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Sample records for chain antibody constructs

  1. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  2. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  3. Charge-modified single chain antibody constructs of monoclonal antibody CC49: generation, characterization, pharmacokinetics, and biodistribution analysis

    International Nuclear Information System (INIS)

    A novel strategy was developed in which an antibody scFv fragment of the monoclonal antibody (MAb) CC49 was modified by engineering DNA coding sequences to lower its isoelectric point. Negatively charged amino acids were added to the carboxy terminus of the CC49 VH region by adding nucleotide sequences in a polymerase chain reaction (PCR) amplification of the coding sequence of CC49 scFv. Two new DNA constructs coding for CC49 scFv with lower isoelectric points of 5.8 and 5.2 were engineered. These novel strategy-generated, charge-modified antibody constructs were compared for their immunological, pharmacokinetic, and biodistribution properties in athymic mice bearing LS-174T human colon carcinoma xenografts

  4. Construction of Large Human Single-chain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half-nest PCR, and assembly by two-way fusion PCR. In this stud y, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other rese arches.

  5. Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

    OpenAIRE

    DAN, ZHAOKUI; Tan, Zui; Xia, Hongli; WU, GAN

    2013-01-01

    The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing...

  6. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    Science.gov (United States)

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated. PMID:26945728

  7. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud;

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  8. Single Chain Fv Constructs of Anti-Ganglioside GD2 Antibodies for Radioimaging and Radioimmunotherapy

    International Nuclear Information System (INIS)

    of its broad and usually homogeneous distribution in human solid tumors, and most importantly, their absence on cell membranes of normal human tissues. In separate experiments, we have shown that T-cells transduced with the herpes simplex viral thymidine kinase (HSV-tk) gene can be radiolabeled with 131I-FIAU to a safe nuclear radiation dose. Using a dicistronic construct we are inserting chimeric immune receptor plus HSV-tk into T-cells to allow such their trafficking to be radioactively monitored. We plan to study the role of cytokines, chemoreceptors and CD4 helper T-cells in recruiting CD8+ transduced T-cells to the tumor site. These studies should provide us with an adoptive cell therapy approach to target cytotoxicity to human tumors, and a lymphocyte tracking tool to study delivery to the tumor sites, to determine if they proliferate locally and/or recirculate. Such pharmacologic information is crucial for optimizing gene-modified T-cells in future clinical trials. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging

  9. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    Science.gov (United States)

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples. PMID:26693887

  10. Generation and characterization of heavy chain antibodies derived from Camelids

    OpenAIRE

    Schmidthals, Katrin

    2013-01-01

    Antibodies and antibody fragments are essential tools in basic research, diagnostics and therapy. Conventional antibodies consist of two heavy and two light chains with both chains contributing to the antigen-binding site. In addition to these conventional antibodies, camelids (llamas, alpacas, dromedaries and camels) possess so-called heavy chain antibodies (hcAbs) that lack the light chains. The antigen binding site of these unusual antibodies is formed by one single domain only, the so cal...

  11. Method for preparation of single chain antibodies

    Science.gov (United States)

    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.

  12. Construction and characterization of a non-immune Llama variable heavy chain phage display antibody library%羊驼非免疫重链单域抗体库的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    吴标; 王树军; 夏立亮; 季萍; 葛海良; 赵国屏; 王颖

    2011-01-01

    本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础.我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性.结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性.上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础.%To construct a non-immune Llama variable domain of heavy chain antibody(VHH) phage display antibody library (VHH antibody library). Llama peripheral blood mononuclear lymphocytes were isolated from whole blood by Ficoll-hypaque density gradient centrifugation. Total RNA was extracted from PBMCs and reverse-transcripted into cDNA by using specific primers. VHH were amplified by nested PCR. PCR products of VHH fragments were then purified and ligated with phagemid vector pCANTAB5E by a modified two-step ligation method. Recombinant pCANTAB5E-VHH vectors were electroporated into competent TGI E.coli cells to obtain the primary VHH antibody library. The library capacity was titrated through limited dilution. Recombination efficacy and diversity of VHH antibody library was determined by sequencing analysis. Alignment a-nalysis was performed to compare the homology between Llama VHH domain and human/mouse variable regions of heavy chains. By using a modified strategy, we have constructed a non-immune Llama VHH antibody library with 1. 5

  13. Pharmacokinetics and biodistribution of 177Lu-labeled multivalent single-chain Fv construct of the pancarcinoma monoclonal antibody CC49

    International Nuclear Information System (INIS)

    Lutetium-177 (177Lu) is a radionuclide of interest for radioimmunoimaging (RII) and radioimmunotherapy (RIT) on account of its short half-life (161 h) and the ability to emit both β and γ radiation. Single-chain Fv (scFv) constructs have shown advancement in cancer diagnosis and therapy due to the pharmacokinetics advantage and seem to be intriguing tools in oncology. The objective of this study was to evaluate the pharmacokinetics and biodistribution characteristics of the 177Lu-labeled tetravalent scFv of CC49 MAb and intact CC49 IgG in vivo. Conjugation and labeling conditions of multivalent scFv with 177Lu were optimized without affecting integrity and immunoreactivity. For this purpose, multivalent scFv constructs dimer, sc(Fv)2; tetramer, [sc(Fv)2]2 of the MAb CC49 were expressed as secretory proteins in Pichia pastoris. The purified scFv constructs and IgG form of CC49 were conjugated with a bifunctional chelating agent, ITCB-DTPA, and labeled with 177Lu. The comparative biodistribution, blood clearance, and tumor-targeting characteristics of 177Lu-labeled tetravalent [sc(Fv)2 ]2 construct of CC49 MAb and intact CC49 IgG were investigated in the athymic mice bearing LS-174T xenografts. Approximately, 90% of 177Lu incorporation was achieved using ITCB-DTPA chelator, and the labeled immunoconjugates maintained integrity and immunoreactivity. Blood clearance studies demonstrated an alpha half-life (t1/2 α) of 177Lu-labeled [sc(Fv)2 ]2 and IgG of CC49 at 4.40 and 9.50 min and a beta half-life (t1/2 β) at 375 and 2,193 min, respectively. At 8 h post administration, the percent of the injected dose accumulated/gram (%ID/g) of the LS-174T tumor was 6.4 ±1.3 and 8.9 ±0.6 for 177Lu-labeled [sc(Fv)2 ]2 and IgG of CC49, respectively, in the absence of l-lysine. The corresponding values were 8.0 ±0.6 and 8.4 ±1.2 in the presence of l-lysine. Renal accumulation of [sc(Fv)2 ]2 was significantly (p <0.005) reduced in the presence of l-lysine. (orig.)

  14. Pharmacokinetics and biodistribution of {sup 177}Lu-labeled multivalent single-chain Fv construct of the pancarcinoma monoclonal antibody CC49

    Energy Technology Data Exchange (ETDEWEB)

    Chauhan, Subhash C.; Jain, Maneesh; Moore, Erik D.; Wittel, Uwe A.; Batra, Surinder K. [University of Nebraska Medical Center, Department of Biochemistry and Molecular Biology, Omaha, NE (United States); Li, Jing; Gwilt, Peter R. [University of Nebraska Medical Center, College of Pharmacy, Omaha, NE (United States); Colcher, David [Beckman Research Institute at City of Hope National Medical Center, Department of Radioimmunotherapy, Duarte, CA (United States)

    2005-03-01

    Lutetium-177 ({sup 177}Lu) is a radionuclide of interest for radioimmunoimaging (RII) and radioimmunotherapy (RIT) on account of its short half-life (161 h) and the ability to emit both {beta} and {gamma} radiation. Single-chain Fv (scFv) constructs have shown advancement in cancer diagnosis and therapy due to the pharmacokinetics advantage and seem to be intriguing tools in oncology. The objective of this study was to evaluate the pharmacokinetics and biodistribution characteristics of the {sup 177}Lu-labeled tetravalent scFv of CC49 MAb and intact CC49 IgG in vivo. Conjugation and labeling conditions of multivalent scFv with {sup 177}Lu were optimized without affecting integrity and immunoreactivity. For this purpose, multivalent scFv constructs dimer, sc(Fv){sub 2}; tetramer, [sc(Fv){sub 2}]{sub 2} of the MAb CC49 were expressed as secretory proteins in Pichia pastoris. The purified scFv constructs and IgG form of CC49 were conjugated with a bifunctional chelating agent, ITCB-DTPA, and labeled with {sup 177}Lu. The comparative biodistribution, blood clearance, and tumor-targeting characteristics of {sup 177}Lu-labeled tetravalent [sc(Fv){sub 2} ]{sub 2} construct of CC49 MAb and intact CC49 IgG were investigated in the athymic mice bearing LS-174T xenografts. Approximately, 90% of {sup 177}Lu incorporation was achieved using ITCB-DTPA chelator, and the labeled immunoconjugates maintained integrity and immunoreactivity. Blood clearance studies demonstrated an alpha half-life (t{sub 1/2} {alpha}) of {sup 177}Lu-labeled [sc(Fv){sub 2} ]{sub 2} and IgG of CC49 at 4.40 and 9.50 min and a beta half-life (t{sub 1/2} {beta}) at 375 and 2,193 min, respectively. At 8 h post administration, the percent of the injected dose accumulated/gram (%ID/g) of the LS-174T tumor was 6.4 {+-}1.3 and 8.9 {+-}0.6 for {sup 177}Lu-labeled [sc(Fv){sub 2} ]{sub 2} and IgG of CC49, respectively, in the absence of l-lysine. The corresponding values were 8.0 {+-}0.6 and 8.4 {+-}1.2 in the

  15. Improving construction performance through supply chain management

    Institute of Scientific and Technical Information of China (English)

    王要武; 薛小龙

    2004-01-01

    A typical model of construction supply chain (CSC) is presented firstly. Then the characteristics and problems of CSC are identified. The authors also describe the progress towards adoption of supply chain management (SCM) in the construction industry. Traditional construction management approaches (CMAs) are compared with new SCM-based CMAs. The authors suggest that integrated SCM based on cooperating competition is crucial to improve construction performance. Based on relevant results from related research, a framework for integrated construction supply chain management (CSCM) is developed to guide the effective implementation of SCM in construction.

  16. Understanding Construction Supply Chains: An Alternative Interpretation

    OpenAIRE

    Vrijhoef, Ruben; Koskela, Lauri; Howell, Greg

    2001-01-01

    Much research work has assessed that construction is ineffective and many problems can be observed. Analysis of these problems has shown that a major part of them are supply chain problems, originating at the interfaces of different parties or functions. There have been several kinds of initiatives aiming at improvement and renewal of construction supply chains, but only few have a track record of consequent and significant successes. Here construction supply chains are approached from an alt...

  17. A recombinant single chain antibody interleukin-2 fusion protein.

    OpenAIRE

    Savage, P; So, A; Spooner, R A; Epenetos, A. A.

    1993-01-01

    Recombinant interleukin-2 (rIL-2) therapy has been shown to be of value in the treatment of some cases of melanoma and renal cell carcinoma. However its use can be limited by severe systemic toxicity. Targeting rIL-2 to the tumour should improve the anti-tumour immune response and decrease the systemic toxicity. With this aim we have employed recombinant DNA techniques to construct a single chain antibody interleukin-2 fusion protein (SCA-IL-2). The protein used in this model system comprises...

  18. Construction of human single-chain variable fragment antibodies of medullary thyroid carcinoma and single photon emission computed tomography/computed tomography imaging in tumor-bearing nude mice.

    Science.gov (United States)

    Liu, Qiong; Pang, Hua; Hu, Xiaoli; Li, Wenbo; Xi, Jimei; Xu, Lu; Zhou, Jing

    2016-01-01

    Medullary thyroid carcinoma (MTC) is a rare tumor of the endocrine system with poor prognosis as it exhibits high resistance against conventional therapy. Recent studies have shown that monoclonal antibodies labeled with radionuclide have become important agents for diagnosing tumors. To elucidate whether single-chain fragment of variable (scFv) antibody labeled with 131I isotope is a potential imaging agent for diagnosing MTC. A human scFv antibody library of MTC using phage display technique was constructed with a capacity of 3x10(5). The library was panned with thyroid epithelial cell lines and MTC cell lines (TT). Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to identify the biological characteristics of the panned scFv. Methyl thiazolyl tetrazolium (MTT) assay was also used to explore the optimal concentration of the TT cell proliferation inhibition rate. They were categorized into TT, SW480 and control groups using phosphate-buffered saline. Western blotting showed that molecular weight of scFv was 28 kDa, cell ELISA showed that the absorbance of TT cell group was significantly increased (P=0.000??) vs. the other three groups, and MTT assay showed that the inhibition rate between the two cell lines was statistically significantly different (P<0.05) when the concentration of scFv was 0.1, 1 and 10 µmol/l. The tumor uptake of 131I-scFv was visible at 12 h and clear image was obtained at 48 h using the single photon emission computed tomography. scFv rapidly and specifically target MTC cells, suggesting the potential of this antibody as an imaging agent for diagnosing MTC. PMID:26498224

  19. Supply Chain Management in the Construction Industry

    OpenAIRE

    Olsson, Fredrik

    2000-01-01

    In this licentiate thesis the implications of a deliberate use of logistics knowledge have been investigated by describing and analyzing the use of a supply chain management approach in the construction industry.
    A five-step approach is used in the thesis. First, theoretical models are developed from the supply chain management literature. Two models are elaborated; One model identifying supply chain criteria and the other comparing traditional to supply chain-based.

  20. Stability of llama heavy chain antibody fragments under extreme conditions

    OpenAIRE

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The applications in which these VHHs are to be used, require functionality in non-physiological environments. High temperature, anionic and non-ionic surfactants in shampoo, and the low pH and digestive ...

  1. Recombinant anti-tenascin antibody constructs

    Energy Technology Data Exchange (ETDEWEB)

    ZALUTSKY, MICHAEL R

    2006-08-29

    The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

  2. Achieving Supply Chain Integration within Construction Industry

    OpenAIRE

    Peter McDermotti; Malik Khalfan

    2012-01-01

    The main driver behind the adoption of supply chain management (SCM) philosophy into the construction industry was the successes within other industry sectors. SCM can be defined as network of different organisations, linked upstream and downstream in a chain, aiming to produce quality and value in the services and products for the end consumers through integrated processes and activities. In order to achieve the optimised level of integration of the whole supply chain, the industry has respo...

  3. Chain specificity assignment of monoclonal antibodies to human laminins by using recombinant laminin beta1 and gamma1 chains.

    Science.gov (United States)

    Geberhiwot, T; Wondimu, Z; Salo, S; Pikkarainen, T; Kortesmaa, J; Tryggvason, K; Virtanen, I; Patarroyo, M

    2000-05-01

    In the present study, the chain specificity of 16 commonly used monoclonal antibodies to human laminin(s) was analysed by using recombinant laminin beta1 and gamma1 chains. By ELISA, all antibodies reacted with purified placenta laminin, and most antibodies recognised either recombinant beta1 or gamma1 chains. Reactivity and chain specificity was confirmed against the recombinant chains in Western blotting under non-reducing conditions, and only a few antibodies were reactive under reducing conditions. Most antibodies were able to immunoprecipitate associated laminin beta1/gamma1 chains from platelet lysates. Based on these results and data from the literature, a tentative epitope map is presented. PMID:10842099

  4. Quality in Construction- A Supply Chain Perspective

    DEFF Research Database (Denmark)

    Koch, Christian; Larsen, Casper Schultz

    2006-01-01

    transformed into the material stream. The paper proposes initiatives to strengthen partnerships in supply chains, especially at mixed stable and project configured types. The contradiction between permanent enterprise organisations potentially capable of handling purchasing and the role of the project manager......Often the construction supply chain is perceived as consisting of two intertwined streams; a stream of materials and a stream of immaterial informational character. Moreover the supply delivery would be characterized by configuration by project. That this configuration is not always successful can...

  5. Construction of human Alzheimer's disease specific single chain antibodies library%人源性阿尔茨海默病噬菌体单链抗体库的构建

    Institute of Scientific and Technical Information of China (English)

    李楠; 王建平

    2015-01-01

    Objective To construct the human Alzheimer's disease (AD) specific single chain anti‐bodies (scFv) library for sceening human AD scFv to Aβ1‐42 oligomers .Methods RNA was isola‐ted from 40 ml peripheral blood taken from 18 AD patients .Variable heavy (V H ) and variable light (VL ) genes were amplified by RT‐PCR and linked to the scFv fragments that were then cloned into the phage vector pCANTAB5E .The scFv library was constructed by electroporating E .coli TG1 cells into the pCANTAB5E and rescuing the assistant phage M13K07 .Results The total RNA was extracted .The VH and VL genes were amplified .Electropharesis showed that the length of VH and VL genes was 360bp and 300bp respectively and the length of linked scFv was 750bp .The 2 .4 × 109 scFv library was thus constructed and identified by BstN I digestion .Elec‐tropharesis showed that the length of BstN I‐digested scFv fragments varied ,indicating that the library is of a good diversity .Conclusion The human AD phage scFv library we constructed lays a foundation for screening the antibodies to Aβ1‐42 oligomers and the treatment of AD .%目的:构建人源性阿尔茨海默病(AD)噬菌体单链抗体(scFv)库,为筛选β淀粉样蛋白(Aβ1‐42)的人源性特异性抗体奠定基础。方法采集18例AD患者的外周血40 ml ,提取总RNA ,应用RT‐PCR法得到人抗体可变区重链(V H )和可变区轻链(V L )基因。将 V H 和 V L 由连接肽连接得到 scFv 片段,将所得片段双酶切后,克隆至pCANTAB5E噬菌体载体,大肠埃希菌TG1感受态细胞经电击转化,辅助噬菌体 M13K07拯救后构建scFv型噬菌体抗体库。结果总RNA经逆转录PCR扩增VH和VL可变区基因的凝胶电泳显示,PCR产物长度分别为360 bp和300 bp ,其连接形成的scFv片段长度为750 bp ,最终构建了库容为2.4×109的scFv库。BstNⅠ酶切鉴定构建的scFv库,经凝胶电泳可见,酶切片段长度差异性大,

  6. Optimization modeling of single-chain antibody against hepatoma based on similarity algorithm.

    Science.gov (United States)

    Zhao, Zhi-Jun; Chen, Jing-Tao; Yuan, Jia-Ying; Yin, Xiao-Xiang; Song, Hua-Yong; Wang, Xin-Chun

    2015-01-01

    and construction of single-chain antibody. PMID:26628964

  7. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lin-Xu [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Mellon, Michael [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Bowder, Dane [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Quinn, Meghan [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Shea, Danielle; Wood, Charles [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Xiang, Shi-Hua, E-mail: sxiang2@unl.edu [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States)

    2015-01-15

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.

  8. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    International Nuclear Information System (INIS)

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture

  9. Construction of a human antibody domain (VH) library

    OpenAIRE

    Chen, Weizao; Zhu, Zhongyu; Xiao, Xiaodong; Dimitrov, Dimiter S.

    2009-01-01

    Highly diverse antibody (Fab or scFv) libraries have become vital sources to select antibodies with high affinity and novel properties. Combinatorial strategies provide efficient ways of creating antibody libraries containing a large number of individual clones. These strategies include the reassembly of naturally occurring genes encoding the heavy and light chains from either immune or nonimmune B-cell sources, or introduction of synthetic diversity to either the framework regions (FRs) or t...

  10. Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

    Institute of Scientific and Technical Information of China (English)

    FANG Min; JIANG Xin; YANG Zhi; YIN Changcheng; LI Hua; ZHAO Rui; ZHANG Zhong; LIN Qing; HUANG Hualiang

    2003-01-01

    Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequences on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominated as Fc, HSA, 205C′, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E. coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression levelof scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequences had difference in the antigen-binding activities and terminal half-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of interlinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biologicalproperties more suitable for clinical application.

  11. Characterization of single chain antibody targets through yeast two hybrid

    Directory of Open Access Journals (Sweden)

    Vielemeyer Ole

    2010-08-01

    Full Text Available Abstract Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID, efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise

  12. Antibodies against the light chain of tetanus toxin in human sera.

    OpenAIRE

    Lin, C S.; Habig, W H; Hardegree, M C

    1985-01-01

    Tetanus toxoid elicits protective antibodies against tetanus toxin in humans and animals. It has been reported that antitoxin from immunized humans contains no anti-light chain antibodies, based on immunodiffusion and quantitative precipitin analyses. We confirmed the absence of precipitating anti-light chain antibodies in tetanus immune globulin. However, the presence of antibodies against the light chain of the toxin was shown by direct binding and inhibition analyses, using enzyme-linked i...

  13. Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Jiong Cai; Fang Li; Shizhen Wang

    2008-01-01

    BACKGROUND: Studies have shown that monoclonal or polyclonal antibody injections ofamyloid β peptide arc effective in removing amyloid β peptide overload in the brain.OBJECTIVE: Based on successful screening of a human single-chain fragment variable antibody specific to amyloid β peptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide.DESIGN, TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006.MATERIALS: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library.METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a Teasy plasmid for pT-seFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA β was cut by EcoRl and Notl endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvA β expression vector, which was confirmed by gene sequencing. Linearized pPICgK-scFvA β was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5 % methanol to express human single-chain fragment variable antibody specific to amyloid β peptide.MAIN OUTCOME MEASURES: Protein electrophoresis was used to identify PCR products, gene sequencing was uscd to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris.RESULTS: Gene sequencing confirmed pPICgK-scFvA β orientation. Rccomhinants were obtained by lineadzed pPIC9K-scFvA β transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size

  14. Embending Sustainability Dynamics in the Lean Construction Supply Chain Management

    Directory of Open Access Journals (Sweden)

    Sertyesilisik Begum

    2016-07-01

    Full Text Available The world’s habitat is being deteriorated despite of the precautions taken. Construction industry is among the industries which highly effect the environment adversely not only through its outputs but also through the construction process and its inputs. The main focus in dealing with the reduction of its footprint has been on sustainable building certificates which mainly analyse the output of the construction activies. There is need to analyse the construction supply chain as a whole and to embed sustainability dynamics in construction supply chain management. Lean construction project management contributes to the reduction of the environmental footprint of the construction industry, enabling reduction in waste, and increasing value added activities. For this reason, based on an in depth literature review, this paper analyses and establishes the principles of the integration of the sustainability dynamics into lean construction supply chain management.

  15. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2008-07-01

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks resulting in a panel of scFvs specific for the target antigen.

