    2016-10-01

    Initial screening of donors and population at high risk of infection with blood transmitted diseases involves a number of analyses using monospesific diagnostic systems, and therefore is expensive labor- and time-consuming process. The goal of this work is to construct a multiplex test enabling to carry out rapid initial complex testing at a low price. The paper describes a kit making it possible to detect simultaneously antibodies to six agents of the most significant blood transmitted diseases: HIV virus, hepatitis B and C viruses, cytomegalovirus, T. pallidum and T. gondii in blood products. The kit comprises multiplex dot-immunoassay based on plane protein arrays (immune chips) using colloidal gold conjugates and silver development. It provides an opportunity to carry out complex analysis within 70min at room temperature, and there is no need of well-qualified personnel. We compared laboratory findings of the kit with monospecific kits for ELISA produced by two Russian commercial companies. Dot-assay results correlate well with data obtained using commercial kits for ELISA. Furthermore, multiplex analysis is quicker and cheaper in comparison with ELISA and can be carried out in non-laboratory conditions. The kit for multiplex dot-immunoassay of antibodies to blood transmitted agents can significantly simplify initial complex testing. PMID:27497868

  13. Calibration of Hydraulic Conductivities by the Kalman Filtered Double Constraint Method

    NASA Astrophysics Data System (ADS)

    Zijl, Wouter; El-Rawy, Mustafa; Batelaan, Okke

    2014-05-01

    To assess the consequences of a changing environment for future management decisions we need quantitative techniques validated by case studies. In this context dealing with the limited data availability and inherent uncertainty is a major challenge. In this contribution we present a combination of two techniques (the Double Constraint Method and the Kalman Filter) exemplified by case studies. The techniques assist in the calibration of hydraulic grid block conductivities as well as in finding the reliability of the result. To focus on the basic principles we exemplify our approach for flow in which storage by water compressibility and pore space deformation is negligible. Only storage by water table movements plays a role. Such conditions hold for most flow problems in the relatively shallow aquifer-aquitard systems of deltaic regions. In a forward problem the conductivity is specified in all grid blocks. In addition, on each point of the boundary and in each well only one type of boundary condition has to be specified: either head, or flux. Our approach is based on the principle that calibration of the initial conductivities is meaningful only if we can specify both head and flux at a number of boundary points or wells, including no-flux monitoring wells. In general a hydrogeological model is a "flux model," i.e., the model is as much as possible based on specified ("measured") fluxes through the boundaries and in the wells. An exception is Tóth's flow systems analysis where, instead of the usual recharge fluxes, heads are specified on the water table. This suggests building a second forward model, a "head model," that is as much as possible based on specified (measured) heads on the boundaries and in the wells. The initial conductivities are then updated by applying Darcy's law K = -q/(¶h/¶x) to the fluxes q obtained by the "flux model" and the head gradients ¶h/¶x obtained by the "head model." This so-called "double constraint method" (DCM) leads to a

  14. 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. PMID:24854328

  15. Double ionization of helium by fast electrons with the Generalized Sturmian Functions method

    NASA Astrophysics Data System (ADS)

    Ambrosio, M. J.; Colavecchia, F. D.; Gasaneo, G.; Mitnik, D. M.; Ancarani, L. U.

    2015-03-01

    The double ionization of helium by high energy electron impact is studied. The corresponding four-body Schrödinger equation is transformed into a set of driven equations containing successive orders in the projectile-target interaction. The first order driven equation is solved with a generalized Sturmian functions approach. The transition amplitude, extracted from the asymptotic limit of the first order solution, is equivalent to the familiar first Born approximation. Fivefold differential cross sections are calculated for (e, 3e) processes within the high incident energy and small momentum transfer regimes. The results are compared with other numerical methods, and with the only absolute experimental data available. Our cross sections agree in shape and magnitude with those of the convergent close coupling method for the (10+10) eV and (4+4) eV emission energies. To date this had not been achieved by any two different numerical schemes when solving the three-body continuum problem for the fast projectile (e, 3e) process. Though agreement with the experimental data, in particular with respect to the magnitude, is not achieved, our findings partly clarify a long standing puzzle.

  16. 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.