  16. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2009-08-02

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magneticactivated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

  17. Y-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

    Institute of Scientific and Technical Information of China (English)

    王字玲; 荣康泰; 杨日芳; 恽榴红

    2000-01-01

    A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitro-phenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcatt= 198 min-1 and the rate enhancement kcatkuncat = 122 419. When 0.16 mg · mL-1

  18. Recent advances in the construction of antibody-drug conjugates

    Science.gov (United States)

    Chudasama, Vijay; Maruani, Antoine; Caddick, Stephen

    2016-02-01

    Antibody-drug conjugates (ADCs) comprise antibodies covalently attached to highly potent drugs using a variety of conjugation technologies. As therapeutics, they combine the exquisite specificity of antibodies, enabling discrimination between healthy and diseased tissue, with the cell-killing ability of cytotoxic drugs. This powerful and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers with two US Food and Drug Administration approved ADCs currently on the market (Adcetris and Kadcyla) and approximately 40 currently undergoing clinical evaluation. However, most of these ADCs exist as heterogeneous mixtures, which can result in a narrow therapeutic window and have major pharmacokinetic implications. In order for ADCs to deliver their full potential, sophisticated site-specific conjugation technologies to connect the drug to the antibody are vital. This Perspective discusses the strategies currently used for the site-specific construction of ADCs and appraises their merits and disadvantages.

  19. Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein.

    Science.gov (United States)

    Yuan, Ruosen; Chen, Xiaoxu; Chen, Yan; Gu, Tiejun; Xi, Hualong; Duan, Ye; Sun, Bo; Yu, Xianghui; Jiang, Chunlai; Liu, Xintao; Wu, Chunlai; Kong, Wei; Wu, Yongge

    2014-02-01

    Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes. PMID:24241896

  20. Construction of a rationally designed antibody platform for sequencing-assisted selection.

    Science.gov (United States)

    Larman, H Benjamin; Xu, George Jing; Pavlova, Natalya N; Elledge, Stephen J

    2012-11-01

    Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ~10(12)-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform. PMID:23064642

  1. Synthesised genes of VH and VL of single-chain antibody of human cardiac myosin heavy chain

    International Nuclear Information System (INIS)

    Objective: To synthesize genes of the heavy chain variable region (VH) and light chain variable region (VL) of single-chain antibody of human cardiac myosin heavy chain for the development of myocardial imaging agents: single-chain antibody of human cardiac myosin heavy chain. Methods: To extracted total RNA of the anti-HCMHC McAb hybridoma using TRIZOL reagent, synthesize the first-strand cDNA, using this first-strand cDNA as template, with specific primers, DNA polymerase and four single nucleotides, amplify the genes of the heavy chain variable region (VH) and light chain variable region (VL) by PCR. To identify products of each step and study relationship between RNA stability and storage temperature and optimize cycle selection temperature with MgCl2 concentration. Results: The purity of the first-strand cDNA reached 95%, PCR products by agarose gel electrophoresis showed a single band with a bright swimming, molecular weight of VH and VL genes were 340bp and 320bp, which was consistent with literature reports. Conclusion: Synthesized genes of VH and VL, laid the foundation for the development of myocardial imaging agents: single-chain antibody of human cardiac myosin heavy chain. (authors)

  2. Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

    Science.gov (United States)

    Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

    1994-05-01

    Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

  3. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao

    2001-01-01

    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  4. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  5. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs).

    Science.gov (United States)

    Maass, David R; Sepulveda, Jorge; Pernthaner, Anton; Shoemaker, Charles B

    2007-07-31

    Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor alpha (TNFalpha) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFalpha. PMID:17568607

  6. New Genetic Constructs for Generation of Stable Therapeutic Antibodies to Organophosphorus Toxins in Methylotrophic Yeasts Pichia Pastoris.

    Science.gov (United States)

    Mokrushina, Yu A; Stepanova, A V; Bobik, T V; Smirnov, I V; Gabibov, A G

    2016-05-01

    We propose a new method of obtaining of stable Fab-fragments of antibodies in Pichia pastoris expression system. Recently, we obtained Fab-fragments of antibodies neutralizing organophosphorus toxins. However, high yield of the target products was not attained because of high level of proteolytic degradation. In the present study, we identified sites of proteolytic degradation in Fab-fragments and endogenous proteases performing degradation, which allowed obtaining optimized genetic constructs for expression of antibody heavy chains (IgGγ1) and kappa and lambda isotypes of light chains. Co-transformation of these vectors allowed obtaining Fab-fragments of antibodies to organophosphorus toxins without proteolytic degradation of the product. PMID:27270933

  7. A repertoire of monoclonal antibodies with human heavy chains from transgenic mice

    International Nuclear Information System (INIS)

    The introduction of human immunoglobulin gene segments in their unrearranged configuration into the germ line of mice might allow the production of a repertoire of human antibodies. Such transgenic mice could be used for the production of human monoclonal antibodies against human antigens. To test the feasibility of this approach, mice were created that carry a human heavy-chain minilocus comprising unrearranged immunoglobulin variable, diversity, and joining elements linked to a human μ-chain gene. The gene segments of this minilocus are rearranged in a large proportion of cells in thymus and spleen but not in nonlymphoid tissue. Some 4% of the B lymphocytes synthesize human μ chains resulting in a serum titer of about 50 μg of transgenic IgM antibody per ml. Hybridomas were established from the transgenic mice that stably secreted several micrograms of antibodies containing human μ heavy chains per milliliter

  8. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs)

    OpenAIRE

    Maass, David R.; Sepulveda, Jorge; Pernthaner, Anton; Shoemaker, Charles B.

    2007-01-01

    Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the...

  9. Construct validation of supply chain management in cooperative

    OpenAIRE

    Idris, Nurjihan; Arshad, Fatimah Mohamed; Radam, Alias; Ali, Noor Azman

    2009-01-01

    This study attempts to analyze construct in supply chain and to determine which construct contribute to performance of agricultural cooperatives in Malaysia. The primary data is collected via questionnaire from top level management of agricultural cooperatives using 5-item Likert scale. Factor analysis and structural equations modeling were used to analyze the data. Findings show that cooperatives places importance on quality and technology, logistic, supplier and governance. As a whole, supp...

  10. The production of recombinant single chain antibody fragments for the detection of illicit drug residues

    OpenAIRE

    Brennan, Joanne

    2005-01-01

    Recombinant antibodies represent a more sensitive and specific detection tool for immunoanalysis. The research carried out for this thesis describes the production of genetically-derived single chain antibody fragments to detect illicit drugs. A variety of novel recombinant antibody fragments against morphine-3-glucuronide, a metabolite of heroin has been produced. A monomeric, dimeric and enzyme-labelled scFv were characterised with respect to their binding abilities and cross reactiviti...

  11. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available BACKGROUND: Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  12. The molecular spin filter constructed from 1D organic chain

    International Nuclear Information System (INIS)

    We proposed a molecular spin filter, which is constructed from the 1D metallic organic chain (Fen+1(C6H4)n). The spin-polarized transport properties of the molecular spin filter are explored by combining density functional theory with nonequilibrium Green's function formalism. Theoretical results reveal that Fen+1(C6H4)n molecular chain exhibits robust spin filtering effect, and only the spin-down electrons can transmit through the molecular chain. At the given bias voltage window [−1 eV,1 eV], the calculated spin filter efficiency is close to 100% in the case of n≥3. We find that the effect of spin polarization origin from both Fen+1 and (C6H4)n. In addition, negative difference resistance behavior appears in Fen+1(C6H4)n molecular chain. The results can help us understand the spin transport properties of organic molecular chain. - Highlights: • Theoretical results reveal that Fen+1(C6H4)n molecular chain exhibits robust spin filtering effect. • The effect of spin polarization origin from both of Fen+1 and (C6H4)n. • Negative difference resistance behavior appears in Fen+1(C6H4)n molecular chain

  13. Construction and expression of a recombinant antibody-targeted plasminogen activator

    International Nuclear Information System (INIS)

    Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin β chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the γ2b constant region and the catalytic β chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

  14. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  15. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Science.gov (United States)

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  16. Induction of antiphosphocholine antibodies utilizing lambda light chains in BALB/c mice.

    Science.gov (United States)

    Hall, T J

    1987-01-01

    BALB/c mice immunized with 1 or 100 micrograms of phosphocholine (PC)-keyhole-limpet hemocyanin absorbed on aluminum hydroxide gel (alum) produced up to 50% lambda 1-light-chain-containing anti-PC antibodies in their serum. Only 10% lambda 1 antibodies were seen when complete Freund's adjuvant or bentonite were used as the adjuvant or if PC-bovine serum albumin was used with alum. Thus, the induction of large lambda-antibody responses to PC (a response usually dominated by kappa-antibodies) was both adjuvant- and carrier-dependent. PMID:3108165

  17. Constructing 1/ωα noise from reversible Markov chains

    Science.gov (United States)

    Erland, Sveinung; Greenwood, Priscilla E.

    2007-09-01

    This paper gives sufficient conditions for the output of 1/ωα noise from reversible Markov chains on finite state spaces. We construct several examples exhibiting this behavior in a specified range of frequencies. We apply simple representations of the covariance function and the spectral density in terms of the eigendecomposition of the probability transition matrix. The results extend to hidden Markov chains. We generalize the results for aggregations of AR1-processes of C. W. J. Granger [J. Econometrics 14, 227 (1980)]. Given the eigenvalue function, there is a variety of ways to assign values to the states such that the 1/ωα condition is satisfied. We show that a random walk on a certain state space is complementary to the point process model of 1/ω noise of B. Kaulakys and T. Meskauskas [Phys. Rev. E 58, 7013 (1998)]. Passing to a continuous state space, we construct 1/ωα noise which also has a long memory.

  18. Last Planner and Critical Chain in Construction Management: Comparative Analysis

    OpenAIRE

    Koskela, Lauri; Stratton, Roy; Koskenvesa, Anssi

    2010-01-01

    This paper endeavours to compare the Last Planner System of production control and the Critical Chain production management method. This comparison is carried out in the context of construction management. The original prescription and the evolution of the practice are examined regarding both approaches, and the similarities and differences are noted. Based on these considerations, gaps in the two approaches are identified and the potential of a synthesis of them is explored.

  19. CONSTRUCTION AND EXPRESSION OF A HUMAN-MOUSE CHIMERIC ANTIBODY AGAINST HUMAN BLADDER CANCER

    Institute of Scientific and Technical Information of China (English)

    白银; 王琰; 周丽君; 俞莉章

    2001-01-01

    To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

  20. Porter's value chain (construction, deconstruction, reconstruction and values management

    Directory of Open Access Journals (Sweden)

    E.V. Krykavskyy

    2015-06-01

    Full Text Available The aim of the article. The phases of the Porter's value chain are distinguished: construction of chain value – Porters model (Stage 1; deconstruction – identifying contradictions, disorganizing elements of unnecessary processes that do not add value (Stage 2; reconstruction (synthesis – creates a new value chain (Stage 3. The results of the analysis. The principles of convergence of value and supply chains are identified and the need to focus on supply chain performance is proved. The dependence of design value chain/supply chain on the chosen general strategy and the need to subject its reconstruction process of deconstruction + reconstruction in case of changes or modifications as to this strategy is grounded. The impacts on the creation of value in the Porter’s chain are accepted. The need to identify for each working block of micro factors that affect the financial factors (macro factors that is net and operating profit, investments into fixed and working capital, the cost of companies capital is proved. Such classification allows to determine what types of management actions or motivations for managers of lower level must be selected. It is revealed that in addition to measurable factors-generators on cost to the value chain the indirect (mediated factors have an impact, especially when the management is replaced by objectives and values of enterprise management use. It is proved that the problem of environment and society in the concept of sustainable development, social responsibility, marketing value of 3.0 outlines another group of factors influencing the Porter’s value chain, as evidenced by the current trend of prioritization of human values. It is proved that companies, especially global ones, give the increasing domination of human values in B2B and B2C transactions at the maturity stage in their life cycle in the structure of forced strategic objectives and make adjustments by modifying the cited above vision and mission

  1. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  2. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  3. Enzymic construction of maltosaccharide chains on a heart protein

    International Nuclear Information System (INIS)

    The authors have reported that when 100,000 g pellets of rabbit-heart and rabbit-muscle homogenates are incubated with UDP(14C)glucose, the sugar is incorporated into a protein with Mr 40 KDa. They suggested that these in vitro observations corresponded to the initial stage in the synthesis of glycogen on a protein that they have named glycogenin and which in rabbit muscle appears to be covalently linked to the glycogen via tyrosine residues. The following new observations support the role of a protein as the precursor of glycogen and suggest that glycogen-free glycogenin is present in heart tissue. (1) The (14C)glucose residues added to the heart protein can be removed with glycogenolytic enzymes that hydrolyse 1,4-alpha-glucosidic bonds and therefore constitute synthetic maltosaccharide chains. (2) The newly added glucose residues appear to be attached to pre-existing glucose residues on the protein. Chain elongation does not proceed beyond a few glucose residues. (3) The further relevance of these observations to glycogen synthesis shown by a Western blot in which the radioglucosylated heart protein was found to cross-react with polyclonal antibody to glycogenin obtained from rabbit-muscle glycogen

  4. Generation and characterization of a human single-chain fragment variable (scFv antibody against cytosine deaminase from Yeast

    Directory of Open Access Journals (Sweden)

    Tombesi Marina

    2008-09-01

    Full Text Available Abstract Background The ability of cytosine deaminase (CD to convert the antifungal agent 5-fluorocytosine (5-FC into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT aiming to improve the therapeutic ratio (benefit versus toxic side-effects of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

  5. Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies.

    Science.gov (United States)

    Li, Xinyang; Duan, Xiaobo; Yang, Kai; Zhang, Wei; Zhang, Changjiang; Fu, Longfei; Ren, Zhe; Wang, Changxi; Wu, Jinghua; Lu, Ruxue; Ye, Yanrui; He, Mengying; Nie, Chao; Yang, Naibo; Wang, Jian; Yang, Huanming; Liu, Xiao; Tan, Wen

    2016-01-01

    Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data. PMID:27588755

  6. How to construct the spin chains with perfect state transfer

    CERN Document Server

    Vinet, Luc

    2011-01-01

    It is shown how to systematically construct the $XX$ quantum spin chains with nearest-neighbor interaction that allow perfect state transfer (PST). Sets of orthogonal polynomials (OPs) are in correspondence with such systems. The key observation is that for any admissible one-excitation energy spectrum, the weight function of the associated OPs is uniquely prescribed. This entails the complete characterization of these PST models with the mirror symmetry property arising as a corollary. A simple and efficient algorithm to obtain the corresponding Hamiltonians is presented. A new model connected to a special case of the symmetric $q$-Racah polynomials is offered. It is also explained how additional models with PST can be derived from from a parent system by removing energy levels from the one-excitation spectrum of the latter. This is achieved through Christoffel transformations and is also completely constructive in regards to the Hamiltonians.

  7. A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells

    NARCIS (Netherlands)

    Ailor, E; Pathmanathan, J; Jongbloed, JDH; Betenbaugh, MJ

    1999-01-01

    The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the sig

  8. Optimization of the crystallizability of a single-chain antibody fragment

    Czech Academy of Sciences Publication Activity Database

    Škerlová, Jana; Král, Vlastimil; Fábry, Milan; Sedláček, Juraj; Veverka, Václav; Řezáčová, Pavlína

    2014-01-01

    Roč. 70, č. 12 (2014), s. 1701-1706. ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : single-chain antibody fragment * Thermofluor assay * differential scanning fluorimetry * crystallizability optimization * oligomerization * crystallization Subject RIV: CE - Biochemistry Impact factor: 0.527, year: 2014

  9. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA level

  10. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.

    Science.gov (United States)

    Duan, Ye; Gu, Tiejun; Zhang, Xizhen; Jiang, Chunlai; Yuan, Ruosen; Li, Zhuang; Wang, Dandan; Chen, Xiaoxu; Wu, Chunlai; Chen, Yan; Wu, Yongge; Kong, Wei

    2014-06-01

    Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies. PMID:24598312

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

    Science.gov (United States)

    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.

  12. Antibodies against major histocompatibility complex class I-related chain A in transplant recipients

    Institute of Scientific and Technical Information of China (English)

    Yizhou Zou; Peter Stastny

    2011-01-01

    Objective To review the role of polymorphism of major histocompatibility complex class I-related chain A (MICA) gene and antibodies against MICA antigens in transplant immunology.Data sources The data used in this review were mainly from our own results and from the relevant English language literatures published from 1999 to 2010. Some data presented in this review are in press.Study selection Articles regarding MICA gene discovery and pioneering finding of antibodies against MICA antigen and allograft rejection were selected. This review chronicles the development of our understanding of the role that MICA antigens and antibodies may play in organ transplantation.Results Polymorphic glycoprotein MICA antigens were detected on freshly isolated human umbilical cord endothelial cells, but not on peripheral lymphocytes. Antibodies were found and typing of recipients and donors by sequencing the MICA alleles has established that de novo antibodies produced in kidney transplant recipients are directed at mismatched MICA epitopes and are associated with acute rejection and chronic transplant failure. The specificity of antibodies against the epitopes of MICA antigens were well characterized by donor MICA typing, single antigen array testing with antibody absorption and elution. Acute graft-versus-host disease was observed in stem-cell recipients who were mismatched for MICA.Conclusions Immunization against mismatched MICA epitopes encountered in donor organs after transplantation may result in antibodies against MICA alleles. Testing for MICA donor-specific antibodies (DSA) which are associated with early failure of kidney transplants may be helpful for identifying some of the targets of antibodies against antigens other than the human leukocyte antigen (HLA) and for improving transplantation outcome.

  13. Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2011-11-01

    Full Text Available Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Results Single-chain variable fragments of immunoglobulins (scFvs were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Conclusions Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in

  14. Demand uncertainty in construction supply chains: a discrete event simulation study

    OpenAIRE

    C Vidalakis; J.E. Tookey; Sommerville, J

    2013-01-01

    The delivery of construction projects is typically an assembly operation involving a high number of subassemblies and materials brought on site by the supply chain. However, although supply chain management in construction has attracted significant attention, paradoxically little focus has been placed on construction supply networks and operations. This paper places emphasis on supply chain operations by looking at the logistics function of construction material suppliers. Specifically, the p...

  15. Characterization of monoclonal antibodies against MHC class II-associated p41 invariant chain fragment

    International Nuclear Information System (INIS)

    Mouse monoclonal antibodies directed against human MHC class II-associated p41 invariant chain fragment have been generated. Mice were immunized with human recombinant Ii-isoform p26. For hybridoma production mouse splenocytes and myeloma cells were fused. Hybridoma cells were screened using ELISA and immunoblotting. Three cell lines (42B10, 42G11 and 43C8) were used for production of specific antibodies, which reacted with p41 fragment and did not bind to cathepsins L or S or their proenyzmes. As primary antibody for immunofluorescence staining of lymph node tissue sections clone 2C12 MAb was selected. Specific localization of p41 fragment in certain cells in lymph nodes was observed. (author)

  16. Selection of bisphenol A - single-chain antibodies from a non-immunized mouse library by ribosome display.

    Science.gov (United States)

    Zhao, Li; Ning, Baoan; Bai, Jialei; Chen, Xiang; Peng, Yuan; Sun, Siming; Li, Guimin; Fan, Xianjun; Liu, Yuanyuan; Liu, Jianqing; Sun, Yanan; Gao, Zhixian; Zhang, Juankun

    2015-11-01

    Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KDS) of one clone was 1.76μM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies. PMID:24269893

  17. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    Science.gov (United States)

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. PMID:21277981

  18. In silico design, construction and cloning of Trastuzumab humanized monoclonal antibody: A possible biosimilar for Herceptin

    OpenAIRE

    Soudabeh Akbarzadeh-Sharbaf; Bagher Yakhchali; Zarrin Minuchehr; Mohammad Ali Shokrgozar; Sirous Zeinali

    2012-01-01

    Background: There is a novel hypothesis in that antibodies may have specificity for two distinct antigens that have been named "dual specificity." This hypothesis was evaluated for some defined therapeutic monoclonal antibodies (mAbs) such as Trastuzumab, Pertuzumab, Bevacizumab, and Cetuximab. In silico design and construction of expression vectors for trastuzumab monoclonal antibody also in this work were performed. Materials and Methods: First, in bioinformatics studies the 3D structur...

  19. PURE mRNA display for in vitro selection of single-chain antibodies.

    Science.gov (United States)

    Nagumo, Yu; Fujiwara, Kei; Horisawa, Kenichi; Yanagawa, Hiroshi; Doi, Nobuhide

    2016-05-01

    mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications. PMID:26711234

  20. Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Frank; Martiniuk; Seth; Pincus; Sybille; Müller; Heinz; Kohler; Kam-Meng; Tchou-Wong

    2010-01-01

    AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.

  1. Target-selective joint polymerase chain reaction: A robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells

    Directory of Open Access Journals (Sweden)

    Isobe Masaharu

    2011-07-01

    Full Text Available Abstract Background During the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies. Results We have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR, and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days. Conclusion Our system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.

  2. Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol

    NARCIS (Netherlands)

    Schouten, A.; Roosien, J.; Bakker, J.; Schots, A.

    2002-01-01

    Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fra

  3. Fuzzy Optimization of Construction Engineering Project Schedule Based on Critical Chain Management

    OpenAIRE

    Liu, Jianbing; Ren, Hong; Junshu DU

    2013-01-01

    Critical chain management was the very important theory innovation in the development of construction project schedule management field in recent years. Compared with the traditional methods of construction project schedule management, the methods of critical chain management considered resource constraints into construction project schedule management, the construction project cycle was shortened and the efficiency was enhanced by using the dynamic thinking and circulation pattern to manage ...

  4. Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody(VHH)Yeast Two-Hybrid Library%双峰驼天然重链抗体可变区酵母双杂交文库的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    胡湘云; 高小龙; 付向晶; 刘丹丹; 范文涛; 王燕平; 杜恩岐; 王兴龙; 党如意

    2015-01-01

    构建双峰驼天然重链抗体可变区(VHH)酵母双杂交文库,并对文库质量进行鉴定.采集未经免疫的6月龄雌性双峰驼外周血、骨髓、脾和淋巴结,并从外周血中分离淋巴细胞.抽提总RNA,反转录为cDNA,用2对引物经过2轮PCR扩增得到400 bp左右的双峰驼VHH片段.根据Clontech公司Mate&PlateTM library construction system使用手册,将带有同源臂的VHH片段与线性化的pGADT7-Rec质粒共转化Y187酵母感受态细胞,转化产物涂布SD/-Leu平板,30℃倒置培养3~5 d后,收集平板上的所有克隆,即为双峰驼天然重链抗体可变区酵母双杂交文库.对文库的滴度、重组效率及多样性进行鉴定.结果显示,本研究构建的双峰驼天然重链抗体可变区酵母双杂交文库库容为2.07×107,滴度为7.6×108 cfu·mL-1.随机挑取47个独立克隆进行菌落PCR鉴定,43个含有VHH片段,重组率为91.5%;选取其中19个测序,均为独立克隆,且多样性好.该研究成功构建双峰驼天然重链抗体可变区酵母双杂交文库,且文库质量满足要求,为进一步获得特定抗原的VHH奠定了基础.

  5. Porous silicon biosensor for detection of variable domain of heavy-chain of HCAb antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong-yan; Lü Xiao-yi; JIA Zhen-hong; LI Jiang-wei; ZHANG Fu-chun

    2012-01-01

    In this paper,we produce porous silicon (PSi) by electrochemical etching,and it is the first time to evaluate the performance of label-free porous silicon biosensor for detection of variable domain of heavy chain of heavy-chain antibody (VHH).The binding of hen egg white lysozyme (HEWL) and VHH causes a red shift in the reflection spectrum of the biosensor.The red shift is proportional to the VHH concentration in the range from 14 μg · ml-1 to 30μg·ml-1 with a detection limit of 0.648 ng ·ml-1.The research is useful for the development of label-free biosensor applied in the rapid and sensitive determination of small molecules.

  6. Rapid specific amplification of rat antibody cDNA from nine hybridomas in the presence of myeloma light chains.

    Science.gov (United States)

    Brady, Jamie L; Corbett, Alexandra J; McKenzie, Brent S; Lew, Andrew M

    2006-08-31

    Most monoclonal antibodies to mouse antigens have been derived from rat spleen-mouse myeloma fusions. Many resultant hybridomas express one of several myeloma kappa chain transcripts, even though the parent myeloma may have been ascribed as not expressing light chain protein. Previous reports have only differentiated against one of these mouse light chains. We have found at least three different myeloma kappa transcripts in the panel of nine hybridomas that were derived from four different myeloma parents. We have designed an amplification strategy that differentiates the rearranged rat kappa chain from all mouse light chains. Moreover, this method is expedient as it requires minimal downstream manipulation. PMID:16901500

  7. IgG subclass and light chain distribution of anticardiolipin and anti-DNA antibodies in systemic lupus erythematosus.

    OpenAIRE

    Gharavi, A E; Harris, E N; Lockshin, M D; Hughes, G R; Elkon, K B

    1988-01-01

    The IgG subclass and light chain distribution of anticardiolipin and anti-DNA antibodies were determined in serum samples from patients with systemic lupus erythematosus. With an enzyme linked immunosorbent assay (ELISA) and mouse monoclonal antibodies to individual subclasses, significant differences in the distributions of IgG2, IgG3, and IgG4 subclasses were observed between anticardiolipin and anti-DNA antibodies. Whereas anti-DNA antibodies were predominantly IgG1 and IgG3, all subclasse...

  8. Construction of Network Management Information System of Agricultural Products Supply Chain Based on 3PLs

    OpenAIRE

    Gao, Shujin; Yang, Qiangxian

    2010-01-01

    The necessity to construct the network management information system of 3PLs agricultural supply chain is analyzed, showing that 3PLs can improve the overall competitive advantage of agricultural supply chain. 3PLs changes the homogeneity management into specialized management of logistics service and achieves the alliance of the subjects at different nodes of agricultural products supply chain. Network management information system structure of agricultural products supply chain based on 3PL...

  9. The milk delivery chain and presence of Brucella spp. antibodies in bulk milk in Uganda.

    Science.gov (United States)

    Rock, Kim Toeroek; Mugizi, Denis Rwabiita; Ståhl, Karl; Magnusson, Ulf; Boqvist, Sofia

    2016-06-01

    This study examined the influence of informal milk delivery chains on the risk of human exposure to Brucella spp. through milk consumption in two regions of Uganda (Gulu and Soroti Districts). The work involved describing milk delivery chains, investigating brucellosis awareness amongst milk deliverers and determining the presence of Brucella spp. antibodies in cattle milk on delivery to primary collection points (boiling points and dairies). Milk samples (n = 331) were collected from deliverers at primary collection points and from street vendors at point of sale and analysed using indirect enzyme-linked immunosorbent assay (I-ELISA). A written questionnaire was used to collect data from deliverers (n = 279) on their milk delivery chains and their brucellosis awareness. The most common delivery points in Gulu District were small dairies and in Soroti District boiling points. The presence of Brucella spp. antibodies in milk samples was higher in Soroti (40 %) than in Gulu (11 %) (P < 0.0001). There are possible public health risk consequences of this finding as 42 % of deliverers in Soroti District reported drinking raw milk, compared with 15 % in Gulu District (P < 0.0001). Awareness of brucellosis was low, with 70 % of all milk deliverers reporting not having heard of the disease or the bacterium. Application of quality controls for milk (colour and odour) along the delivery chain varied depending upon supply and demand. This study provides evidence of the diversity of informal milk markets in low-income countries and of the potential public health risks of consuming unpasteurised milk. These results can be useful to those planning interventions to reduce brucellosis. PMID:27026231

  10. Baculovirus display of single chain antibody (scFv using a novel signal peptide

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  11. Measuring Safety Attitude Differences in the Construction Supply Chain

    OpenAIRE

    Saunders, Lance Walter

    2013-01-01

    Construction worker safety is normally a construction activity in the United States, even though there is an emerging body of literature discussing the positive effects of considering safety earlier in the construction lifecycle.  This literature has discussed the fragmentation in terms of safety attitudes between owners and designers and those carrying out the construction of a project.  Quantitatively identifying the specific areas that the differences exist in terms of safety attitudes bet...

  12. Construction of phage antibody library and screening of anti-FasFab antibody%噬菌体呈现抗体库的构建及抗 Fas-Fab抗体的研究

    Institute of Scientific and Technical Information of China (English)

    王希良; 黄云辉; 陈克敏; 朱锡华

    2001-01-01

    目的构建噬菌体抗体库,获得具有功能的抗Fas-Fab噬菌体抗体。方法以Fas重组蛋白为抗原免疫Balb/c小鼠。取其脾细胞提取mRNA,采用RT-PCR方法扩增抗体基因,构建重链和κ链基因库,用重组Fas抗原对所构建的抗体库进行4轮筛选,并以ELISA法鉴定其功能。结果获得抗体重链Fd基因和κ链基因长度约700bp。构建的重链Fd基因为3.5×106的抗体重链基因库。构建的重链和κ链基因库的容量均为3.1×106。经VCSM13感染得到噬菌体的滴度为8.9×1016cFu/L的噬菌体抗体库,含有抗体重链和κ链基因的噬菌体占27%。用重组人Fas抗原进行4轮筛选,得到100%的富集,说明Fas重组抗原富集了抗Fas-Fab噬菌体抗体,经ELISA检测均有抗Fas抗体的特异性。结论制备的可溶性抗Fas-Fab抗体具有抗Fas抗体的特异性,为进一步的研究奠定了基础。%Aim The phage display antibody library was constructed to obtainfunctional anti-Fas Fab. Methods Balb/c mice were immunized with recombinant Fas protein. mRNA was isolated from splenocytes and antibody heavy chain genes Fd and κ chain genes were amplified by RT-PCR, and combinatorial Fab antibody library. This iuelicated that the screening of antibody library was done with recombinont Fas antigen and the immunifacient function of the antibody was determined by ELISA. Results The amplified products were the desired fragments and contained 700 bp. The amplified antibody heavy chain Fd genes were cloned into pcomb3 vector at first, and transformed into E.coli XL1-blue to construct an antibody heavy chain library with a size of 3.5× 106 members. The κ chain genes were then randomly combined with heavy chain genes to generate a combinatorial vector encoding both chains and capable of generating Fab fragments. These combinatorial vectors were transformed to E.coli XL1-Blue,and the combinatorial Fab antibody library was 3.7× 106. The phage antibody library was

  13. The Rural Information-based Construction under the Perspective of Expanding Agricultural Industrial Chain

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    On the basis of expounding connotation and significance of expansion of agricultural industrial chain, coupled with the connotation of rural informatization connotation, this article analyses the role of rural informatization in expanding agricultural industrial chain: it can enhance market competitiveness of industry chain, improve the operational efficiency of industry chain, and promote the income and quality of farmers in industry chain. Under the perspective of expanding agricultural industrial chain, this article puts forwards thinking about the construction of rural informatization as follows: first, give full play to the leading role of the government; second, strengthen the construction of information-based network facility; third, integrate information resources in rural areas, and improve the quality of information; fourth, build comprehensive information service platform in rural areas; fifth, improve organizational level of production and management of individual farmers; sixth, strengthen the construction of information-based personnel in rural areas; seventh, strengthen publicity and training, promote overall cultural quality and information awareness of farmers.

  14. Construction, expression and tumor targeting of a single-chain Fv against human colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jin Fang; Hong-Bin Jin; Jin-Dan Song

    2003-01-01

    AIM: A single-chain antibody fragment, ND-1scFv, against human colorectal carcinoma was constructed and expressed in E.coli, and its biodistribution and pharmacokinetic properties were studied in mice bearing tumor.METHODS: VH and VL genes were amplified from hybridoma cell IC-2, secreting monoclonal antibody ND-1, by RT-PCR,and connected by linker (Gly4Ser)3 to form scFv gene, which was cloned into expression vector pET 28a(+) and finally expressed in E.coli. The expressed product ND-1scFv was purified by metal affinity chromatography using Ni-NTA, its purity and biological activity were determined using SDSPAGE and ELISA. ND-1scFv was labeled with 99mTc, and then injected into mice bearing colorectal carcinoma xenograft for phamacokinetic study in vivo.RESULTS: SDS-PAGE analysis showed that the relative molecular weight of recombinant protein was 30kDa with purity of 94%. ELIAS assay revealed that ND-1scFv retained the immunoactivity of parent mAb, being capable of binding specifically to human colorectal carcinoma cell line expressing associated antigen. Radiolabeled ND-1scFv exhibited rapid tumor targeting, with specific distribution in mice bearing colorectal carcinoma xenograft observed as early as 1 h following injection. In vivo pharmacokinetic studies also demonstrated that ND-1scFv had very rapid plasma clearance (T1/2α of 5.7 min, T1/2β of 2.6 h).CONCLUSION: ND-1scFv shows significant immunoactivity,and better pharmacokinetic and biodistribution characteristics compared with intact mAbs, demonstrating the possibility as a carrier for tumor-imaging.

  15. Schistosoma japonicum:construction of phage display antibody library and its application in the immunodiagnosis of infection

    Institute of Scientific and Technical Information of China (English)

    陈代雄; 何蔼; 詹希美; 俞慕华; 雷智刚; 孟锦绣; 李卓雅; 梁瑜; 张瑞琳

    2004-01-01

    Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×106 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conclusions The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

  16. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    Science.gov (United States)

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. PMID:26772159

  17. Construction and Application of a Refined Hospital Management Chain.

    Science.gov (United States)

    Lihua, Yi

    2016-01-01

    Large scale development was quite common in the later period of hospital industrialization in China. Today, Chinese hospital management faces such problems as service inefficiency, high human resources cost, and low rate of capital use. This study analyzes the refined management chain of Wuxi No.2 People's Hospital. This consists of six gears namely, "organizational structure, clinical practice, outpatient service, medical technology, and nursing care and logistics." The gears are based on "flat management system targets, chief of medical staff, centralized outpatient service, intensified medical examinations, vertical nursing management and socialized logistics." The core concepts of refined hospital management are optimizing flow process, reducing waste, improving efficiency, saving costs, and taking good care of patients as most important. Keywords: Hospital, Refined, Management chain PMID:27180468

  18. Supply chain for new construction: buy where we build

    International Nuclear Information System (INIS)

    A global supply strategy has been placed by Westinghouse in order to face the current and future constructions. And in this way, Westinghouse will be able to take advantage of benefit of Local Supply. this article shows the Westinghouse Global Supply Strategy, Local Supply considerations, the current state of supplier calcifications, and finally Memorandums of Understanding and Alliances agreed with local suppliers around the world to provide a suitable solution for the new construction needs. (Author)

  19. An Adaptively Constructed Algebraic Multigrid Preconditioner for Irreducible Markov Chains

    OpenAIRE

    Brannick, James; Kahl, Karsten; Sokolovic, Sonja

    2014-01-01

    The computation of stationary distributions of Markov chains is an important task in the simulation of stochastic models. The linear systems arising in such applications involve non-symmetric M-matrices, making algebraic multigrid methods a natural choice for solving these systems. In this paper we investigate extensions and improvements of the bootstrap algebraic multigrid framework for solving these systems. This is achieved by reworking the bootstrap setup process to use singular vectors i...

  20. Fuzzy Optimization of Construction Engineering Project Schedule Based on Critical Chain Management

    Directory of Open Access Journals (Sweden)

    Jianbing LIU

    2013-07-01

    Full Text Available Critical chain management was the very important theory innovation in the development of construction project schedule management field in recent years. Compared with the traditional methods of construction project schedule management, the methods of critical chain management considered resource constraints into construction project schedule management, the construction project cycle was shortened and the efficiency was enhanced by using the dynamic thinking and circulation pattern to manage whole project. In view of the progress of critical chain management of the article, fuzzy theory  was used to calculate and optimize the buffer zone sizes with the determination of the buffer size of  the existing resources in certain critical chain management so as to shorten the cycle..

  1. Strategies for integrating design and construction and operations and maintenance supply chains in Singapore

    OpenAIRE

    Ling, FYY; Toh, BGY; Kumaraswamy, MM; Wong, KWK

    2014-01-01

    Purpose – The purpose of this paper is to investigates strategies for achieving better integration between the design and construction (DC) and operation and maintenance (OM) supply chains in Singapore. The specific objectives are to: discover the goals that stakeholders want to achieve in integrating the supply chains; identify the stakeholders that play important integration role in each supply chain; and investigate the effective strategies that may yield better integration of the supply ...

  2. Identification of internalizing human single chain antibodies targeting brain tumor sphere cells

    Science.gov (United States)

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C. David; Berger, Mitchel S.; Liu, Bin

    2010-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor and there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive media, and exhibit enhanced tumor initiating ability and resistance to therapy. We report here the identification of internalizing human single chain antibodies (scFvs) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133 positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular non-selective media. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. PMID:20587664

  3. Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The c-erbB-2 proto-oncogene encodes a 185kDa protein p185, which belongs to epidermal growth factorreceptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis forcertain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibitsproliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. Inorder to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed thesingle-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichiacoli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cellimmunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain(ECD) of p185. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion moleculewith a homodimer form and the recombinant product was expressed in mammalian cells. In a series ofsubsequent analysis this fusion protein showed identical antigen binding site and activity with the parentantibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targetingdrugs, with a view of application in the diagnosis and treatment of human breast cancer.

  4. Preparation and Identification of a Single-chain Variable Fragment Antibody Against Canine Distemper Virus.

    Science.gov (United States)

    Yi, Li; Cheng, Shipeng

    2015-08-01

    The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 1N8, which secretes the monoclonal antibody against CDV N protein (aa 277-471). The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/1N8). After sequence analysis, the scFv/1N8 gene was cloned into the prokaryotic expression vector PET32a with a His-tag. The recombinant scFv/1N8 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the binding activity and specificity of the scFv were determined by indirect ELISA (His-tag) and competitive ELISA. The recombinant scFv/1N8 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule. PMID:26301925

  5. Constructing Chains of Enablers for Alternative Economic Futures

    DEFF Research Database (Denmark)

    Hull Kristensen, Peer

    2016-01-01

    that have made possible novel forms of work-organization and business models. These institutional enablers are capacitating on the "supply side" by enabling labor to take active part in shaping enterprises supported by social welfare services (training, child- and eldercare, support for housing, etc...... economies. This article illustrates a way of researching alternative economic futures by identifying chains of enablers in Denmark and other Nordic countries by which society and business can co-develop and capture capabilities to take on new roles in globalization. Focus is on institutional enablers.......). Being generally inclusive of social movements, welfare states has also helped identify new needs on the "demand side" such as child- and eldercare, environmental protection, alternative energy and energy-saving, health, and city planning. This is illustrated by a number of firms that supply products...

  6. On Construction of Supply Chain System for China’s Modern Agricultural Products

    Institute of Scientific and Technical Information of China (English)

    Wanjiang; WANG

    2015-01-01

    In view of drawbacks in supply chain of China’s traditional agricultural products,this paper proposed building supply chain system for modern agricultural products: taking informationization as basis,channel system as core,organization system as support,and service system and safety system as guarantee,to promote high efficient operation of the supply chain system. The channel system stresses alliance and integration of channel system,informationization of channel management,and terminalization of channel operation; the organization system stresses organization,large scale,group,and brand of participant entities; service system stresses construction of service means,service platform,and operation mechanism; safety system stresses building quality safety based agricultural product supply chain management mode. In order to ensure high efficient operation of supply chain for modern agricultural products,it is required to straighten out supply chain management system,actively cultivate core enterprises of supply chain,strengthen information construction of supply chain,select suitable supply chain mode,and improve benefit allocation mechanism.

  7. Network design and operational modelling for construction green supply chain management

    Directory of Open Access Journals (Sweden)

    Pengfei Zhou Dong Chen

    2013-01-01

    Full Text Available Based on studying organizational structure of Construction Green Supply Chain Management (CGSCM, a mathematical programming model of CGSCM was proposed. The model aimed to maximize the aggregate profits of normalized construction logistics, the reverse logistics and the environmental performance. Numerical experiments show that the proposed approach can improve the aggregate profit effectively. In addition, return ratio, subsidies from governmental organizations, and environmental performance were analyzed for CGSCM performance. Herein, the proper return, subsidy and control strategy could optimize construction green supply chain.

  8. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    International Nuclear Information System (INIS)

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to

  9. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  10. Antibody

    Science.gov (United States)

    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 ...

  11. Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

    Directory of Open Access Journals (Sweden)

    Yang J

    2014-05-01

    Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

  12. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-06-01

    Full Text Available Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

  13. Novel human single chain antibody fragments that are rapidly interalizing effectively target epithelioid and sarcomatoid mesotheliomas

    Science.gov (United States)

    Iyer, Arun K.; Lan, Xiaoli; Zhu, Xiaodong; Su, Yang; Feng, Jinjin; Zhang, Xiaoju; Gao, Dongwei; Seo, Youngho; VanBrocklin, Henry F.; Broaddus, V. Courtney; Liu, Bin; He, Jiang

    2011-01-01

    Human antibodies targeting all subtypes of mesothelioma could be useful to image and treat this deadly disease. Here we report tumor targeting of a novel internalizing human single chain antibody fragment (scFv) labeled with 99mTc (99mTc-M40) in murine models of mesothelioma of both epithelioid (M28) and sarcomatoid (VAMT-1) origins. 99mTc-M40 was taken up rapidly and specifically by both subtype tumor cells in vitro, with 68–92% internalized within 1h. The specificity of binding was evidenced by blocking (up to 95%) with 10-fold excess of unlabeled M40. In animal studies, tumors of both subtypes were clearly visualized by SPECT/CT as early as 1h post-injection of 99mTc-M40. Tumor uptake measured as percent of injected dose per gram tissue (%ID/g) at 3h was 4.38 and 5.84 for M28 and VAMT-1 tumors respectively, significantly greater than all organs or tissues studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and 60:1, for M28 and VAMT-1 respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma. PMID:21447742

  14. DIRECTIONS FOR FUTURE CONSTRUCTION SUPPLY CHAIN MANAGEMENT RESEARCH IN NEW ZEALAND: A REAL OPTIONS PERSPECTIVE

    Directory of Open Access Journals (Sweden)

    Tran, Van

    2012-04-01

    Full Text Available Real Options (RO has been a universally accepted concept in a number of major industries. However, its use in the construction supply chain management (CSCM sector has been limited. Some rare supply chain management RO studies have shown a number of limitations. First, there is a lack of a rigorous theoretical RO framework pertaining specifically to CSCM. All such supply chain management RO studies are based off RO theories or models developed for other sectors (engineering, infrastructure, natural resources. And second, attempts to extend real option to wider uses in CSCM seem premature at the present. This paper reviews all recent literature pertaining to real options and real options applied specifically to the construction supply chain management area. The study proposes a research programme pertaining to CSCM in New Zealand in order to enhance the current understanding of RO in this area and in the process develop a comprehensive theory for the RO application in New Zealand CSCM.

  15. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

    Directory of Open Access Journals (Sweden)

    Kazuhisa Sugimura

    2013-02-01

    Full Text Available An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.

  16. Quantitative determination of type I myosin heavy chain in bovine muscle with anti myosin monoclonal antibodies.

    Science.gov (United States)

    Picard, B; Leger, J; Robelin, J

    1994-01-01

    Bovine type I muscle fibers were characterized by enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for slow myosin heavy chains (MHC 1). Two bovine muscles, the Masseter and Cutaneus trunci, were analyzed by different complementary techniques: electrophoresis, immunoblotting and immunohistiology. The results showed that the two muscles have extreme characteristics. The Masseter contains only slow MHC and the Cutaneus trunci is composed solely of rapid MHC (MHC 2a and 2b). A standard for this ELISA was obtained by mixing the two muscles and was used as a reference in the determination of the percentage of MHC 1 in a given muscle. In this study, the Longissimus thoracis of 27 Charolais cattle were examined. The different conditions under which assays were carried out were described and the accuracy of the measurement was calculated. In view of the results, ELISA was chosen for the analysis of muscle fiber types in large numbers of animal specimens. This technique could be used in several research projects to study the muscle characteristics that determine beef quality. PMID:22061628

  17. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains.

    Science.gov (United States)

    Hu, Zu-Quan; Li, He-Ping; Zhang, Jing-Bo; Huang, Tao; Liu, Jin-Long; Xue, Sheng; Wu, Ai-Bo; Liao, Yu-Cai

    2013-02-18

    Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv-AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10(-2) μg mL(-1), superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10(-3) mg g(-1) of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food. PMID:23374219

  18. Construction of Network Management Information System of Agricultural Products Supply Chain Based on 3PLs

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The necessity to construct the network management information system of 3PLs agricultural supply chain is analyzed,showing that 3PLs can improve the overall competitive advantage of agricultural supply chain.3PLs changes the homogeneity management into specialized management of logistics service and achieves the alliance of the subjects at different nodes of agricultural products supply chain.Network management information system structure of agricultural products supply chain based on 3PLs is constructed,including the four layers (the network communication layer,the hardware and software environment layer,the database layer,and the application layer) and 7 function modules (centralized control,transportation process management,material and vehicle scheduling,customer relationship,storage management,customer inquiry,and financial management).Framework for the network management information system of agricultural products supply chain based on 3PLs is put forward.The management of 3PLs mainly includes purchasing management,supplier relationship management,planning management,customer relationship management,storage management and distribution management.Thus,a management system of internal and external integrated agricultural enterprises is obtained.The network management information system of agricultural products supply chain based on 3PLs has realized the effective sharing of enterprise information of agricultural products supply chain at different nodes,establishing a long-term partnership revolving around the 3PLs core enterprise,as well as a supply chain with stable relationship based on the supply chain network system,so as to improve the circulation efficiency of agricultural products,and to explore the sales market for agricultural products.