  17. A blind image detection method for information hiding with double random-phase encoding

    NASA Astrophysics Data System (ADS)

    Sheng, Yuan; Xin, Zhou; Jian-guo, Chen; Yong-liang, Xiao; Qiang, Liu

    2009-07-01

    In this paper, a blind image detection method based on a statistical hypothesis test for information hiding with double random-phase encoding (DRPE) is proposed. This method aims to establish a quantitative criterion which is used to judge whether there is secret information embedded in the detected image. The main process can be described as follows: at the beginning, we decompose the detected gray-scale image into 8 bit planes considering it has 256 gray levels, and suppose that a secret image has been hidden in the detected image after it was encrypted by DRPE, thus the lower bit planes of the detected image exhibit strong randomness. Then, we divide the bit plane to be tested into many windows, and establish a statistical variable to measure the relativity between pixels in every window. Finally, judge whether the secret image exists in the detected image by operating the t test on all statistical variables. Numerical simulation shows that the accuracy is quite satisfactory, when we need to distinguish the images carrying secret information from a large amount of images.

  18. 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.

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

    PubMed

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

    2016-01-01

    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

  20. Evaluation of a method for monoclonal antibody HLA-B27 analysis with the CELL-DYN Sapphire haematology analyser.

    PubMed

    Aarsand, A K; Johannessen, H B; Scott, C S

    2007-12-01

    Analysis of HLA-B27 is usually performed by flow cytometry using commercial single or two colour fluorescence reagents. The CELL-DYN Sapphire (CD-Sapphire) is a high-volume routine haematology analyser that allows cell population analysis by monoclonal antibody fluorochromes analogous to flow cytometry. In this study, in-house flow cytometry analysis (n = 96, HLA-B27, One Lambda) performed on routine patient samples was used as the comparison method for analysis of HLA-B27, One Lambda (n = 40) and HLA-B27/HLA-B7, Immunotech (n = 96) reagents on the CD-Sapphire. The One Lambda results agreed 100% with the comparison method and offered clear population discrimination. The Immunotech combination also had a high level of agreement, but interpretation was more complex because of the wider cross-reactivity of the ABC-m3 antibody with B7 and other HLA-B alleles. When analysing HLA-B27 with antibodies showing nonspecific reactivity, a cut-off staining level yielding high specificity should be chosen, as the primary diagnostic value of HLA-B27 is as a 'rule-out' test for ankylosing spondylitis. The CD-Sapphire incorporates automated sampling and lysis, and medical scientists familiar with the instrument would require little additional technical training to perform the analysis. The reduced preanalytical work and total turnaround time constitute an important step towards automation of HLA-B27 and similar simple high-volume flow cytometry analysis. PMID:17988301

  1. Automated microaneurysm detection method based on double ring filter in retinal fundus images

    NASA Astrophysics Data System (ADS)

    Mizutani, Atsushi; Muramatsu, Chisako; Hatanaka, Yuji; Suemori, Shinsuke; Hara, Takeshi; Fujita, Hiroshi

    2009-02-01

    The presence of microaneurysms in the eye is one of the early signs of diabetic retinopathy, which is one of the leading causes of vision loss. We have been investigating a computerized method for the detection of microaneurysms on retinal fundus images, which were obtained from the Retinopathy Online Challenge (ROC) database. The ROC provides 50 training cases, in which "gold standard" locations of microaneurysms are provided, and 50 test cases without the gold standard locations. In this study, the computerized scheme was developed by using the training cases. Although the results for the test cases are also included, this paper mainly discusses the results for the training cases because the "gold standard" for the test cases is not known. After image preprocessing, candidate regions for microaneurysms were detected using a double-ring filter. Any potential false positives located in the regions corresponding to blood vessels were removed by automatic extraction of blood vessels from the images. Twelve image features were determined, and the candidate lesions were classified into microaneurysms or false positives using the rule-based method and an artificial neural network. The true positive fraction of the proposed method was 0.45 at 27 false positives per image. Forty-two percent of microaneurysms in the 50 training cases were considered invisible by the consensus of two co-investigators. When the method was evaluated for visible microaneurysms, the sensitivity for detecting microaneurysms was 65% at 27 false positives per image. Our computerized detection scheme could be improved for helping ophthalmologists in the early diagnosis of diabetic retinopathy.

  2. A simple in vitro method of evaluating monoclonal antibody binding to tumor tissue

    SciTech Connect

    Rusckowski, M.; Hnatowich, D.J.; Doherty, P.W.; Virzi, F.; Bogden, A.E.