  19. Optimisation of recombinant protein production in Pichia pastoris:single-chain antibody fragment model protein

    OpenAIRE

    Khatri, N. K. (Narendar Kumar)

    2011-01-01

    Abstract Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the most common diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), which can be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, is generally considered an interesting production system candidate, as it can secrete properly folded proteins. These proteins accumulate in high concentrations during fermentation, reducing the cost for...

  20. PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明

    2002-01-01

    Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti -Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL)genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13KO7 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. Thetypesoftheanti-IdScFvdisplayedontheselectedphagecloneswerepreliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γtype anti-Id ScFv.Conclsion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

  1. Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

    Institute of Scientific and Technical Information of China (English)

    Yan-Wei Zhong; Jun Cheng; Gang Wang; Shuang-Shuang Shi; Li Li; Ling-Xia Zhang; Ju-Mei Chen

    2002-01-01

    AIM: To screen human single chain Fv antibody (scFv)against hepatitis C virus E2 antigen and identify its applicationin immunohistochemistry.METHODS: The phage antibody library was panned by HCVE2 antigen, which was coated in microtiter plate. After fiverounds of biopanning,56 phage clones were identified specificto HCV E2 antigen. The selected scFv clones were digestedby SfiI/NotI and DNA was sequenced. Then it was subclonedinto the vector pCANTABSE for expression as E-taggedsoluble scFv. The liver tissue sections from normal personand patients with chronic hepatitis B and chronic hepatitis Cwere immunostained with HCV E2 scFv antibody.RESULTS: The data of scFv-E2 DNA digestion and DNAsequencing showed that the scFv gene is composed of 750bp. ELISA and immunohistochemistry demonstrated that thehuman single chain Fy antibody against hepatitis C E2 antigenhas a specific binding character with hepatitis virus E2 antigenand paraffin-embedded tissue, but did not react with liver tissuesfrom healthy persons or patients with chronic hepatitis B.CONCLUSION: We have successfully screened andidentified HCV E2 scFv and the scFv could be used in theimmunostaining of liver tissue sections from patients withchronic hepatitis C.

  2. Preconceptions and Relations Used by Children in the Construction of Food Chains.

    Science.gov (United States)

    Gallegos, Leticia; And Others

    1994-01-01

    Examines the predator-prey relations and the preconceptions held by children (n=506) on the construction of food chains. Results show that the classification of herbivores and carnivores is based on children's preconceptions of size and ferocity and sheds light on the difficulties students have at higher education levels in the resolution of food…

  3. Not As Easy As It Seems: Automating the Construction of Lexical Chains Using Roget's Thesaurus

    CERN Document Server

    Jarmasz, Mario

    2012-01-01

    Morris and Hirst present a method of linking significant words that are about the same topic. The resulting lexical chains are a means of identifying cohesive regions in a text, with applications in many natural language processing tasks, including text summarization. The first lexical chains were constructed manually using Roget's International Thesaurus. Morris and Hirst wrote that automation would be straightforward given an electronic thesaurus. All applications so far have used WordNet to produce lexical chains, perhaps because adequate electronic versions of Roget's were not available until recently. We discuss the building of lexical chains using an electronic version of Roget's Thesaurus. We implement a variant of the original algorithm, and explain the necessary design decisions. We include a comparison with other implementations.

  4. Green nanotechnology in Nordic Construction: Eco-innovation strategies and Dynamics in Nordic Window Value Chains

    DEFF Research Database (Denmark)

    Andersen, Maj Munch

    2010-01-01

    This project analyzes Nordic trends in the development and industrial uptake of green nanotechno-logy in construction. The project applies an evolutionary economic perspective in analyzing the innovation dynamics and firm strategies in the window value chains in three Nordic countries, Denmark......, Finland and Sweden. Hence the project investigates two pervasive parallel market trends: The emergence of the green market and the emergence of nanotechnology. The analysis investigates how a traditional economic sector such as the construction sector reacts to such major trends. Conclusions are multiple...... of nanotechnology in the construction sector in the Nordic countries we do find quite a high number of nanotech applications in the Nordic window chains. Eco-innovation is influencing strongly on the nanotech development. We see several examples of nano-enabled smart, multifunctional green solutions in the Nordic...

  5. Green Nanotechnology in Nordic Construction - Eco-innovation strategies and Dynamics in nordic Window Chains

    DEFF Research Database (Denmark)

    Andersen, Maj Munch; Sandén, Björn A.; Palmberg, Christopher

    This project analyzes Nordic trends in the development and industrial uptake of green nanotechno-logy in construction. The project applies an evolutionary economic perspective in analyzing the innovation dynamics and firm strategies in the window value chains in three Nordic countries, Denmark......, Finland and Sweden. Hence the project investigates two pervasive parallel market trends: The emergence of the green market and the emergence of nanotechnology. The analysis investigates how a traditional economic sector such as the construction sector reacts to such major trends. Conclusions are multiple...... of nanotechnology in the construction sector in the Nordic countries we do find quite a high number of nanotech applications in the Nordic window chains. Eco-innovation is influencing strongly on the nanotech development. We see several examples of nano-enabled smart, multifunctional green solutions in the Nordic...

  6. Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library.

    Science.gov (United States)

    Xu, Yang; Xiong, Liang; Li, Yanping; Xiong, Yonghua; Tu, Zhui; Fu, Jinheng; Chen, Bo

    2015-07-01

    Compared with peptide-based mimotope, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one β-type AI alpaca heavy chain single domain antibody (β-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phage-displayed VHH library. The affinity constant (K D) of obtained β-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to anti-CIT McAb (68 nM). The developed VHH-based enzyme-linked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10% (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n = 3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 = 96.2 ng/mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISA was evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other β-AI VHH toward small organic molecules from a naive VHH library. PMID:25910884

  7. A framework for measuring the supply chain's agility of mass construction industry in Iran

    Directory of Open Access Journals (Sweden)

    Keyvan Poloie

    2012-10-01

    Full Text Available Impotent planning system in house providing process is the result of inadequate housing management system in theoretical, empirical, and operational fields. In addition, Mass Construction industry in Iran confronts with other problems such as instability in raw material prices, unsteadiness in production and investment laws and regulations, frailty of transportation infrastructure, international sanctions and etc. Furthermore, customers’ needs, lower costs, and greater customizations lead mass producing to search for new solutions and novel producing system. Agility is offered as a strategy to enable Mass Construction associations to be maintained in the competition of constantly changing market in Iran. In such a market, previous approaches lose their capabilities in supply chain. Thus to achieve agility by Mass Construction association is the chief aim of this study. This study is descriptive-analytic and can be identified as developmental –functional considering its target. After surveying previous research literature and using experts’ opinions, we investigated final agile sub criteria of supply chain and then we used interpretive- structural modeling approach to determine the relation among sub criteria and to offer an agile supply chain model. Surveying research literature and experts’ opinions lead us to identify 8 criteria (society, government, financial, information technology, market, partnership, quality and technology and also 22 sub criteria for supply chain’s agility. Then the results were analyzed through interpretive-structural approach and relation of criteria and sub criteria and their consequence were achieved. These relations showed that government and infrastructure investment, culture, regulations and responses to social and environmental issues are the basis of agility in mass housing productions’ supply chain. This model helps supply chain managers to have strategic planning to enhance agility in supply chain

  8. Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.

    Science.gov (United States)

    Säll, Anna; Persson, Helena; Ohlin, Mats; Borrebaeck, Carl A K; Wingren, Christer

    2016-09-25

    Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner. The sensitivity of GPS, and other methods relying on affinity enrichment, is largely affected by the activity of the exploited affinity reagent. We here present an improvement of the GPS platform by utilizing an antibody immobilization approach which ensures a controlled immobilization process of the antibody to the magnetic bead support. More specifically, we make use of an antibody format that enables site-directed biotinylation and use this in combination with streptavidin coated magnetic beads. The performance of the expanded GPS platform was evaluated by profiling yeast proteome samples. We demonstrate that the oriented antibody immobilization strategy increases the ability of the GPS platform and results in larger fraction of functional antibodies. Additionally, we show that this new antibody format enabled in-solution capture, i.e. immobilization of the antibodies after sample incubation. A workflow has been established that permit the use of an oriented immobilization strategy for the GPS platform. PMID:26703809

  9. THE CONSTRUCTION OF MULTITYPE CANONICAL MARKOV BRANCHING CHAINS IN RANDOM ENVIRONMENTS

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The investigation for branching processes has a long history by their strong physics background, but only a few authors have investigated the branching processes in random environments. First of all, the author introduces the concepts of the multitype canonical Markov branching chain in random environment (CMBCRE) and multitype Markov branching chain in random environment (MBCRE) and proved that CMBCRE must be MBCRE, and any MBCRE must be equivalent to another CMBCRE in distribution. The main results of this article are the construction of CMBCRE and some of its probability properties.

  10. The effects of the global financial crisis on the Australian building construction supply chain

    Directory of Open Access Journals (Sweden)

    Ram Karthikeyan Thangaraj

    2012-09-01

    Full Text Available This study involves a financial analysis of 43 publicly listed and large private companies in the building and construction supply chain from 2005 to 2010; straddling the period of the global financial crisis (GFC; and examines the impact of the GFC on the performance of these companies. The construction supply chain was divided into four sectors – material suppliers, construction companies, property developers and real estate investment trusts (REITs. The findings indicate that the impact was minimal for both material suppliers and construction companies, but especially severe for the more leveraged property developers and REITs. Building material suppliers and construction companies have benefitted substantially from the building economic stimulus package provided by the Australian government to mitigate the effects of the GFC. Decreases in the valuation of assets have, to a large extent, reduced the profitability of property developers and REITs during the GFC but these companies have recovered quickly from these adverse conditions to return to a sound financial position by the end of the 2010 financial year. The results will inform investors, managers and construction professionals in devising strategies for prudent financial management and for weathering future financial crises.

  11. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Minenkova, Olga; Hartung, John

    2016-03-01

    'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications. PMID:26744234

  12. Production and characterization of a biotinylated single-chain variable fragment antibody for detection of parathion-methyl.

    Science.gov (United States)

    Wang, Huimin; Zhao, Fengchun; Han, Xiao; Yang, Zhengyou

    2016-10-01

    In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM. PMID:27181246

  13. Important fission product nuclides identification method for simplified burnup chain construction

    International Nuclear Information System (INIS)

    A method of identifying important fission product (FP) nuclides which are included in a simplified burnup chain is proposed. This method utilizes adjoint nuclide number densities and contribution functions which quantify the importance of nuclide number densities to the target nuclear characteristics: number densities of specific nuclides after burnup. Numerical tests with light water reactor (LWR) fuel pin-cell problems reveal that this method successfully identifies important FP nuclides included in a simplified burnup chain, with which number densities of target nuclides after burnup are well reproduced. A simplified burnup chain consisting of 138 FP nuclides is constructed using this method, and its good performance for predictions of number densities of target nuclides and reactivity is demonstrated against LWR pin-cell problems and multi-cell problem including gadolinium-bearing fuel rod. (author)

  14. Constructing Agri-food Industry Input-Output Data: A Value Chain Modelling Approach

    OpenAIRE

    Xayavong, Xila; Islam, Nazrul

    2009-01-01

    Various methods can be used to construct input-output data for sectoral modelling. These methods are broadly classified in the literature as commodity based survey, non-survey and hybrid approaches. Each approach has its own strengths and weaknesses. This paper presents an alternative approach to map the flows of goods and services. The case study of generating inputoutput data for agricultural industries in Western Australia is used to show how value chain modelling can accurately and reliab...

  15. The design and construction of an electronic model of the chain reaction process

    International Nuclear Information System (INIS)

    The design and construction of an electronic model simulating the nuclear chain reaction process up to three successive neutron generations is briefly described. This model is equipped with a special sound effect. However, a tape recorder can be attached and replayed in synchronism with the display with minimum hardware modifications. In this manner, it can function as a useful educational aid. The circuit is built around easily available ttl integrated circuits. (author)

  16. Constructions of the Average Rate of Return of Pension or Investment Funds Based on Chain Indices

    Directory of Open Access Journals (Sweden)

    Jacek Białek

    2014-12-01

    Full Text Available In this paper we consider the problem of the proper construction of the average rate of return of pension (or investment funds. We refer to some economical postulates given by Gajek and Kaluszka (2000. We present, discuss and compare several measures of the average rate of return of funds. We also present alternative measures based on original chain indices. We take into consideration discrete and continuous time stochastic models.

  17. Constructions of the Average Rate of Return of Pension or Investment Funds Based on Chain Indices

    OpenAIRE

    Jacek Białek

    2014-01-01

    In this paper we consider the problem of the proper construction of the average rate of return of pension (or investment) funds. We refer to some economical postulates given by Gajek and Kaluszka (2000). We present, discuss and compare several measures of the average rate of return of funds. We also present alternative measures based on original chain indices. We take into consideration discrete and continuous time stochastic models.

  18. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. PMID:26232710

  19. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

    Science.gov (United States)

    Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

  20. Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein

    Energy Technology Data Exchange (ETDEWEB)

    Yazaki, Paul J. [Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)], E-mail: pyazaki@coh.org; Kassa, Thewodros; Cheung, Chia-wei; Crow, Desiree M. [Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Sherman, Mark A. [Division of Information Sciences, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Bading, James R.; Anderson, Anne-Line J.; Colcher, David; Raubitschek, Andrew [Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)

    2008-02-15

    Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [{sup 125}I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [{sup 111}In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [{sup 64}Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents.

  1. The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies.

    Science.gov (United States)

    Hardingham, T E; Fosang, A J; Hey, N J; Hazell, P K; Kee, W J; Ewins, R J

    1994-03-01

    We have used progressive chondroitinase digestion of pig aggrecan in conjunction with ELISA assays and disaccharide analysis to derive information about the pattern of 4- and 6-sulphation in chondroitin sulphate chains. Digestion with chondroitinase ABC resulted in the release of mainly disaccharides from the nonreducing terminal of chondroitin sulphate chains but there was also the release of some tetra- and hexa-saccharides which were degraded to disaccharides with more extensive digestion. Chondroitinase ACII, in contrast, released only disaccharides. Analysis of the disaccharide composition of the intact and digested products at different stages of digestion showed that there was a slight increase in 6-sulphate content of the chains as they were shortened. Reaction of the partially digested proteoglycans with monoclonal antibodies 3-B-3 and 3-D-5 which recognise chains terminating in 6- or 4-sulphated disaccharides, respectively, showed major differences between chondroitinase ABC and ACII products. The results suggested that chondroitinase ABC preferentially cleaved next to 4-sulphated, rather than 6-sulphated disaccharides and this resulted in some oligosaccharides as well as disaccharide being released. Chondroitinase ACII also cleaved an additional disaccharide next to the linkage to protein of chondroitin sulphate, which was not removed by chondroitinase ABC and this disaccharide was mainly nonsulphated. PMID:7514097

  2. Production of the recombinant single chain anti-B cell lymphoma antibody and evaluation of immunoreactivity

    International Nuclear Information System (INIS)

    Recombinant ScFv lym-1 was produced, using pET vector system for large scale production. ScFv lym-1 gene inserted pET-22b (+) vector, was expressed in E. coli BL-21 strain. ScFv lym-1 antibody extracted from periplasm, was purified with His-Taq column. To evaluated immunoreactivity with Raji cell, ScFv lym-1 was labeled with I-125 and I-125 ScFv lym-1 was purified with desalting column. Raji cell was injected into the C57BR/cdJ SCID mice. Gamma camera imaging were taken time point at 1, 8, 24 and 48 hr with 8 mm pinhole collimator. An active scFv lym-1 could be produced in E. coli with soluble from using pET vector system. Immunoreactivity and affinity constant of lgG lym-1 were 54% and 1.83 x 109 M-1, respectively, and those of scFv lym-1 were 53.7% and 1.46 x 109 M-1, respectively. Biodistribution of I-125 scFv lym-1 antibody showed faster clearance in blood, spleen, kidney and than I-125 lgG lym-1 antibody. Gamma camera image of I-125 scFv lym-1 antibody showed faster clearance and tumor targeting liver than I-125 lgG lym-1 antibody. In vitro properties of scFv lym-1 were similar to those of lgG lym-1. ScFv lym-1 showed faster blood clearance than lgG lym-1. These results suggest that scFv lym-1 antibody can be useful for tumor imaging agent

  3. An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.

    Science.gov (United States)

    Cogotzi, Laura; Giampetruzzi, Annalisa; Nölke, Greta; Orecchia, Martin; Elicio, Vito; Castellano, Maria Antonietta; Martelli, Giovanni P; Fischer, Rainer; Schillberg, Stefan; Saldarelli, Pasquale

    2009-01-01

    Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection. PMID:19082687

  4. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  5. Information Sharing and Channel Construction of Supply Chain under Asymmetric Demand Information

    Directory of Open Access Journals (Sweden)

    Guangdong Wu

    2014-01-01

    Full Text Available Information sharing and marketing channel building have become an important problem of supply chain management theory and practice. The research of information sharing focused on traditional channel of supply chain between upstream and downstream enterprises; however, the research ignores the behavior of information sharing with potential entrants and composite structure characteristics about traditional marketing channel with the direct channel. This paper uses the model to research the effects brought about sharing demand information with potential entrants and building marketing channel, which reveals information sharing and channel building mechanism in the supply chain. The study found that the five-force model of Porter regards potential entrants only as a threat that is one-sided. When the channel competitiveness meets certain conditions, manufacturer and retailer will share demand information with potential entrants. Building composite marketing channel is the manufacturer's absolute dominant strategy. Channel construction will increase the entry barriers for potential entrants and weaken the effect of double marginalization; meanwhile, the performance of supply chain will be augmented.

  6. Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2.

    Directory of Open Access Journals (Sweden)

    Xiangjing Fu

    Full Text Available Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6 cfu/3 µg and 2×10(9 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2 Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

  7. Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

    Science.gov (United States)

    Fu, Xiangjing; Gao, Xiaolong; He, Shengfang; Huang, Di; Zhang, Peng; Wang, Xinglong; Zhang, Shuxia; Dang, Ruyi; Yin, Shuanghui; Du, Enqi; Yang, Zengqi

    2013-01-01

    Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy. PMID:23469171

  8. Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment.

    Science.gov (United States)

    Asmus, Lutz R; Grimshaw, John P A; Richle, Philipp; Eicher, Barbara; Urech, David M; Gurny, Robert; Möller, Michael

    2015-09-01

    Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10 weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500 g/mol, 2500 g/mol, 3500 g/mol and 5000 g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6-14 weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS-PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment. PMID:25779352

  9. Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis.

    Science.gov (United States)

    Guo, L; Wang, J; Qian, S; Yan, X; Chen, R; Meng, G

    2000-11-20

    In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme. PMID:11005928

  10. Eliciting neutralizing antibodies with gp120 outer domain constructs based on M-group consensus sequence.

    Science.gov (United States)

    Qin, Yali; Banasik, Marisa; Kim, SoonJeung; Penn-Nicholson, Adam; Habte, Habtom H; LaBranche, Celia; Montefiori, David C; Wang, Chong; Cho, Michael W

    2014-08-01

    One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development. PMID:25046154

  11. Design and Selection of a Camelid Single-Chain Antibody Yeast Two-Hybrid Library Produced De Novo for the Cap Protein of Porcine Circovirus Type 2 (PCV2)

    OpenAIRE

    Xiangjing Fu; Xiaolong Gao; Shengfang He; Di Huang; Peng Zhang; Xinglong Wang; Shuxia Zhang; Ruyi Dang; Shuanghui Yin; Enqi Du; Zengqi Yang

    2013-01-01

    Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VH...

  12. Characterization of monoclonal antibodies recognizing immunoglobulin kappa and lambda chains in pigs by flow cytometry

    Czech Academy of Sciences Publication Activity Database

    Šinkora, Jiří; Řeháková, Zuzana; Šamánková, Lucie; Haverson, K.; Butler, J. E.; Zwart, R.; Boersma, W.

    2001-01-01

    Roč. 80, - (2001), s. 79-91. ISSN 0165-2427 R&D Projects: GA ČR GA524/00/1280; GA MŠk ME 339 Institutional research plan: CEZ:AV0Z5020903 Keywords : immunoglobulin * light chain * pigs Subject RIV: EC - Immunology Impact factor: 1.389, year: 2001

  13. Characterization of the fine specificity of peptide antibodies to HLA-DQ beta-chain molecules

    DEFF Research Database (Denmark)

    Petersen, J S; Atar, D; Karlsen, Alan E;

    1990-01-01

    In an attempt to produce epitope specific antisera which could distinguish two closely associated HLA-DQ beta-chain alleles, we immunized 20 rabbits with synthetic peptides representing sequences from the first domain of the HLA-DQw8 and -DQw7 beta-chain molecules, differing only by one amino acid...... in position 57. Several of the antisera in immunoblotting specifically recognized either the HLA-DQw7 or the HLA-DQw8 beta-chain allele as previously reported. The fine specificity of the antisera was tested in ELISA using synthetic peptides of varying length as solid phase antigen. Two out of the 20 antisera...... specifically recognized DQw7 beta peptides and two antisera bound only to DQw8 beta peptides from the region containing the amino acid in position 57. To analyze whether the antisera bound to native HLA-DQ beta-chain molecules, FACS analysis was carried out. Seven of the 20 antisera bound to intact EBV...

  14. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

    Science.gov (United States)

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei

    2016-07-01

    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes. PMID:27262204

  15. Detection of activated platelets in a mouse model of carotid artery thrombosis with 18F-labeled single-chain antibodies

    International Nuclear Information System (INIS)

    Introduction: Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel 18F PET radiotracer based on this antibody. Methods: ScFvanti-LIBS and control antibody mut-scFv were reacted with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl3 induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied. Results: After incubation with increasing concentrations of 18F-scFvanti-LIBS clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled 18F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6). Conclusions: Our results show that the novel antibody radiotracer 18F-scFvanti-LIBS is useful for the sensitive detection of activated platelets and thrombosis. Advances in knowledge and implications for patient care: We describe the first 18F variant of a scFvanti-LIBS against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future

  16. Occupational risk induced by construction of energy-production chains. Methodology and evaluation in France

    International Nuclear Information System (INIS)

    The Centre d'etude sur l'evaluation de la protection dans le domaine nucleaire has been conducting studies for several years on the comparison of health and social effects in various electricity production chains; the global results of this research are presented in paper IAEA-SM-254/51, by Fagnani et al., at this Symposium. Attention should be paid to a particular area in risk evaluation: the calculation of effects during the construction stage. The method used, the Leontieff macro-economic analysis, should be examined in detail, for it is a powerful tool whose uses go well beyond risk calculation. Moreover, it is at this stage that the richness and ambiguities of occupational risk measurement appear most clearly. As this is a major factor in the evaluation of risks generated by techniques of electricity production, it is important to show how it is calculated and, especially, how comparative evaluations have been interpreted. Three conventional production technologies are examined (PWR, oil and coal) and two solar-powered (a thermal system extrapolated from the Themis plant built in France and a photovoltaic process). The production scenarios are adapted to the French context, with the location of the installations and the origin of the fuel clearly spelled out. The results of this evaluation are striking: with the exception of coal, which is a labour-intensive industry, a very large portion of occupational risk can be traced to the construction phase (from 67% for the PWR system to 97% for the photovoltaic process). Even in total health risk (occupational and public), construction accounts for a preponderant share. In fact, this risk should be compared to average occupational risk in the economy at large, i.e. that to which workers in electricity production chains would be exposed if they worked elsewhere. This is the meaning of the 'reference level' pointed out in the study. (author)

  17. Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

    Science.gov (United States)

    Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

    2016-01-01

    A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

  18. Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity.

    Directory of Open Access Journals (Sweden)

    Marcela Torres

    Full Text Available Mouse-human chimeric antibodies composed of murine variable (V and human (C chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of C(H glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by C(H region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the C(H domains through an allosteric effect involving networks of highly connected amino acids.

  19. Improving the accuracy of the structure prediction of the third hypervariable loop of the heavy chains of antibodies.

    KAUST Repository

    Messih, Mario Abdel

    2014-06-13

    MOTIVATION: Antibodies are able to recognize a wide range of antigens through their complementary determining regions formed by six hypervariable loops. Predicting the 3D structure of these loops is essential for the analysis and reengineering of novel antibodies with enhanced affinity and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. RESULTS: We describe a method, based on the Random Forest automatic learning technique, to select structural templates for H3 loops among a dataset of candidates. These can be used to predict the structure of the loop with a higher accuracy than that achieved by any of the presently available methods. The method also has the advantage of being extremely fast and returning a reliable estimate of the model quality. AVAILABILITY AND IMPLEMENTATION: The source code is freely available at http://www.biocomputing.it/H3Loopred/ .

  20. A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG

    OpenAIRE

    Rumfelt, Lynn L; Avila, David; Diaz, Marilyn; Bartl, Simona; McKinney, E. Churchill; Flajnik, Martin F.

    2001-01-01

    In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM1gj, from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells (“germline-joined”). Such H chain V genes were discovered over...

  1. Immunoreactivity and Radioimmunoscintigraphy of 4-Lysine Single Chain (Fv) Lym-1 Antibody for the Radiometal Chelation

    International Nuclear Information System (INIS)

    Small size of recombinant scFv, composed of VH and VL region of IgG, has many advantages such as faster blood clearance, improved tumor localization and reduced human anti-mouse antibody (HAMA) response. On the other hand, owing to small size, number of amino group, which was not involved in binding site, of ScFv lym-1 was insufficient in conjugation with CITC-DTPA chelator for radio metal labeling. The goal of this study is to introduce 4-lysine tag to the end of ScFv lym-1 sequence for radio metal conjugation and to evaluate the immunoreactivity and radioimmunoscintigraphy of chelator conjugated 4-lysine taq scFv lym-1 (4-lys scFv)

  2. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    Science.gov (United States)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  3. SCM and extended integration at the lower tiers of the construction supply chain: An explorative study in the Dutch construction industry

    OpenAIRE

    Pryke, S. D.; Broft, R.; Badi, S. M.

    2014-01-01

    Several studies have underlined the potential of Supply Chain Management (SCM) in meeting the formidable challenges associated with fragmentation, adversarial relationships and insufficient customer focus in the delivery of construction projects (e.g. Dainty et al., 2001; Cox and Ireland, 2002; Gadde and Dubois, 2010). However, there remains a paucity of properly documented examples of successfully implemented SCM initiatives, particularly at the lower tiers of the supply chain. This study se...

  4. Light chain editing generates polyreactive antibodies in chronic graft-versus-host reaction

    OpenAIRE

    Witsch, Esther J.; Cao, Hong; Fukuyama, Hidehiro; Weigert, Martin

    2006-01-01

    The chronic graft-versus-host (cGvH) reaction is a model of induced lupus caused by alloreactive CD4+ T cells from a Bm-12 mouse in a C57BL/6 recipient. We used this cGvH reaction in C57BL/6 anti-DNA H chain transgenic mice, 56R/B6, to understand the structure, specificity, and origin of the induced autoantibodies (auto-Abs). We found anti-DNA Abs that reacted to several different antigens, such as phosphatidylserine, myelin basic protein, thyroglobulin, histone, insulin, cytochrome C, and β-...

  5. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

    Science.gov (United States)

    Yang, Ju-Lun; Pan, Xin-Yan; Zhao, Wen-Xing; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; Li, Gui-Yun; Luo, Ying

    2016-03-01

    Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression. PMID:26780944

  6. Anti-CD20 single chain variable antibody fragment–apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas

    Science.gov (United States)

    Crosby, Natasha M.; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A.; Kamei, Ayako; Simonsen, Jens B.; Luo, Bing; Gordon, Leo I.; Forte, Trudy M.; Ryan, Robert O.

    2015-01-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents. PMID:25994015

  7. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    Science.gov (United States)

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents. PMID:25994015

  8. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    Directory of Open Access Journals (Sweden)

    Benevolo Maria

    2006-09-01

    Full Text Available Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu, a member of the human Epidermal Growth Factor (EGF receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs to ErbB-2 that involves their functional expression in (a bacteria, (b transient as well as stable transgenic tobacco plants, and (c a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6 was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T, displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1 only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology

  9. Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody

    Directory of Open Access Journals (Sweden)

    Saeed M

    2016-03-01

    Full Text Available Mesha Saeed,1 Mandy van Brakel,2 Sara Zalba,1 Erik Schooten,2 Joost AP Rens,1 Gerben A Koning,1,† Reno Debets,2 Timo LM ten Hagen1 1Laboratory of Experimental Surgical Oncology, Section Surgical Oncology, Department of Surgery, Erasmus MC, Rotterdam, the Netherlands; 2Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands †Dr Gerben A Koning passed away on December 29, 2015 Abstract: Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs directed against a tumor-selective cancer testis antigen (CTA NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1 presented by human leukocyte antigen A1 (HLA-A1, in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with

  10. Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody.

    Science.gov (United States)

    Saeed, Mesha; van Brakel, Mandy; Zalba, Sara; Schooten, Erik; Rens, Joost Ap; Koning, Gerben A; Debets, Reno; Ten Hagen, Timo Lm

    2016-01-01

    Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting. PMID:27022262

  11. Targeting Prostate Cancer Cells In Vivo Using a Rapidly Internalizing Novel Human Single-Chain Antibody Fragment

    Science.gov (United States)

    He, Jiang; Wang, Yong; Feng, Jinjin; Zhu, Xiaodong; Lan, Xiaoli; Iyer, Arun K.; Zhang, Niu; Seo, Youngho; VanBrocklin, Henry F.; Liu, Bin

    2010-01-01

    Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma. Methods A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with 99mTc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37°C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection. Results The UA20 scFv was labeled in 55%–65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37°C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 sc

  12. Heavy-chain isotype patterns of human antibody-secreting cells induced by Haemophilus influenzae type b conjugate vaccines in relation to age and preimmunity

    DEFF Research Database (Denmark)

    Barington, T; Juul, Lars; Gyhrs, A;

    1994-01-01

    The influence of preexisting immunity on the heavy-chain isotypes of circulating antibody-secreting cells (AbSC) induced by vaccination with Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP) coupled to tetanus toxoid (TT) or diphtheria toxoid (DT) and by vaccination with TT or DT...

  13. Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.

    Science.gov (United States)

    Almagro, Juan Carlos; Raghunathan, Gopalan; Beil, Eric; Janecki, Dariusz J; Chen, Qiang; Dinh, Thai; LaCombe, Ann; Connor, Judy; Ware, Mark; Kim, Paul H; Swanson, Ronald V; Fransson, Johan

    2012-03-01

    Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles. PMID:22407976

  14. Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

    OpenAIRE

    Su, Yu-Cheng; Al-Qaisi, Talal S; Tung, Hsin-Yi; Cheng, Tian-Lu; Chuang, Kuo-Hsiang; Chen, Bing-Mae; Roffler, Steve R.

    2014-01-01

    Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-i...

  15. The Construction of Cold-Chain Logistics Park of Agricultural Products in Sanshui from Two-stage Perspective

    Institute of Scientific and Technical Information of China (English)

    Jianhui; HUANG; Wei; WANG; Quan; LI

    2015-01-01

    This paper firstly analyzed the operation model,market positioning,market demand forecast as well as market competition and challenges,park site selection,and transportation conditions for construction of the Cold-Chain Logistics Park of Agricultural Products in Sanshui.Then,it presented the overall planning scheme for construction of the Cold-Chain Logistics Park of Agricultural Products from a progressive two-stage perspective of overall planning and stage-by-stage implementation.The first stage mainly performs the function as a transaction platform of agricultural products and meanwhile provides customers with agricultural products storage and inspection services.The second stage adds value-added services such as distribution processing,modified atmosphere storage,freezing and refrigeration,market price information distribution,E-commerce of agricultural products and personalized services.It is expected to provide references and suggestions for the construction of the Cold-Chain Logistics Park of Agricultural Products.

  16. The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma

    Science.gov (United States)

    Iyer, Arun K.; Su, Yang; Feng, Jinjin; Lan, Xiaoli; Zhu, Xiaodong; Liu, Yue; Gao, Dongwei; Seo, Youngho; VanBrocklin, Henry F.; Broaddus, V. Courtney; Liu, Bin; He, Jiang

    2011-01-01

    Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with 111In (111In-IL-M1), along with control non-targeted liposomes (111In-CL). Incubation of 111In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell-line (BPH-1) at 37°C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor bearing mice intravenous (i.v.) injection of 111In-IL-M1 led to remarkable tumor accumulation: 4 % and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of 111In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of 111In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting. PMID:21255833

  17. Harnessing T cells to fight cancer with BiTE(®) antibody constructs - past developments and future directions.

    Science.gov (United States)

    Klinger, Matthias; Benjamin, Jonathan; Kischel, Roman; Stienen, Sabine; Zugmaier, Gerhard

    2016-03-01

    Bispecific T-cell engager (BiTE(®) ) antibody constructs represent a novel immunotherapy that bridges cytotoxic T cells to tumor cells, thereby inducing target cell-dependent polyclonal T-cell activation and proliferation, and leading to apoptosis of bound tumor cells. Anti-CD19 BiTE(®) blinatumomab has demonstrated clinical activity in Philadelphia chromosome (Ph)-negative relapsed or refractory (r/r) acute lymphoblastic leukemia (ALL) eventually resulting in conditional approval by the U.S. Food and Drug Administration in 2014. This drug is currently further developed in pediatric and Ph(+) r/r, as well as in minimal residual disease-positive ALL, and might also offer clinical benefit for patients with non-Hodgkin's lymphoma, especially for those with aggressive forms like diffuse large B-cell lymphoma. Another BiTE(®) antibody construct in hemato-oncology designated AMG 330 targets CD33 on acute myeloid leukemia blast cells. After showing promising ex vivo activity, this drug candidate has recently entered phase 1 clinical development, and has further indicated potential for combination with checkpoint inhibitors. In solid tumor indications, three BiTE(®) antibody constructs have been tested in phase 1 studies so far: anti-EpCAM BiTE(®) AMG 110, anti-CEA BiTE(®) MEDI-565/AMG 211, and anti-PSMA BiTE(®) BAY2010112/AMG 212. Pertinent questions comprise how to maximize BiTE(®) penetration and T-cell infiltration of the tumor while simultaneously minimizing any adverse events, which is currently explored by a continuous intravenous infusion approach. Thus, BiTE(®) antibody constructs will hopefully provide new treatment options for patients in several indications with high unmet medical need. PMID:26864113

  18. High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector.

    Science.gov (United States)

    Gurkan, Cemal; Symeonides, Stefan N; Ellar, David J

    2004-02-01

    The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene ( AOX1 ). For example, pIB4 is an Escherichia coli - P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae alpha-factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4alpha, and its successful use for the high-level expression and secretion of a functional single-chain antibody fragment (scFv), C6.5, which targets p185(HER-2), a cell-surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4alpha also introduced a short DNA sequence coding for a C-terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized-metal-affinity chromatography, to a yield of 70 mg x l(-1) of shake-flask culture. In conclusion, our results suggest that the secretion expression vector pIB4alpha not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors. PMID:12962542

  19. Construction of a prokaryotic expression system of vacA gene and detection of vatA gene,VacA protein in Helicobacter pylori isolates and ant-VacA antibody in patients'sera

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Ya-Fei Mao

    2004-01-01

    AIM: To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein,and to determine prevalence of vacA-carryinglVacA expressing Hpyloriisolates and seroprevalence of specific ant-VacA antibody in H pyloriinfected patients.METHODS: Polymerase chain reaction technique was used to amplify complete vacA gene of H pyloristrain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDSPAGE. Western blot using commercial antibodies against whole cell of H pyloriand an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pyloriisolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori.RESULTS: In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pyloriand to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pyloriisolates carried vacA gene, but only 66.1% expressed Vac A protein. Of the serum samples tested,42.4% were positive for specific anti-VacA antibody.CONCLUSION: A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high

  20. Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.

    Science.gov (United States)

    McMillan, J R; McGrath, J A; Pulkkinen, L; Kon, A; Burgeson, R E; Ortonne, J P; Meneguzzi, G; Uitto, J; Eady, R A

    1997-06-01

    The anchoring filament protein laminin 5 is composed of three polypeptide chains (alpha 3, beta 3 and gamma 2) each encoded by separate genes (LAMA3, LAMB3 and LAMC2, respectively). Mutations in any of these three genes may give rise to the autosomal recessive blistering skin disease, junctional epidermolysis bullosa. At present, there is no easy way of predicting which of these three genes might harbour the pathogenetic laminin 5 mutations in a case of junctional epidermolysis bullosa. In this study, we assessed whether immunohistochemistry might be helpful in this regard. We performed immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies in 11 patients with junctional epidermolysis bullosa, in whom the laminin 5 mutations had been previously delineated. Although, labelling for the laminin 5 chain bearing the mutations was attenuated or undetectable in all cases, a complete absence of labelling or a reduction in the staining intensity for the other two chains was also seen in all cases. The results showed that immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies is not a specific indicator for which of the laminin 5 chain genes contains the pathogenetic mutations, and is therefore unreliable in screening for individual laminin 5 gene mutations in cases of junctional epidermolysis bullosa. PMID:9217810

  1. Characterization of the Recognition Specificity of BH2, a Monoclonal Antibody Prepared against the HLA-B27 Heavy Chain

    Directory of Open Access Journals (Sweden)

    Hui-Chun Yu

    2015-04-01

    Full Text Available BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC, can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS. However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.

  2. Antiperiodic XXZ Chains with Arbitrary Spins: Complete Eigenstate Construction by Functional Equations in Separation of Variables

    Science.gov (United States)

    Niccoli, Giuliano; Terras, Véronique

    2015-07-01

    Generic inhomogeneous integrable XXZ chains with arbitrary spins are studied by means of the quantum separation of variables (SOV) method. Within this framework, a complete description of the spectrum (eigenvalues and eigenstates) of the antiperiodic transfer matrix is derived in terms of discrete systems of equations involving the inhomogeneity parameters of the model. We show here that one can reformulate this discrete SOV characterization of the spectrum in terms of functional T - Q equations of Baxter's type, hence proving the completeness of the solutions to the associated systems of Bethe-type equations. More precisely, we consider here two such reformulations. The first one is given in terms of Q-solutions, in the form of trigonometric polynomials of a given degree , of a one-parameter family of T - Q functional equations with an extra inhomogeneous term. The second one is given in terms of Q-solutions, again in the form of trigonometric polynomials of degree but with double period, of Baxter's usual (i.e., without extra term) T - Q functional equation. In both cases, we prove the precise equivalence of the discrete SOV characterization of the transfer matrix spectrum with the characterization following from the consideration of the particular class of Q-solutions of the functional T - Q equation: to each transfer matrix eigenvalue corresponds exactly one such Q-solution and vice versa, and this Q-solution can be used to construct the corresponding eigenstate.

  3. First steps toward the construction of a hyperphase diagram that covers different classes of short polymer chains

    Science.gov (United States)

    Sabeur, Sid

    2014-06-01

    We present the results of a multicanonical Monte Carlo study of flexible and wormlike polymer chains, where we investigate how the polymer structures observed during the simulations, mainly coil, liquid, and crystalline structures, can help to construct a hyperphase diagram that covers different polymer classes according to their thermodynamic behavior.

  4. Nanoscale superstructures assembled by polymerase chain reaction (PCR): programmable construction, structural diversity, and emerging applications.

    Science.gov (United States)

    Kuang, Hua; Ma, Wei; Xu, Liguang; Wang, Libing; Xu, Chuanlai

    2013-11-19

    Polymerase chain reaction (PCR) is an essential tool in biotechnology laboratories and is becoming increasingly important in other areas of research. Extensive data obtained over the last 12 years has shown that the combination of PCR with nanoscale dispersions can resolve issues in the preparation DNA-based materials that include both inorganic and organic nanoscale components. Unlike conventional DNA hybridization and antibody-antigen complexes, PCR provides a new, effective assembly platform that both increases the yield of DNA-based nanomaterials and allows researchers to program and control assembly with predesigned parameters including those assisted and automated by computers. As a result, this method allows researchers to optimize to the combinatorial selection of the DNA strands for their nanoparticle conjugates. We have developed a PCR approach for producing various nanoscale assemblies including organic motifs such as small molecules, macromolecules, and inorganic building blocks, such as nanorods (NRs), metal, semiconductor, and magnetic nanoparticles (NPs). We start with a nanoscale primer and then modify that building block using the automated steps of PCR-based assembly including initialization, denaturation, annealing, extension, final elongation, and final hold. The intermediate steps of denaturation, annealing, and extension are cyclic, and we use computer control so that the assembled superstructures reach their predetermined complexity. The structures assembled using a small number of PCR cycles show a lower polydispersity than similar discrete structures obtained by direct hybridization between the nanoscale building blocks. Using different building blocks, we assembled the following structural motifs by PCR: (1) discrete nanostructures (NP dimers, NP multimers including trimers, pyramids, tetramers or hexamers, etc.), (2) branched NP superstructures and heterochains, (3) NP satellite-like superstructures, (4) Y-shaped nanostructures and DNA

  5. Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine.

    Science.gov (United States)

    Ohshima, Motohiro; Inoue, Kazuyuki; Hayashi, Hideki; Tsuji, Daiki; Mizugaki, Michinao; Itoh, Kunihiko

    2010-11-01

    DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens. PMID:20876190

  6. Reshaping Human Antibodies: Grafting an Antilysozyme Activity

    Science.gov (United States)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

    1988-03-01

    The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

  7. Value Chain Analysis of Construction Enterprise in China%国内建筑施工企业的价值链分析

    Institute of Scientific and Technical Information of China (English)

    邓正位; 叶敏

    2011-01-01

    在分析建筑行业现状的基础上,结合价值链理论和多年的建筑施工经验,构建施工企业的价值链系统,系统介绍施工企业如何对企业的行业价值链、内部价值链和相关单位价值链进行分析,最后对如何提高企业价值链管理水平提出建议.%Based on current situation of construction industry, value chain theory and building construction experience, this paper constructs value chain system of construction enterprise, presents how to analyze industry value chain. intemal value chain and related units value chain. Finally, some suggestions are put forward to enhance value chain management for construction enterprise.

  8. Chlamydia trachomatis-specific antibodies in patients with pelvic inflammatory disease: comparison with isolation in tissue culture or detection with polymerase chain reaction.

    OpenAIRE

    Theunissen, J J; Minderhoud-Bassie, W; Wagenvoort, J H; Stolz, E; Michel, M F; Huikeshoven, F.J.

    1994-01-01

    OBJECTIVE--The detection of acute phase antibodies against C trachomatis and its comparison with tissue culture or polymerase chain reaction (PCR) on samples of cervix and urethra obtained from patients with pelvic inflammatory disease (PID). METHODS--In the academic hospital Dijkzigt, Rotterdam, The Netherlands, prospective investigations were performed on 49 consecutive patients who were admitted with the diagnosis of PID. Infections with C trachomatis were traced using tissue culture, PCR ...

  9. Production and characterization of a single-chain antibody of anti-CD3%抗CD3单链抗体基因克隆表达及生物活性鉴定

    Institute of Scientific and Technical Information of China (English)

    陈秀芹; 阎锡蕴

    2003-01-01

    The genes encoding antibody heavy and light chain variable regions(VH and VL)were cloned by RT-PCR from OKT3 hybridoma cells,which produced anti-CD3 moleclonal antibody.The VH and VL genes were fused and become a single chain Fv(scFv).The scFv gene was cloned into pCANTAB5E vector and expressed on bacterial phage surface.By three panning rounds,we have obtained two single-chain antibodys that specific for CD3.The antiCD3 scFv wil be a reagent fox diagnosis and therapy of immuno-disorder.

  10. Immunoprophylaxis in fish by injection of mouse antibody genes

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Cupit, P.M.; Einer-Jensen, Katja; Lorenzen, Ellen; Ahrens, Peter; Secombes, C.J.; Cunningham, C.

    2000-01-01

    infectious diseases. To test this in a fish model, a gene construct encoding a neutralizing single-chain antibody to the fish-pathogenic rhabdovirus VHSV (viral hemorrhagic septicemia virus) was administered to rainbow trout by intramuscular injection of plasmid DNA, Circulating recombinant antibodies could...

  11. Expression of the p70 chain of the IL-2 receptor on human lymphoid cells. Analysis using a monoclonal antibody and high-sensitivity immunofluorescence

    International Nuclear Information System (INIS)

    Using monoclonal antibody (MoAb) against the p70(β) chain of the interleukin-2receptor (IL-2R) and a high-sensitivity immunofluorescence procedure, p70 can be demonstrated on the majority of lymphocytes from normal blood samples, without in vitro stimulation. A subpopulation of cells bears a high concentration of receptor, and these cells have the physical properties of large granular leucocytes (LGL). The smaller lymphocytes, although weaker, are quite clearly positive. T8 cells stain relatively strongly, while T4 cells and B cells stain relatively weakly. Leukaemic cells showed a variety of phenotypes when examined for expression of the p55 and p70 chains of the IL-2R. The demonstration of IL-2R chains directly by immunofluorescence on unstimulated lymphocytes has potential clinical applications, since this technique can be used in a diagnostic setting. 22 refs., 5 figs

  12. Application of a Trifunctional Reactive Linker for the Construction of Antibody-Drug Hybrid Conjugates

    OpenAIRE

    Thomas, Joshua D.; Hofer, Thomas; Rader, Christoph; Burke, Terrence R

    2008-01-01

    A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin α4β1-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells express...