    1984-01-01

    Application of hybridoma technology has resulted in the availability of many monoclonal antibodies (MoAbs) which are potentially useful for in vivo tumor localization in cancer diagnosis and therapy. What is now required is a simple and predictive assay which will select those MoAbs with superior properties for clinical trials. The authors have developed a simple in vitro means of evaluating the relative binding properties of a panel of MoAbs to human tumor tissue. Fresh and frozen surgical explants of human colorectal and breast tumor tissue as 1 mm cubes were incubated in microtiter-plate wells containing media and radio-labelled MoAb. A MoAb specific for carcinoembryonic antigen (anti-CEA) was used as a known colorectal-specific MoAb and an anti-prostatic acid phosphatase MoAb (anti-PAP) was chosen to measure non-specific binding. The MoAbs were coupled with DTPA and labeled with /sup 111/In. Kinetic parameters were measured which define the rate of MoAb uptake and levels of saturation in tissue. In colorectal tumor tissue the anti-CEA:anti-PAP binding ratio was about 10 while in breast tumor tissue this value was obtained by saturating the tumor antigenic sites with excess unlabeled anti-CEA: the levels of binding of specific MoAb was reduced to that of the control MoAb. Equivalent results were obtained with the same tissue which had been stored at -70/sup 0/C for 24 hours. Evaluation of 7 anti-breast and 4 anti-colorectal MoAbs in their respective tumor tissues showed good reproducibility of repeat measurements and 1-15 fold differences in binding between different antibodies in the same tissue. The authors' results suggest that this in vitro assay may help to identify superior MoAb for human trials.

  3. Development and Evaluation of a Direct Fluorescent Antibody Method for the Diagnosis of Pneumocystis carinii Infections in Experimental Animals

    PubMed Central

    Lim, Sook Kyung; Eveland, Warren C.; Porter, Richard J.

    1973-01-01

    Because no fully satisfactory diagnostic method has been available for use in pneumocystis infection, an attempt was made to apply the fluorescent antibody technique in the identification of Pneumocystis carinii. Hyperimmune sera were prepared in rabbits against P. carinii from human and rat sources. After proper adsorption, these antisera were conjugated with fluorescein isothiocyanate and used as reagents in a direct fluorescent antibody procedure. Each of the two reagents was found to stain trypsin-treated P. carinii organisms from either human or rat sources, indicating the presence of common antigens. Stained organisms were demonstrated in the hypopharyngeal material from rats in which pneumocystis infection had been activated by the administration of corticosteroid. From the results reported here, the procedure outlined is considered sufficiently sensitive and specific to justify tests on pneumocystis infections in man. The findings in a series of specimens from human subjects will be reported separately. The method also provides an extended approach to related research problems. The need for controls of the procedure at all points is emphasized. Images PMID:4202704

  4. Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody.

    PubMed

    Schneeweis, Lumelle A; Obenauer-Kutner, Linda; Kaur, Parminder; Yamniuk, Aaron P; Tamura, James; Jaffe, Neil; O'Mara, Brian W; Lindsay, Stuart; Doyle, Michael; Bryson, James

    2015-12-01

    Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy. PMID:26343417

  5. 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.

  6. 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.

  7. 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.

  8. Preparation and in vitro evaluation of ethyl cellulose microspheres containing stavudine by the double emulsion method.

    PubMed

    Sahoo, S K; Mallick, A A; Barik, B B; Senapati, P C

    2007-02-01

    The aim of this study was to formulate and evaluate microspheres of stavudine by water-in-oil-in-oil (w/o/o) double emulsion solvent diffusion method using ethyl cellulose and ethyl cellulose in combination with polyvinyl pyrrolidone. A mixed solvent system consisting of acetonitrile and dichloromethane in an 1: 1 ratio and light liquid paraffin was chosen as primary and secondary oil phase, respectively. Span 80 was used as surfactant for stabilizing the secondary oil phase. The influence of formulation factors like stirring speed, surfactant concentration on particle size and polymer:drug ratio and combination of polymers on drug release characteristics of the microspheres was investigated. The prepared microspheres characterized by micrometric properties, drug loading, Fourier transform infrared spectroscopy, X-ray powder difractometry and scanning electron microscopy. The prepared microspheres were white, free flowing and spherical in shape, stable in nature, with 41-65% of drug entrapment efficiency. The best-fit release kinetics was achieved with Higuchi plot followed by first order and zero order. The release of stavudine was influenced by the drug to polymer ratio, particle size and polymer combination. PMID:17341031

  9. 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.

  10. Radiation dose determines the method for quantification of DNA double strand breaks.

    PubMed

    Bulat, Tanja; Keta, Otilija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-03-01

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. PMID:26959322