  13. Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

    Indian Academy of Sciences (India)

    Ming Ni; Bing Yu; Y U Huang; Zhenjie Tang; Ping Lei; Xin Shen; Wei Xin; Huifen Zhu; Guanxin Shen

    2008-12-01

    We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïve phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.

  14. Construction of a biotinylated cameloid-like antibody for lable-free detection of apolipoprotein B-100.

    Science.gov (United States)

    Li, Henan; Yan, Junrong; Ou, Weijun; Liu, Hong; Liu, Songqin; Wan, Yakun

    2015-02-15

    Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15 kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL(-1) with a detection limit of 0.03 ng mL(-1). This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis. PMID:25203942

  15. The role of the supply chain in the elimination and reduction of construction rework and defects: an action research approach

    OpenAIRE

    Taggart, Martin; Koskela, Lauri; Rooke, John

    2014-01-01

    Since 2007, Ireland has suffered a circa 80% reduction in construction output. This has resulted in bankruptcy, unemployment and bad debt. Contractors have attached greater emphasis to production efficiency and cost reduction as a means of survival. An action research (AR) strategy was used to improve processes adopted by a small/medium enterprise (SME) contractor for the control of defects in its supply chain. It is conservatively estimated that rework, typically, accounts for circa 5% of to...

  16. Information Sharing and Channel Construction of Supply Chain under Asymmetric Demand Information

    OpenAIRE

    Guangdong Wu; Qingshan Kong; Jian-gang Shi; Hamid Reza Karimi; Wei Zhang

    2014-01-01

    Information sharing and marketing channel building have become an important problem of supply chain management theory and practice. The research of information sharing focused on traditional channel of supply chain between upstream and downstream enterprises; however, the research ignores the behavior of information sharing with potential entrants and composite structure characteristics about traditional marketing channel with the direct channel. This paper uses the model to research the eff...

  17. Click ‘Like’ and Post It on Your Wall! Chain Posts on Facebook – Identity Construction and Values

    Directory of Open Access Journals (Sweden)

    Piret Voolaid

    2013-04-01

    Full Text Available The article focuses on chain posts that were collected in the years 2010–2012 and spread predominantly among girls of ten to twelve on Facebook (facebook.com – a social network that has a membership of over 450,000 in Estonia. The source material comprising approximately 220 texts is similar by form and content to chain letters known from earlier tradition; yet, the web environment with its specific technical structure allows the texts to turn into a peculiar Facebook-like phenomenon.The author takes a closer interest in the changes in form adapted to chain letters as a genre in Facebook environment as well as thematic categories of these letters. The analysis of the epistolary cultural phenomenon focuses on the socio-folkloric nature of texts with its communicative and socio-cultural aspects. The main focus is on how socio-cultural environment influences changes in the genre, what kind of global and local impacts occur in Estonian-language chain posts and how this everyday genre reflects the realities of the era and the values intrinsic to this age group. The levels of personal and collective identity construction of chain posters as a special age group have been analysed against the identity motivation theory known from social psychology.

  18. Preparation and Identification of a Human Single Chain Fv Antibody Against Ascaris lumbricoides%抗蛔虫人源单链抗体库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    何光志; 田维毅; 高英; 王平; 王文佳; 奚锦; 俞琦; 王乾宇; 黄高

    2011-01-01

    Objective To construct humanize phage antibody library against Ascaris lumbricoides and to screen specificity scFv to Ascaris lumbricoides. Methods Total RNA was abstracted from peripheral blood lympho-cytes of 20 persons, and was used to amplify VH and VL gene by RT-PCR. The amplified VH and VL genes were spliced to form scFv gene which was cloned into pCANTAB-SE phagemid, and the constructed recombinant phage-mid was transformed to E. Coli TC1 to construct human natural single-chain antibody library from which positive clones were screened. Results A primary library of 1.5 × 106 and a second library of 1.2 × 106 were constructed. Conclusion This study was to provide us the basis for radionuclide imaging and therapy for ascariasis.%目的:构建抗蛔虫人源单链抗体库,从中筛选建抗蛔虫人源特异性单链抗体.方法:分离10个患蛔虫病人的淋巴细胞,提取总RNA反转录为cDNA,PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,采用SOE-PCR法将VH和VL片段随机拼接成scFv片段,并克隆入噬菌粒载体pCANTAB5E中,构建噬菌体单链抗体库.结果:初级库库容量为1.8×106,在大肠杆菌TG1中重组后得到1.6×106的次级抗体库.结论:本研究成功构建抗蛔虫人源单链抗体库,拟在为蛔虫病的预防、诊断、治疗奠定基础.

  19. Polymorphisms of the T cell receptor CD3delta and CD3varepsilon chains affect anti-CD3 antibody binding and T cell activation

    DEFF Research Database (Denmark)

    Boding, Lasse; Nielsen, Martin Weiss; Bonefeld, Charlotte Menné;

    2010-01-01

    suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms...... CD3delta and varepsilon ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for......T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of...

  20. Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.

    Science.gov (United States)

    Zhao, Fengchun; Tian, Yuan; Wang, Huimin; Liu, Jiye; Han, Xiao; Yang, Zhengyou

    2016-09-01

    Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides. PMID:27411546

  1. Supply chain partnership in construction a field study on project team level factors

    NARCIS (Netherlands)

    Koolwijk , J.S.J.; Van Oel, C.J.; Wamelink , J.W.F.

    2015-01-01

    People and their relationship are at the heart of supply chain partnerships, however there is a lack of qualitative studies focusing on how integrated relationships may be developed. Therefore, the purpose of this study was to conduct field research to deepen our understanding of team level variable

  2. Improving supply chain management in construction: what can be learned from the aerospace industry?

    NARCIS (Netherlands)

    Voordijk, H.; Vrijhoef, R.; Greenwood, D.J.

    2003-01-01

    In order to provide for controllable delivery, reliable lead times and efficient customer response, lean manufacturing and platform assembly practices play an important role in supply chains in the aerospace industry. The adoption of lean manufacturing practices ensures an efficient delivery of prod

  3. 2D warp-and-woof interwoven networks constructed by helical chains with different chirality.

    Science.gov (United States)

    Feng, Yuhua; Guo, Yang; OuYang, Yan; Liu, Zhanquan; Liao, Daizheng; Cheng, Peng; Yan, Shiping; Jiang, Zonghui

    2007-09-21

    Two unprecedented 2D entangled layers of warp-and-woof threads interwoven by left- and right-handed helical chains, {[Mn(salen)Au(CN)2]4(H2O)}n (salen = N,N'-ethylenebis(salicylideneaminato)) and {Mn(acacen)Ag(CN)2}n (acacen = N,N'-ethylenebis(acetylacetonylideneiminate)) 2, have been synthesized and characterized. PMID:17728880

  4. Modularity in supply chains: a multiple case study in the construction industry

    NARCIS (Netherlands)

    Voordijk, Hans; Meijboom, Bert; Haan, de Job

    2006-01-01

    Purpose – The objective of this study is to assess the applicability of Fine's three-dimensional modularity concept as a tool to describe and to analyze the alignment of product, process, and supply chain architectures. Fine claims that the degree of modularity in the final output product has a one-

  5. In silico design, construction and cloning of Trastuzumab humanized monoclonal antibody: A possible biosimilar for Herceptin

    Directory of Open Access Journals (Sweden)

    Soudabeh Akbarzadeh-Sharbaf

    2012-01-01

    Conclusions: This study was based on a recently developed technology for mAb expression in mammalian cells. The obtained constructs could be successfully used for biosimilar recombinant mAb production in CHO DG44 dihydrofolate reductase (DHFR gene deficient cell line in the suspension culture medium.

  6. Hydrothermal synthesis, crystal structure and properties of a novel chain coordination polymer constructed by tetrafunctional phosphonate anions and cobalt ions

    International Nuclear Information System (INIS)

    A novel cobalt phosphonate, [Co(HL)(H2O)3]n (1) (L=N(CH2PO3H)33−) has been synthesized by hydrothermal reaction at 150 °C and structurally characterized by X-ray diffraction, infrared spectroscopy, elemental and thermogravimetric analysis. Complex 1 features a 1D chain structure with double-channel built from CoO6 octahedra bridged together by the phosphonate groups. Each cobalt ion is octahedrally coordinated by three phosphonate oxygen atoms and three water molecules. The coordinated water molecules can form the hydrogen bonds with the phosphonate oxygen atoms to link the 1D chains, building a 2D layered structure, further resulting in a 3D network. The luminescence spectrum indicates an emission maximum at 435 nm. The magnetic susceptibility curve exhibits a dominant antiferromagnetic behavior with a weakly ferromagnetic component at low temperatures. - Graphical abstract: The connectivity between cobalt ions and the ligands results in a chain structure with a 1D double-channel structure, which is constructed by A-type subrings and B-type subrings. - Highlights: • The tetrafunctional phosphonate ligand was used as the ligand. • A novel chain structure can be formed by A-type rings and B-type rings. • Two types of rings can form a 1D double-channel structure, along the c-axis

  7. Hydrothermal synthesis, crystal structure and properties of a novel chain coordination polymer constructed by tetrafunctional phosphonate anions and cobalt ions

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Lei, E-mail: gl_coord@163.com [School of Chemistry and Materials Science, Liaoning University of Petroleum and Chemical Engineering, Fushun 113001 (China); Wang, Ying [Center of Experiment, College of Chemistry, Chemical Engineering and Environmental Engineering, Liaoning University of Petroleum and Chemical Engineering, Fushun 113001 (China)

    2015-08-15

    A novel cobalt phosphonate, [Co(HL)(H{sub 2}O){sub 3}]{sub n} (1) (L=N(CH{sub 2}PO{sub 3}H){sub 3}{sup 3−}) has been synthesized by hydrothermal reaction at 150 °C and structurally characterized by X-ray diffraction, infrared spectroscopy, elemental and thermogravimetric analysis. Complex 1 features a 1D chain structure with double-channel built from CoO{sub 6} octahedra bridged together by the phosphonate groups. Each cobalt ion is octahedrally coordinated by three phosphonate oxygen atoms and three water molecules. The coordinated water molecules can form the hydrogen bonds with the phosphonate oxygen atoms to link the 1D chains, building a 2D layered structure, further resulting in a 3D network. The luminescence spectrum indicates an emission maximum at 435 nm. The magnetic susceptibility curve exhibits a dominant antiferromagnetic behavior with a weakly ferromagnetic component at low temperatures. - Graphical abstract: The connectivity between cobalt ions and the ligands results in a chain structure with a 1D double-channel structure, which is constructed by A-type subrings and B-type subrings. - Highlights: • The tetrafunctional phosphonate ligand was used as the ligand. • A novel chain structure can be formed by A-type rings and B-type rings. • Two types of rings can form a 1D double-channel structure, along the c-axis.

  8. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  9. Research on the Construction of Project Critical Chain Model in South-to-North Water Diversion Project with Multi-Resource Constraints Based on Portfolio Technology

    Directory of Open Access Journals (Sweden)

    Jing-chun FENG

    2011-06-01

    Full Text Available Recently, the critical chain study has become the hot spot within the project management research field, so as the construction of it with multi-resource constraints become the new research subject. Referring to System Analysis Theory and Project Portfolio Theory, this paper discusses the creation of project portfolio based on project similarity, and definition of priority in multi-resource allocation based on quantitative analysis. Then according to the theory on critical chain construction, the author made relevant research and proposed the construction model with the multi-resource constraints, which to be applied to the critical chain construction of A-bid section in South-to-North Water Diversion Project. And necessary contrast analysis with the Comprehensive Treatment Construction Method and Aggressive Treatment Construction Method also has been made within this paper.

  10. Construction and Design of Post-Tensioned Pearl-Chain Bridges using SL-Technology

    DEFF Research Database (Denmark)

    Halding, Philip Skov

    2016-01-01

    a filling layer of lower stiffness above the arches to level the road surface. The present Ph.D. thesis is part of a larger development project about Pearl‐Chain Bridges funded by Innovationsfonden. The project also included another Ph.D. study about the developed materials used in Pearl‐Chain Bridges......, and numerical modelling. Despite of high levels of normal force in PC‐Bridges, the result showed that a Mesnager inspired hinge type had a response similar to what was predicted in the literature. A specially designed saddle bearing also had elastic and plastic rotational resistance, but this hinge type......) A test to fracture to observe the ductility in the system, and fracture type. The collapse occurred after two plastic hinges were formed in the 3/8, and 5/8 points of the span. Several warnings signs were observed when approaching the maximum loading....

  11. Spontaneous construction of photoactive hollow TiO2 microspheres and chains

    International Nuclear Information System (INIS)

    Discrete and chain-like aggregates of well-defined hollow TiO2 microspheres are reproducibly synthesized in high yield by a modified fluoride-mediated self-transformation strategy using urea as a base catalyst. The shell walls are composed of agglomerated polyhedral anatase nanocrystals, and exhibit hierarchical porosity. The addition of urea tunes the nucleation dynamics and surface states of the elementary TiO2 building blocks, which together promote the formation of metastable solid microparticles of uniform size that subsequently transform into morphologically invariant hollow microspheres and chain-like aggregates. The high surface area, bimodal mesoporosity of the shell walls, and increased band gap of the hollow TiO2 microspheres give rise to increases in photocatalytic activity when compared with anatase nanoparticles and aggregates prepared in the absence of urea.

  12. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    Science.gov (United States)

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-01-01

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. Images PMID:493112

  13. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    Science.gov (United States)

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. PMID:493112

  14. The use of a cocktail of single chain Fv antibody fragments to improve the in vitro and in vivo targeting of melanoma

    International Nuclear Information System (INIS)

    Radio scintigraphy using single chain antibody fragments (scFvs) offers a potenti al means of early detection of melanoma metastases. However, previous studies have shown suboptimal levels of tumour localization and nonspecific background accumulation which may be due to antigen heterogeneity. We aimed to improve tumour localization by using a cocktail of different scFvs targeting different epitopes on melanoma cells. We have previously developed three scFvs against distinct and highly tumour-specific melanoma cell-surface antigens by chain shuffling and antibody phage selection on melanoma cells. Three scFvs, RAFT3, B3 and B4 were labeled with 125Iodine and tested both individually and as a cocktail in a nude mouse xenograft model far human melanoma. Results demonstrated improved tumour localization in vivo when compared to the individual scFvs. Tumour uptake of the cocktail at l hour was 24.220% ID/g (injected dose/gram) compared with 2.854%, 2.263% and 1.355% far B4, RAFT3 and B3, respectively, when injected individually. In addition, the cocktail exhibited significantly superior tumour to normal tissue ratios far muscle and spleen (p<0.05). A combination or cocktail of scFv clones may have an advantage aver individual scFvs far melanoma targeting in patients because of heterogeneity in the expression of different epitopes of antigens on melanoma cells

  15. Identification of the specificity of isolated phage display single-chain antibodies using yeast two-hybrid screens

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik

    2009-01-01

    A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast...

  16. A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

    Science.gov (United States)

    Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H; Shoemaker, Charles B

    2015-03-01

    Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. PMID:25564615

  17. Optimizing the supply chain for passive materials in network construction projects of a telecom operator

    OpenAIRE

    Bergman, Leo Bergman

    2016-01-01

    Manufacturing Planning and Control (MPC) is a hierarchical manufacturing paradigm that has not been studied much in a project-oriented production environment. The few articles that have covered the field have come to a conclusion that it is either not feasible at all or that some aspects of MPC can be applied also in project business. In this thesis, MPC is tested in a project environment by conducting a case study on the supply chain of passive materials in optical network projects of a ...

  18. Brief Communication: Earthquake sequencing: analysis of time series constructed from the Markov chain model

    Science.gov (United States)

    Cavers, M. S.; Vasudevan, K.

    2015-10-01

    Directed graph representation of a Markov chain model to study global earthquake sequencing leads to a time series of state-to-state transition probabilities that includes the spatio-temporally linked recurrent events in the record-breaking sense. A state refers to a configuration comprised of zones with either the occurrence or non-occurrence of an earthquake in each zone in a pre-determined time interval. Since the time series is derived from non-linear and non-stationary earthquake sequencing, we use known analysis methods to glean new information. We apply decomposition procedures such as ensemble empirical mode decomposition (EEMD) to study the state-to-state fluctuations in each of the intrinsic mode functions. We subject the intrinsic mode functions, derived from the time series using the EEMD, to a detailed analysis to draw information content of the time series. Also, we investigate the influence of random noise on the data-driven state-to-state transition probabilities. We consider a second aspect of earthquake sequencing that is closely tied to its time-correlative behaviour. Here, we extend the Fano factor and Allan factor analysis to the time series of state-to-state transition frequencies of a Markov chain. Our results support not only the usefulness of the intrinsic mode functions in understanding the time series but also the presence of power-law behaviour exemplified by the Fano factor and the Allan factor.

  19. Need of a consistent and convenient nucleus identification in ENDF files for the automatic construction of the depletion chains

    Science.gov (United States)

    Mosca, Pietro; Mounier, Claude

    2016-03-01

    The automatic construction of evolution chains recently implemented in GALILEE system is based on the analysis of several ENDF files : the multigroup production cross sections present in the GENDF files processed by NJOY from the ENDF evaluation, the decay file and the fission product yields (FPY) file. In this context, this paper highlights the importance of the nucleus identification to properly interconnect the data mentioned above. The first part of the paper describes the present status of the nucleus identification among the several ENDF files focusing, in particular, on the use of the excited state number and of the isomeric state number. The second part reviews the problems encountered during the automatic construction of the depletion chains using recent ENDF data. The processing of the JEFF-3.1.1, ENDF/B-VII.0 (decay and FPY) and the JEFF-3.2 (production cross section) points out problems about the compliance or not of the nucleus identifiers with the ENDF-6 format and sometimes the inconsistencies among the various ENDF files. In addition, the analysis of EAF-2003 and EAF-2010 shows some incoherence between the ZA product identifier and the reaction identifier MT for the reactions (n, pα) and (n, 2np). As a main result of this work, our suggestion is to change the ENDF format using systematically the isomeric state number to identify the nuclei. This proposal is already compliant to a huge amount ENDF data that are not in agreement with the present ENDF format. This choice is the most convenient because, ultimately, it allows one to give human readable names to the nuclei of the depletion chains.

  20. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    OpenAIRE

    Benevolo Maria; Natali Pier; Martayan Aline; Fraioli Rocco; Tornambé Andrea; Sperandei Maria; Di Donato Monica; Novelli Flavia; Pietraforte Immacolata; Lombardi Alessio; Galeffi Patrizia; Mottolese Marcella; Ylera Francisco; Cantale Cristina; Giacomini Patrizio

    2006-01-01

    Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach ...

  1. Eco-innovation Strategies in the Construction Sector: Impacts on Nanotech Innovation in the Window Chain

    Science.gov (United States)

    Andersen, M. M.; Molin, M.

    In this paper we examine the strategic response of the construction companies to the climate agenda and the emerging nanotechnologies. A case is brought on the Danish window industry. The construction industry has for a long time been considered little innovative and this also goes for the window section. It seems that a combination of an intensified focus on climate issues and the potential of nanotechnology is invoking a new innovation potential and pressure in the window industry. Specifically we identify a strategic shift among the major players towards more systemic innovations that places the window as a part of the wider energy system of the house, a trend that isn’t driven by but could be reinforced by the new nanotech opportunities that so far only play a limited strategic role.

  2. Drug Development Value Chain Constructed by Collaboration Between The SOSHO Project and The NPO BIOGRID

    Science.gov (United States)

    Inoue, Tsuyoshi; Kado, Yuji; Tokuoka, Keiji; Matsumura, Hiroyoshi; Kai, Yasushi; Mori, Yusuke; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Fukunishi, Yoshifumi; Nakamura, Haruki; Kinoshita, Takayoshi; Nakanishi, Isao; Okuno, Yasushi; Minakata, Seiji; Sakata, Tsuneaki

    2007-03-01

    We recently established a drug development value chain collaborated by The SOSHO project (http://www.sosho.jp) and The BioGrid Project (http://www.biogrid.jp/) to accelerate new drug development. The SOSHO project provides new crystal growth methods including handling of protein crystals, and The BioGrid Project their developing software necessary for the in silico screening of promising drugs and the simulation of biological responses to proteins. We selected the two target enzymes; human hematopoietic prostaglandin D synthase (H-PGDS) and orotidine 5'-monophosphate decarboxylase from human malaria parasite plasmodium falciparum (PfOMPDC). The optimizing of HQL-79, the inhibitor for human H-PGDS and the screening of a lead compound for PfOMPDC by using in silico method are in study.

  3. Characterization of a Bispecific FLT3 X CD3 Antibody in an Improved, Recombinant Format for the Treatment of Leukemia

    OpenAIRE

    Durben, Michael; Schmiedel, Dominik; Hofmann, Martin; Vogt, Fabian; Nübling, Tina; Pyz, Elwira; Bühring, Hans-Jörg; Rammensee, Hans-Georg; Salih, Helmut R.; Große-Hovest, Ludger; Jung, Gundram

    2015-01-01

    FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies...

  4. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

    Science.gov (United States)

    To improve expression efficiency of the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse in the methylotrophic yeast Pichia pastoris (P. pastoris) GS115, the DNA sequence encoding for CBL-scFv was designed and synthesized based on the codon bias of P. p...

  5. Spherical core-shell magnetic particles constructed by main-chain palladium N-heterocyclic carbenes

    Science.gov (United States)

    Zhao, Huaixia; Li, Liuyi; Wang, Jinyun; Wang, Ruihu

    2015-02-01

    The encapsulation of the functional species on magnetic core is a facile approach for the synthesis of core-shell magnetic materials, and surface encapsulating matrices play crucial roles in regulating their properties and applications. In this study, two core-shell palladium N-heterocyclic carbene (NHC) particles (Fe3O4@PNP1 and Fe3O4@PNP2) were prepared by a one-pot reaction of semi-rigid tripodal imidazolium salts and palladium acetate in the presence of magnetite nanoparticles. The magnetite nanoparticles are encapsulated inside the main-chain palladium, which act as cores. The conjugated effects of triphenyltriazine and triphenylbenzene in the imidazolium salts have important influence on their physical properties and catalytic performances. Fe3O4@PNP2 shows better recyclability than Fe3O4@PNP1. Unexpectedly, Pd(ii) is well maintained after six consecutive catalytic runs in Fe3O4@PNP2, and Pd(0) and Pd(ii) coexist in Fe3O4@PNP1 under the same conditions; moreover, the morphologies of these spherical core-shell particles show no significant variation after six consecutive catalytic runs.The encapsulation of the functional species on magnetic core is a facile approach for the synthesis of core-shell magnetic materials, and surface encapsulating matrices play crucial roles in regulating their properties and applications. In this study, two core-shell palladium N-heterocyclic carbene (NHC) particles (Fe3O4@PNP1 and Fe3O4@PNP2) were prepared by a one-pot reaction of semi-rigid tripodal imidazolium salts and palladium acetate in the presence of magnetite nanoparticles. The magnetite nanoparticles are encapsulated inside the main-chain palladium, which act as cores. The conjugated effects of triphenyltriazine and triphenylbenzene in the imidazolium salts have important influence on their physical properties and catalytic performances. Fe3O4@PNP2 shows better recyclability than Fe3O4@PNP1. Unexpectedly, Pd(ii) is well maintained after six consecutive catalytic runs in

  6. Antiperiodic XXZ chains with arbitrary spins: Complete eigenstate construction by functional equations in separation of variables

    CERN Document Server

    Niccoli, G

    2014-01-01

    Generic inhomogeneous integrable XXZ chains with arbitrary spins are studied by means of the quantum separation of variables (SOV) method. Within this framework, a complete description of the spectrum (eigenvalues and eigenstates) of the antiperiodic transfer matrix is derived in terms of discrete systems of equations involving the inhomogeneity parameters of the model. We show here that one can reformulate this discrete SOV characterization of the spectrum in terms of functional T-Q equations of Baxter's type, hence proving the completeness of the solutions to the associated systems of Bethe-type equations. More precisely, we consider here two such reformulations. The first one is given in terms of Q-solutions, in the form of trigonometric polynomials of a given degree $N_s$, of a one-parameter family of T-Q functional equations with an extra inhomogeneous term. The second one is given in terms of Q-solutions, again in the form of trigonometric polynomials of degree $N_s$ but with double period, of Baxter's ...

  7. Extent and Effect of Horizontal Supply Chain Collaboration among Construction SME

    Directory of Open Access Journals (Sweden)

    Liv Torjussen

    2012-01-01

    Full Text Available The majority of companies involved in value delivery in the Swedish housing industry are Small- and Medium-sized Enterprises (SME. An SME is often managed in an informal way with focus on sales and production. Many SME are also financially vulnerable as they are strongly dependent on a few key customers and key products. As variation will always exist, SME should learn to deal with variation instead of try eliminating it. This paper hypothesises that structural flexibility in SME supply chains through horizontal collaboration leads to a regional environment of economical growth from which all active SME will benefit. The hypothesis is examined through two case studies; a Swedish supplier network that has worked together six years and a four year old Norwegian supplier network. A benefit of collaboration is knowledge sharing that lessens the economical strain of keeping up with the “latest”. Other examples of collaboration are shared production resources in case of low capacity. Collaboration within supplier tier networks is considered to mark the emergence of a “collective strength” that improves individual suppliers bargaining position towards their customers. This evolution is considered an indication of the emergence of a “Lean Enterprise” within the house building sector.

  8. T-cell ligands modulate the cytolytic activity of the CD33/CD3 BiTE antibody construct, AMG 330

    International Nuclear Information System (INIS)

    Preclinical and emerging clinical studies demonstrate that bispecific T-cell engaging (BiTE) antibody constructs can potently lyse targeted tumor cells, but the determinants for their activity remain incompletely understood. Using human acute myeloid leukemia (AML) cell lines engineered to overexpress individual T-cell ligands, we found that expression of the inhibitory ligands, PD-L1 and PD-L2, reduced the cytolytic activity of the BiTE antibody construct targeting CD33, AMG 330; conversely, expression of the activating ligands, CD80 and CD86, augmented the cytotoxic activity of AMG 330. Consistent with these findings, treatment with an activating antibody directed at the co-stimulatory T-cell receptor, CD28, significantly increased AMG 330-induced cytotoxicity in human AML cell lines. Using specimens from 12 patients with newly diagnosed or relapsed/refractory AML, we found that activation of CD28 also increased the activity of AMG 330 in primary human AML cells (P=0.023). Together, our findings indicate that T-cell ligands and co-receptors modulate the anti-tumor activity of the CD33/CD3 BiTE antibody construct, AMG 330. These findings suggest that such ligands/co-receptors could serve as biomarkers of response and that co-treatment strategies with pharmacological modulators of T-cell receptor signaling could be utilized to further enhance the activity of this targeted therapeutic

  9. MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma

    OpenAIRE

    Jinushi, Masahisa; Vanneman, Matthew; Munshi, Nikhil C.; Tai, Yu-Tzu; Prabhala, Rao H.; Ritz, Jerome; Neuberg, Donna; Anderson, Kenneth C; Carrasco, Daniel Ruben; Dranoff, Glenn

    2008-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) is a common disorder of aging and a precursor lesion to full-blown multiple myeloma (MM). The mechanisms underlying the progression from MGUS to MM are incompletely understood but include the suppression of innate and adaptive antitumor immunity. Here, we demonstrate that NKG2D, an activating receptor on natural killer (NK) cells, CD8+ T lymphocytes, and MHC class I chain-related protein A (MICA), an NKG2D ligand induced in malignant p...

  10. The radiosensitizing properties of an anti-epidermal growth factor receptor single-chain antibody isolated from a phage display library

    International Nuclear Information System (INIS)

    Full text: Anti-epidermal growth factor receptor (EGFr) agents have shown promise in the treatment of various malignancies when used as single agents or combined with conventional treatments. These agents include monoclonal antibodies that block the ligand binding site of EGFr. The studies reported herein were performed to isolate single-chain antibodies (scFvs) that target EGFr and to characterize the anti-cancer efficacy of these smaller antibody molecules. It is our hypothesis that therapeutically effective anti-EGFr scFvs could eventually be delivered in a gene-therapy approach that allows affected tumor cells to secrete the anti-EGFr scFvs thereby impacting multiple neighboring cells. Human scFv phage display libraries were screened for EGFr-binding scFvs. One positive EGFr-specific scFv (clone 45) was tested for its ability to sensitize tumor cells to radiation treatment. The EGFr-over expressing cell line, A431 cells (human squamous cell carcinoma), was used in standard cell proliferation and apoptosis (annexin V) assays. A431 cells were treated with EGFr-specific scFv clone 45 (50 μg/ml), 3 Gy or the combination of the two treatments. Cell proliferation was assessed daily and all treatments inhibited proliferation, however; greater inhibition of cell proliferation was noted for the combination treatment than either individual treatment. Inhibition at 4 days compared to controls: 26% (scFv), 32% (3 Gy), and 54% (combined). Cells treated in a similar fashion were studied for apoptosis 4 days after the initiation of treatment. Although the scFv did not induce apoptosis, it did cause a significant increase in radiation-induced apoptosis. An scFv was isolated from a human scFv phage display library and shown to sensitize human A431 cells to radiation treatment. Further studies to determine the mechanism of radiosensitization are being undertaken

  11. Production and characterization of a recombinant single-chain antibody (scFv) for tracing the σ54 factor of Pseudomonas putida.

    Science.gov (United States)

    Jurado, Paola; Fernández, Luis Angel; de Lorenzo, Víctor

    2012-07-31

    The number of alternative sigma factor molecules per bacterial cell determines either stochasticity or evenness of transcription of cognate promoters. An approach for examining the abundance of sigmas in any sample of bacterial origin is explained here which relies on the production of a recombinant highly specific, high-affinity single-chain variable Fv domain (scFv) targeted towards unique protein sites of the factor. Purposely, a super-binder scFv recognizing a distinct epitope of the less abundant sigma σ(54) of Pseudomonas putida (also known as σ(N)) was obtained and its properties examined in detail. To this end, an scFv library was generated from mRNA extracted from lymphocytes of mice immunized with the purified σ(54) protein of this bacterium. The library was displayed on a phage system and subjected to various rounds of panning with purified σ(54) for capturing extreme binders. The resulting high-affinity anti-σ(54) phage antibody (Phab) clone named C2 strongly attached a small region located between positions 172 and 183 of the primary amino acid sequence of σ(54) that overlaps its core RNA polymerase-binding region. The purified scFv-C2 detected minute amounts of σ(54) in whole cell protein extracts not only of P. putida but also Escherichia coli cells and putatively in other bacteria as well. The affinity constant of the purified antibody was measured by surface plasmon resonance (SPR) and found to have a K(D) (k(off)/k(on)) in the range of 2×10(-9)M. The considerable affinity and specificity of this recombinant antibody makes it a tool of choice for quantitative studies on gene expression of σ(54)-dependent promoters in P. putida and other Gram-negative bacteria. PMID:22206981

  12. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo (U. NAM)

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  13. Monoclonal antibodies to HLA-E bind epitopes carried by unfolded β2 m-free heavy chains.

    Science.gov (United States)

    Tremante, Elisa; Lo Monaco, Elisa; Ingegnere, Tiziano; Sampaoli, Camilla; Fraioli, Rocco; Giacomini, Patrizio

    2015-08-01

    Since HLA-E heavy chains accumulate free of their light β2 -microglobulin (β2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2 m-associated and β2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies. PMID:25982269

  14. BIM技术在施工项目供应链管理中的应用%Application of BIM in Supply Chain Management of Construction Projects

    Institute of Scientific and Technical Information of China (English)

    俞启元; 吕玉惠; 张尚

    2016-01-01

    将供应链管理、系统集成和信息技术交叉起来,研究BIM技术在施工项目供应链管理中的应用。首先,阐述施工项目供应链管理的信息传递方式及BIM技术的特点;然后,基于对信息传递的认识,提出将BIM技术应用于施工项目供应链管理的基本路径;最后,设计基于BIM技术的施工项目供应链管理信息系统基本架构。%This paper integrates the supply chain management,system integration and information technology, studies the BIM application in supply chain management of construction project. Firstly,expounds the information transmission mode in the supply chain management of construction project and the characteristics of BIM. Secondly,based on cognition of information transfer,puts forward the basic path of applying BIM in supply chain management of construction project. Finally,designs the basic structure of supply chain management information system of construction project based on BIM Technology.

  15. Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4 fusion protein

    Directory of Open Access Journals (Sweden)

    Ye Qing

    2012-11-01

    Full Text Available Abstract Background The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S4. In the present study, the anti-TfR single-chain antibody (TfRscFv was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells. Results Fusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4

  16. Single Chain Antibodies as Tools to Study transforming growth factor-β-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

    Science.gov (United States)

    Blokzijl, Andries; Zieba, Agata; Hust, Michael; Schirrmann, Thomas; Helmsing, Saskia; Grannas, Karin; Hertz, Ellen; Moren, Anita; Chen, Lei; Söderberg, Ola; Moustakas, Aristidis; Dübel, Stefan; Landegren, Ulf

    2016-06-01

    The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors

  17. Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

    International Nuclear Information System (INIS)

    Human papillomaviruses (HPV) are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs) are valuable tools in cancer immunotherapy and can be used as 'intracellular antibodies' to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 'phage-display' library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and solubility in comparison with the

  18. Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

    Directory of Open Access Journals (Sweden)

    Accardi Luisa

    2007-01-01

    Full Text Available Abstract Background Human papillomaviruses (HPV are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. Methods The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. Results ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and

  19. 应急物流供应链构建研究%Research on the Construction of Emergency Logistics Supply Chain

    Institute of Scientific and Technical Information of China (English)

    刘玲丽

    2014-01-01

    文中在阐释应急物流及其供应链概念的基础上,分析了应急物流供应链的特点及流程,构建了应急物流供应链结构模型,探讨了应急物流供应链的构建原则、参与主体构成、协同机制的建立及信息平台的构建等相关问题。%Base on the definition of emergency logistics supply chain ,this paper analyses it's characteristic and flow.The paper also structures a model of emergency logistics supply chain and discusses the relevant questions about the emergency logistics supply chain construction principles ,it's main participants ,coordination mechanism ,information platform construction and so on.

  20. Proceedings of the Canadian Institute conference on supply chain management in the oil and gas industry : major capital construction projects, maintenance, repair and operations

    International Nuclear Information System (INIS)

    Many companies are now being forced to focus on careful budgeting to ensure that the capital costs of large-scale construction projects do not exceed their budgets. Operators are now investigating the role of supply chain management in reducing project costs. This conference provided a forum for the discussion of issues related to large construction projects for supply chain management in the oil and gas industry. Participants at the conference discussed methods of negotiating with contractors in order to manage higher prices for steel and other commodities. Best practices for maintaining effective purchaser-contractor relations were discussed along with cost benchmarks in contracts and management planning techniques for supply chain processes. The benefits of adopting vendor-managed inventory systems were also examined. Sourcing strategies were presented and issues related to transportation were reviewed along with various planning models. The conference featured 16 presentations. tabs., figs

  1. Modification and identification of a vector for making a large phage antibody library

    Institute of Scientific and Technical Information of China (English)

    ZHANG Guo-min; CHEN Yü-ping; GUAN Yuan-zhi; WANG Yan; AN Yun-qing

    2007-01-01

    Background The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies.Methods scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC Ⅲ encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated.Results The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression.Conclusions The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

  2. 抗阿尔茨海默病Aβ人-鼠嵌合抗体基因的真核载体构建和表达%Vector construction and expression of anti-Aβ human-mouse chimeric antibody against Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    常德; 张建华; 赵雪梅; 梁平

    2010-01-01

    Objectives To construct and to express a human-mouse chimeric antibody against Aβpeptide involved in Alzheimer disease by genetic antibody engineering with reducing of its human anti-mouse antibody response. Methods Total RNA was extracted from a murine hybridoma cell line that secreted antiAβ monoclonal antibody. The entire gene coding heavy and light chains were amplified using RT-PCR and analyzed by Genebank Blast. The chimeric antibody gene was acquired by variable region gene of the monoclonal antibody with constant region gene of human IgG, in which point mutations were induced by recombinant PCR technology, respectively. The eukaryotic expression vectors established by cloning chimeric antibody genes of the heavy and light chains into 3.1 were co-transfected into COS-7 cells. The expressed products were analyzed using ELISA and immunohistochemistry subsequently. Results Genebank Blast analysis showed that the entire cloned antibody genes were in accordance with the murine antibody genes. DNA sequencing confirmed that the expression vectors of chimeric antibody were constructed successfully after splicing the variable region and constant region sequences. By co-transfecting COS-7 cells,a chimeric antibody was produced and collected in the culture medium. The antibody was humanized and bound Aβ specifically by ELISA and immunohistochemistry evaluations. Conclusions Expression vector of chimeric antibody against Aβ was constructed successfully and expressed in the eukaryotic cells. It provides a solid base for developing diagnostic and therapeutic methods for Alzheimer's disease in clinic and paves a way for a further humanization in the future.beta-protein%目的 通过基因工程抗体技术构建和表达抗β-淀粉样多肽(Aβ)人-鼠嵌合抗体,减低鼠源单克隆抗体在临床应用中引起的人体免疫排斥反应.方法从分泌抗Aβ1-42鼠单克隆抗体杂交瘤细胞株中提取总RNA,用逆转录-聚合酶链反应(RT-PCR)扩增鼠

  3. Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv).

    Science.gov (United States)

    Ferreira, A R; Ataíde, F; von Stosch, M; Dias, J M L; Clemente, J J; Cunha, A E; Oliveira, R

    2012-11-01

    In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75 h, typically between 140 and 160 g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5 % of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0 % boundary for longer periods of time when compared to a traditional proportional-integral-derivative algorithm, which tends to destabilize with increasing cell density. PMID:22610694

  4. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    Science.gov (United States)

    Pascho, R.J.; Chase, D.; McKibben, C.L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 3 109 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 3 104 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  5. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid ovarian fluid.

    Science.gov (United States)

    Pascho, R J; Chase, D; McKibben, C L

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 x 10(9) cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 x 10(4) cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods. PMID:9526862

  6. Isolation of ScFv antibodies of rP27Kip1 from phage display libraries constructed from immunized and non-immunized repertoires

    Institute of Scientific and Technical Information of China (English)

    曹跃琼; 乔守怡; 袁有忠; 黄建生; 赵寿元

    1999-01-01

    Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27Kiplimmunized and non-immunized mice. After only one round of selection with rP27Kipl, clones from each library were chosen randomly and digested by Taq I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL21(DE3)/pET-28b(+) system. ELISA showed that some of the fragments could bind to rP27Kipl specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.

  7. Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.

    Science.gov (United States)

    Singh, Chandra K; Ojha, Abhishek; Kachru, Devendra N

    2007-01-01

    To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach

  8. Reflection on Construction Project's Implementation of Supply Chain Management Model%建筑工程实施供应链管理模式的思考

    Institute of Scientific and Technical Information of China (English)

    刘柄延

    2015-01-01

    Transformed from the old management model which used to be implemented in China's previous construction engineering, the found of supply chain management model is beneficial for the establishment of new enterprise manage⁃ment model in China’s construction enterprises. The article has studied the main idea of comprehensive supply chain management concept, on this basis it puts forward some advices for the practical work in the concrete implementation of supply chain management model in projects, and conducts certain analysis of how to implement supply chain management in detail in engineering projects, hoping to provide some reference for building industry in the establishment of supply chain integration framework and the construction of supply chain management model.%供应链管理模式是对中国过去的建筑工程实施的管理模式的变革,有利于促进我国建筑企业新型企业管理模式的构建。本文研究了综合供应链管理概念的中心思想,对项目中具体实施供应链管理模式的工作提出了一些建议,对工程项目详细实施供应链管理做了一定的分析,以期为建筑业建立供应链整合架构和供应链管理模式提供一定的参考。

  9. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    Science.gov (United States)

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  10. Construction of phage display VHH antibody library against avian H5N1 virus from alpaca%抗H5N1禽流感病毒VHH抗体库的构建

    Institute of Scientific and Technical Information of China (English)

    严安; 熊慧; 王颖; 孙冰玉; 夏立亮; 吴标; 包文静; 车小燕; 孙志伟; 金维荣; 赵国屏

    2011-01-01

    Objective: To construct phage display variable domain of heavy chain antibody library (VHH antibody library) from alpaca immunized with inactivated H5N1 vaccine for the future screening of VHH antibodies against avian H5N1 influenza virus. Methods: The camelid species (alpaca, Lama pacos ) was selected for immunization with inactivated H5N1 vaccine. Hemagglutinin inhibition (HI) assay of serum from immunized alpaca was performed against H5N1 avian influenza virus one week after the fourth inntmization. Peripheral lymphocytes were isolated for the amplification of VHH fragments by RT-PCR. PCR products were then purified and inserted into phagemid vector pCANTAB5E. The VHH antibody gene library was obtained by electroporating recombinant pCANTAB5E-VHH vectors into E. coli TG1 cells.The library capacity and diversity of VHH antibody gene library was determined by sequencing analysis. The HI assay was performed with the culture supernatant of primary phage display VHH antibody library. Results:After four rounds of immunization with inactivated H5N1 vaccine,HI antibody titer of the alpaca serum reached to 1: 2 560, which was higher than those fiom immunized mice. A first set of antibody gene library totalling 3 × l08 members were created after cloning VHH genes into a phagemid vector pCANTAB5E. The sequence of 14 members of the unselected library indicated that the camelid VHH antibody library we constructed possessed high diversity and good capacity. The supematant from the primary phage display library displayed effieient HI effect against avian H5N1 influenza virus. And the titration of our phage display VHH library reached 2.17 × l011. Conclusion: Taken together, phage display VHH antibody library from immunized alpaca is successfully constructed,which provides a platform for VHH antibody preparation against H5N1 virus. This will give light to future study on treatment and diagnosis of avian H5N1 influenza virus.%目的:构建抗H5N1禽流感病毒的小羊驼免

  11. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    OpenAIRE

    Goldman Ellen R; Anderson George P; Liu Jinny L

    2007-01-01

    Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct a...

  12. 绿色供应链进程下建筑材料管理探究%Study on Construction Materials Management in Green Supply Chain Process

    Institute of Scientific and Technical Information of China (English)

    段文凤; 董金辉

    2015-01-01

    Through tracing the origin of green supply chain, this paper analyzed the current situation of material management in construction, discussed the problems of procuring and using building materials in the green construction, recycling the construction waste in green scrap under the green supply chain process in construction industry, proposed the mode to play the initiative of the green supply chain members from the perspective of recycled building materials manufacturers, material suppliers and engineering contractors.%本文通过追溯绿色供应链的起源,并对建筑材料管理现状进行分析,论述了建设行业绿色供应链进程下建筑材料在绿色施工中的采购使用问题和绿色报废中的建筑材料回收问题,并分别从再生建材制造商、材料供应商、工程承包商的角度提出了发挥绿色供应链各成员能动性的途径。

  13. Synthesis and pre-clinical evaluation of an (18)F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer.

    Science.gov (United States)

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an (18)F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of (18)F-labeled scFv-B43.13 ([(18)F]FBz-scFv-B43.13) was studied with PET. [(18)F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  14. Synthesis and pre-clinical evaluation of an 18F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer

    Science.gov (United States)

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an 18F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of 18F-labeled scFv-B43.13 ([18F]FBz-scFv-B43.13) was studied with PET. [18F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  15. Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD.

    Science.gov (United States)

    Furukawa, Hiroshi; Oka, Shomi; Shimada, Kota; Masuo, Kiyoe; Nakajima, Fumiaki; Funano, Shunichi; Tanaka, Yuki; Komiya, Akiko; Fukui, Naoshi; Sawasaki, Tatsuya; Tadokoro, Kenji; Nose, Masato; Tsuchiya, Naoyuki; Tohma, Shigeto

    2015-01-01

    Interstitial lung disease (ILD) is frequently associated with collagen disease. It is then designated as collagen vascular disease-associated ILD (CVD-ILD), and influences patients' prognosis. The prognosis of acute-onset diffuse ILD (AoDILD) occurring in patients with collagen disease is quite poor. Here, we report our investigation of auto-antibody (Ab) profiles to determine whether they may be useful in diagnosing CVD-ILD or AoDILD in collagen disease. Auto-Ab profiles were analyzed using the Lambda Array Beads Multi-Analyte System, granulocyte immunofluorescence test, Proto-Array Human Protein Microarray, AlphaScreen assay, and glutathione S-transferase capture enzyme-linked immunosorbent assay in 34 patients with rheumatoid arthritis (RA) with or without CVD-ILD and in 15 patients with collagen disease with AoDILD. The average anti-major histocompatibility complex class I-related chain A (MICA) Ab levels were higher in RA patients with CVD-ILD than in those without (P = 0.0013). The ratio of the average anti-MICA Ab level to the average anti-human leukocyte antigen class I Ab level (ie, MICA/Class I) was significantly higher in RA patients with CVD-ILD compared with those without (P = 4.47 × 10(-5)). To the best of our knowledge, this is the first report of auto-Ab profiles in CVD-ILD. The MICA/Class I ratio could be a better marker for diagnosing CVD-ILD than KL-6 (Krebs von den lungen-6). PMID:26327779

  16. The single-chain anti-TNF-α antibody DLX105 induces clinical and biomarker responses upon local administration in patients with chronic plaque-type psoriasis.

    Science.gov (United States)

    Tsianakas, Athanasios; Brunner, Patrick M; Ghoreschi, Kamran; Berger, Claudia; Loser, Karin; Röcken, Martin; Stingl, Georg; Luger, Thomas; Jung, Thomas

    2016-06-01

    It is not clear whether TNF-α antagonists used in the treatment of psoriasis need to act systemically, or whether local inhibition of skin-produced TNF-α would be sufficient to silence skin inflammation. To answer this question, we conducted two multicentre, double-blinded, randomized, placebo-controlled clinical trials with the novel single-chain anti-TNF-α-PENTRA(®) -antibody DLX105. Upon intra-dermal injection, DLX105 induced a mean local PASI decrease of 33% over baseline after 2 weeks of treatment, while the placebo response was only 12% (P = 0.001). The clinical response was accompanied by changes in biomarkers such as reductions in K16, Ki67 and epidermal thickness as well as decreased mRNA levels of IL-17, TNF-α, IL-23p19, IL-12p40 and IFN-γ. Next, we applied the drug topically twice daily in a 0.5% hydrogel formulation. While the local PASI did not change, topical DLX105 mediated significant reductions of mRNA levels of key proinflammatory cytokines when compared to placebo, and this effect was further enhanced after weekly tape stripping of plaques to increase drug penetration. These results suggest that longer treatment periods and/or increased local drug concentrations might result in better therapeutic efficacy of topically applied DLX105. In sum, we can show for the first time that local inhibition of TNF-α is sufficient to mediate a biological response in psoriasis that translates into clinical efficacy. PMID:26738450

  17. In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Rob J A Nabuurs

    Full Text Available This study investigated the in vivo properties of two heavy chain antibody fragments (V(HH, ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(HH in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(HH by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(HH showed rapid renal clearance (10-20 min. Twenty-four hours post-injection (99mTc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99mTc-ni3A or DTPA((111In-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(HH, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(HH showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(HH detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

  18. In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

    Science.gov (United States)

    Nabuurs, Rob J A; Rutgers, Kim S; Welling, Mick M; Metaxas, Athanasios; de Backer, Maaike E; Rotman, Maarten; Bacskai, Brian J; van Buchem, Mark A; van der Maarel, Silvère M; van der Weerd, Louise

    2012-01-01

    This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits. PMID:22675537

  19. Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8.

    Directory of Open Access Journals (Sweden)

    Irene Michalk

    Full Text Available There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs. Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.

  20. Analysis on construction of the Supply Chain and Brand of Guangxi Characteristic Economic Agricultural Products——A Case Study of Guilin,China

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Firstly,it is briefly introduced that Guangxi characteristic economic agricultural products are currently characterized by typical regional features and strong competitiveness.Then,Guilin is taken as an example,the problems existing in constructing the supply chain and brand of Guilin characteristic economic agricultural products are pointed out after the analysis of its current situations,the problems include weak agricultural production infrastructure,weak deep products processing capacity,low modernization level of storage and transportation as well as short of fixed marketing channels;meanwhile,brand construction faces the problems of little top brands and few differentiated products.Finally,some measures are proposed to solve these problems from the perspectives of establishing the management idea of agricultural products supply chain and enhancing the awareness to protect agricultural products brand,it is also suggested that these measures be extended to the whole Guangxi autonomous region in order to explore the way of the development of Guangxi characteristic agriculture.

  1. McKinsey 7S Model for Supply Chain Management of Local SMEs Construction Business in Upper Northeast Region of Thailand

    OpenAIRE

    Thanaphan Naipinit; Somkier Kojchavivong; Vorawit Kowittayakorn; Thongphon Promsaka Na Sakolnakorn

    2014-01-01

    The purpose of this study is to study the successful business strategies and the guidelines for the management strategies of supply chain management of local small and medium enterprises (SME) construction shops. We use the McKinsey 7S model for the conception of this study by providing 400 questionnaires to participants and we also used focus groups for the management guideline. From the study we found that of the seven strategies (7S) in the model (structure, strategy, systems, styles, skil...

  2. Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.

    OpenAIRE

    Daugherty, B L; DeMartino, J A; Law, M F; Kawka, D W; Singer, I I; Mark, G E

    1991-01-01

    Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs po...

  3. Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Vigre, Håkan; Sørensen, Vibeke; Møller, Kristian

    2005-01-01

    pigs were 2-4 weeks old maternally derived IgG against L. intracellularis was demonstrated by immunofluorescence antibody test in nine pigs whereas the bacterium was detected by PCR in faeces from six pigs. The maternally derived antibodies did not prevent pigs from becoming infected as seven pigs...... immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracelluaris exposed pigs with or without current proliferative enteropathy. (c) 2004...

  4. 抗黄曲霉毒素单链抗体基因的克隆与表达%Cloning and expression of single chain Fv antibody gene against aflatoxin

    Institute of Scientific and Technical Information of China (English)

    李鑫; 李培武; 张奇; 张文; 李园园

    2012-01-01

    The objective of this study was to reduce the preparation cost of aflatoxin antibody. Based on the hybridoma cell 8F6 w0hich secrete mAbs against aflatoxin, the variable heavy (VH) and light ( VL) region genes of 8F6 were amplified using polymerase chain reaction. The VH - linker - VL genes were constructed by overlap extension, then cloned into a phagemid vector pCANTAB 5E and expressed in E. coli TG1 cells as a fusion with a phage protein. Indirect competitive ELISA was performed for detection of the binding activity, which resulted in IC50 value of the constructed ScFv as 0. 57 ng per milliliter.%为降低黄曲霉毒素抗体制备成本,在已有的能分泌抗黄曲霉毒素单克隆抗体的杂交瘤细胞系8F6的基础上,成功克隆得到了该单克隆抗体的重链(VH)和轻链可变区(VL)基因片段.通过重叠延伸PCR的方法将轻、重链可变区基因连接,并引入连接肽(Linker)编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因,并将该基因克隆到噬菌体表达载体pCANTAB 5E上,使单链抗体以噬菌体展示形式在大肠杆菌TG1中表达.间接竞争ELISA方法检测到该ScFv对黄曲霉毒素B1的抑制率(IC50)值为0.57ng/mL,表明该单链抗体与亲本鼠单抗有相同的抗原结合特异性,且具有很高灵敏度.

  5. Click ‘Like’ and Post It on Your Wall! Chain Posts on Facebook – Identity Construction and Values

    OpenAIRE

    Piret Voolaid

    2013-01-01

    The article focuses on chain posts that were collected in the years 2010–2012 and spread predominantly among girls of ten to twelve on Facebook (facebook.com) – a social network that has a membership of over 450,000 in Estonia. The source material comprising approximately 220 texts is similar by form and content to chain letters known from earlier tradition; yet, the web environment with its specific technical structure allows the texts to turn into a peculiar Facebook-like phenomenon.The aut...

  6. 驼源天然单域重链抗体库的构建与鉴定%Construction and Biopanning of Camelid Naive Single-domain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    涂追; 许杨; 刘夏; 何庆华; 陶勇

    2011-01-01

    从未经主动免疫的健康羊驼(Lamapacos)外周血淋巴细胞中提取总RNA,反转录后作为第一轮PCR的模板.根据重链抗体保守区域设计引物,经巢式PCR法扩增获得了全套重链抗体可变区基因,将其克隆至噬菌粒 pHENl,电转化大肠杆菌TG1得到初级抗体库NAL,含有2×10个独立克隆,菌落PCR和Hinf Ⅰ酶切分析结果显示,克隆效率大于97%,文库的多样性良好.辅助噬菌体救援后,得到噬菌体展示文库命名为NA-PDL,滴度达10CFU/ml.以真菌毒素人工抗原DON-MBSA为目标抗原,对NA-PDL进行了淘选,第二轮洗脱物中,阳性克隆率达36.4%,提示针对目标抗原的噬菌体颗粒得到了有效富集,文库NA-PDL多样性较好,为后续淘选针对特定抗原的单域重链抗体奠定了基础.%The objective is to construct a camelid na(i)ve single-domain heavy chain antibody phage display library. Total RNA was purified from 30ml blood of two healthy non-immune alpacas ( Lama pacos) and directly used for complementary DNA (cDNA) synthesis. Three sets of primers were designed based on the conserved region of heavy-chain antibody. The repertoire of VHH coding sequence was amplified by nested PCR, and the PCR products were cloned into a phagemid vector pHEN1. By electroporation of E. coli TG1 , the primary library (designate NAL) was obtained containing more than 107 independence clones. After helper phage rescue, the phage display library ( designate SNA-PDL) was generated with a titre up to 1013 CFU/ml. The library exhibited high diversity as judged by the Hinf Ⅰ restriction pattern. Solid phage biopanning against artificial antigen DONMBSA showed significant enrichment of binding phage particles. The positive rate of panning round two was 36.4% . The data indicated that a na(i)ve single-domain antibody phage display library was constructed. which has good diversity and would be useful for generating VHHs with specific binding affinity.

  7. Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2

    International Nuclear Information System (INIS)

    An antibody–antigen complex consisting of a single-chain variable fragment of the potential therapeutic antibody chA21 and an N-terminal fragment (residues 1–192) of the human ErbB2 extracellular domain was expressed, purified and crystallized. X-ray diffraction data were collected to 2.45 Å resolution. ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody–antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1–192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 Å resolution from a single flash-cooled crystal; the crystal belonged to space group P212121

  8. 关于医药零售连锁企业信息化建设的探讨%Probe into the Informatization Construction of Pharmaceutical Retail Chains

    Institute of Scientific and Technical Information of China (English)

    剧晓红

    2015-01-01

    This paper expounds the current status of the informatization construction of pharmaceutical retail chains, analyzes the benefits brought about by the informatization construction to enterprises, puts forward some improvement countermeasures, and based on this, briefly probes into the development trends of the informatization construction of pharmaceutical retail chains in the aspects of cloud computing, pharmaceutical e-commerce, and new media, etc. in the Internet era.%阐述了医药零售连锁企业信息化建设的现状,分析了信息化建设给企业带来的效益,提出了改进对策,并在此基础上简单探讨了互联网时代医药零售连锁企业信息化建设在云计算、医药电子商务、新媒体等方面的发展趋势。

  9. Supply chain for new construction: buy where we build; Cadena de suministro para las nuevas construcciones: Buy where we build

    Energy Technology Data Exchange (ETDEWEB)

    Cruz, J. L.; Bueno, S.

    2012-07-01

    A global supply strategy has been placed by Westinghouse in order to face the current and future constructions. And in this way, Westinghouse will be able to take advantage of benefit of Local Supply. this article shows the Westinghouse Global Supply Strategy, Local Supply considerations, the current state of supplier calcifications, and finally Memorandums of Understanding and Alliances agreed with local suppliers around the world to provide a suitable solution for the new construction needs. (Author)

  10. Study on Brand Construction from the Perspective of Supply Chain Management%基于供应链视角的品牌建设研究

    Institute of Scientific and Technical Information of China (English)

    宋爱华

    2016-01-01

    Brand is an important embodiment of the enterprise and even the country's competitive power. Now It has entered the era of brand competition and brand consumption,the brand is more and more become the focus of popular attention. Who can walk in the forefront of the brand construction,create a famous brand and a strong brand,who will be able to win the competition. But the traditional brand-building ideas ignored the supply chain management issues,enterprises should think about the strategy of brand construction from the perspective of supply chain management,strengthen the effective control of supply chain operation,act brand construction as a strategic means to improve the quality of the supply chain operation,enhance the competitive ability of the supply chain,thus can cultivate a famous brand and a strong brand,make sure the enterprise last for a long time.%品牌是一个企业乃至国家竞争实力的重要体现。现在已经进入到品牌竞争和品牌消费的时代,品牌越来越成为人们关注的焦点。谁能够走在品牌建设的前列,打造出知名品牌、强势品牌,谁就能够赢得竞争,但传统的品牌建设思路忽视了供应链管理问题,应从供应链管理的角度思考品牌建设的策略,加强供应链运行有效管控,以品牌建设为战略手段提升供应链运行的质量,增强供应链的竞争能力,才能够培育打造出知名品牌、强势品牌,保证企业持续长久。

  11. Need of a consistent and convenient nucleus identification in ENDF files for the automatic construction of the depletion chains

    Directory of Open Access Journals (Sweden)

    Mosca Pietro

    2016-01-01

    As a main result of this work, our suggestion is to change the ENDF format using systematically the isomeric state number to identify the nuclei. This proposal is already compliant to a huge amount ENDF data that are not in agreement with the present ENDF format. This choice is the most convenient because, ultimately, it allows one to give human readable names to the nuclei of the depletion chains.

  12. How to measure Innovative Supply Chain Strategies (ISCS) in the context of SCM? -Construct development and measurement validation

    OpenAIRE

    Lavastre, Olivier; AGERON, Blandine; Chaze-Magnan, L.

    2014-01-01

    Nowadays, organizations have to innovate either in product or in management (Damanpour and Aravind, 2012). Ifinnovations in product are largely studied (Garcia and Calantone, 2002), managerial innovations are seldom studied(Soosay et al., 2008; Arlbjørn et al., 2011). Simultaneously, organizations develop Supply Chain Management(SCM) to manage their inter-organizational relationships with their partners (Chen and Paulraj, 2004). SCM“constitutes an interesting potential area for creating compe...

  13. McKinsey 7S Model for Supply Chain Management of Local SMEs Construction Business in Upper Northeast Region of Thailand

    Directory of Open Access Journals (Sweden)

    Thanaphan Naipinit

    2014-03-01

    Full Text Available The purpose of this study is to study the successful business strategies and the guidelines for the management strategies of supply chain management of local small and medium enterprises (SME construction shops. We use the McKinsey 7S model for the conception of this study by providing 400 questionnaires to participants and we also used focus groups for the management guideline. From the study we found that of the seven strategies (7S in the model (structure, strategy, systems, styles, skill, staff, and shared values, most entrepreneurs scored highly in strategy; however, most entrepreneurs scored low in several areas: working with software applications, lack of outside training, and most entrepreneurs maintain command as the owner and do not give authority to others. In addition, the Thai government should create a policy by collaborating with Lao PDR to reduce some barriers of international trade to help SME construction shops in Thailand.

  14. Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

    Science.gov (United States)

    Kumada, Yoichi; Ishikawa, Yasuyuki; Fujiwara, Yusuke; Takeda, Rui; Miyamoto, Ryosuke; Niwa, Daisuke; Momose, Shun; Kang, Bongmun; Kishimoto, Michimasa

    2014-09-01

    In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results

  15. Thyroid Antibodies

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  16. Production and Evaluation of Immunoreactivity of Poly Lysine-Tagged Single Chain Fragment Variable (ScFv) Lym-1 Antibody for Direct Conjugation to Fluorescence Dye

    International Nuclear Information System (INIS)

    Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity

  17. Production and Evaluation of Immunoreactivity of Poly Lysine-Tagged Single Chain Fragment Variable (ScFv) Lym-1 Antibody for Direct Conjugation to Fluorescence Dye

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Jae Ho; Choi, Tae Hyun; Woo, Kwang Sun; Chung, Wee Sup; Kang, Joo Hyun; Jeong, Su Young; Choi, Chang Woon; Lim, Sang Moo; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-10-15

    Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity

  18. Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

    Directory of Open Access Journals (Sweden)

    Yuya Isoda

    Full Text Available Antibody-dependent cellular cytotoxicity (ADCC is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296

  19. Uses of monoclonial antibody 8H9

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  20. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

    Directory of Open Access Journals (Sweden)

    Xiaocong Yu

    2016-01-01

    Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

  1. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition.

    Science.gov (United States)

    Yu, Xiaocong; Duval, Mark; Gawron, Melissa; Posner, Marshall R; Cavacini, Lisa A

    2016-01-01

    Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. PMID:27419146

  2. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential.

    Directory of Open Access Journals (Sweden)

    Philipp Kolb

    Full Text Available Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF, and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC. Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc derived capsid-like particles (CLPs to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission.

  3. Modular Synthetic Platform for the Construction of Functional Single-Chain Polymeric Nanoparticles: From Aqueous Catalysis to Photosensitization.

    Science.gov (United States)

    Liu, Yiliu; Pauloehrl, Thomas; Presolski, Stanislav I; Albertazzi, Lorenzo; Palmans, Anja R A; Meijer, E W

    2015-10-14

    Single-chain polymeric nanoparticles (SCPNs) are intriguing systems for multiple applications. In order to arrive at a controlled, but random, positioning of the different side groups to the polymer backbone, alternative synthetic routes have to be developed. Here, a general postpolymerization modification strategy of poly(pentafluorophenyl acrylate) (pPFPA) is presented as a versatile method to rapidly access functional SCPNs. We first show that the sequential addition of a benzene-1,3,5-tricarboxamide-based amine, acting as the supramolecular recognition motif, and water-soluble polyetheramine (Jeffamine) to pPFPA affords random copolymers that fold in water into SCPNs. The scope of the modular platform is illustrated by preparing two types of functional SCPNs. First, we prepared SCPNs designed for bio-orthogonal catalysis by attaching pendant mono(benzimidazoylmethyl)-bis(pyridylmethyl) (Bimpy), phenanthroline (Phen), or 2,2'-bipyridine (BiPy), ligands capable of binding either Cu(I) or Pd(II). The Bimpy- and Phen-containing SCPNs ligated to Cu(I) significantly accelerate azide-alkyne cycloaddition reactions while Bipy-containing SCPNs ligated to Pd(II) efficiently catalyze depropargylation reactions. In all cases, reactions proceeded efficiently in phosphate buffer at a physiological pH and at low substrate concentrations. Next, the potential of SCPNs for photodynamic therapy was evaluated. Introducing porphyrins in SCPNs leads to novel photosensitizers that can produce singlet oxygen ((1)O2) upon photoirradiation. Additionally, by attaching both porphyrins and prodrug models, attached via (1)O2-cleavable amino-acrylate linker, to the SCPNs, we show that irradiation of the SCPNs results in a cascade reaction of (1)O2 generation followed by cleavage of the amino-acrylate linkers, releasing the drug model. The modular synthesis strategy reported here provides rapid and controlled access to SCPNs with tunable amounts of active units that fulfill different

  4. Expression Enhancement in Trastuzumab Therapeutic Monoclonal Antibody Production using Genomic Amplification with Methotrexate

    OpenAIRE

    Akbarzadeh-Sharbaf, Soudabeh; Yakhchali, Bagher; Minuchehr, Zarrin; Shokrgozar, Mohammad Ali; Zeinali, Sirous

    2013-01-01

    Background Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. Methods According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were sele...

  5. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  6. Structure-function relationships for the interleukin 2 receptor: location of ligand and antibody binding sites on the Tac receptor chain by mutational analysis.

    OpenAIRE

    1988-01-01

    The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of three monoclonal antibodies (anti-Tac, GL439, and H31) that block high- and low-affinity Tac-IL-2 interactions, thu...

  7. 双特异性单抗对胃肠癌的体内杀伤作用%In vivo killing effect of bispecific single-chain antibody on gastric and colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    刘洪一; 李荣; 刘巨超; 李力; 梁文涛; 钟苑; 徐迎新

    2012-01-01

    Objective To investigate the killing effect of CIK cells mediated by bispecific single - chain antibody, anti - CD3 and anti - CEA, on gastric and colon cancer cells in vivo. Methods Gastric and colon cancer models in nude mice were established after BGC823 cells from human poorly differentiated gastric adenocarcinoma and SW1116 cells from human moderately differentiated colon adenocarcinoma were planted into nude mice. The mice were divided into 3 groups, which were injected with normal saline, CIK cells and CIK cells plus bispecific antibody, respectively, once every 3 days for 6 times. Then the tumors were removed and weighed, and the inhibition rate in each group was calculated. Results Both the CIK cells and CIK cells plus bispecific single - chain antibody could inhibit tumor growth. Inhibition rates of gastric cancer were 29. 90% and 44. 33% respectively. Inhibition rates of colon cancer were 14. 93% and 38. 81% respectively. Compared with CIK cell group, the size and weight of the tumor were significantly smaller in CIK cells plus bispecific singlechain antibody group. Conclusion CIK cells alone can inhibit tumor cell growth and combination of CIK cells and the bispecific single - chain antibody, anti - CD3 and anti - CEA, is more effective in inhibiting tumor growth.%目的 探讨抗CD3抗CEA双特异性单链抗体介导CIK细胞在体内杀伤胃肠癌的作用.方法 分别将人胃低分化腺癌BGC823细胞和人结肠中分化腺癌SW1116细胞种植裸鼠后,建立裸鼠胃癌及肠癌模型,分为3组分别经尾静脉注入生理盐水、CIK细胞及CIK细胞+双特异性单抗,每3天治疗1次,共6次,取出肿瘤组织称重,并计算出各组的抑瘤率.结果 在体内,CIK组和CIK+双抗组均可抑制肿瘤生长,对胃癌的抑瘤率分别为29.90%和44.33%.对结肠癌的抑瘤率分别为14.93%和38.81%.CIK+双抗组:胃癌及结肠癌的瘤体积、瘤重明显小于CIK组.结论 CIK细胞本身可以抑制肿瘤

  8. Fields From Markov Chains

    DEFF Research Database (Denmark)

    Justesen, Jørn

    2005-01-01

    A simple construction of two-dimensional (2-D) fields is presented. Rows and columns are outcomes of the same Markov chain. The entropy can be calculated explicitly.......A simple construction of two-dimensional (2-D) fields is presented. Rows and columns are outcomes of the same Markov chain. The entropy can be calculated explicitly....

  9. Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies

    DEFF Research Database (Denmark)

    Draborg, Anette H; Lydolph, Magnus C; Westergaard, Marie; Olesen Larsen, Severin; Nielsen, Christoffer T; Duus, Karen; Jacobsen, Søren; Houen, Gunnar

    2015-01-01

    OBJECTIVE: In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore......, FLCs' association with Epstein-Barr virus (EBV) antibodies was examined. METHODS: Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations...... of serum FLCs due to decreased clearance. RESULTS: Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (p<0.0001) also after adjusting for Ig levels (p<0.0001). The concentration of serum FLCs correlated with a global disease activity (SLE disease...

  10. Site-specific antibodies distinguish single amino acid substitutions in position 57 in HLA-DQ beta-chain alleles associated with insulin-dependent diabetes

    DEFF Research Database (Denmark)

    Atar, D; Dyrberg, T; Michelsen, Birgitte;

    1989-01-01

    -chain alleles, we immunized rabbits with 12 to 13 amino acid long peptides representing HLA-DQw7 and -DQw8 allelic sequences, differing only by one amino acid in position 57 being aspartic acid (Asp) and alanine (Ala), respectively. Immunoblot analysis of lymphoblastoid cells showed that several antisera...... recognized a 29-kDa protein, equivalent to the expected molecular size of the HLA-DQ beta-chain to yield two antisera specific for HLA-DQw7 (pos. 57Asp) and three antisera for HLA-DQw8 (pos. 57Ala) positive cells. Analysis of HLA-DR 3/4 positive IDDM patients (n = 24) and controls (n = 19) showed that all...

  11. A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

    Directory of Open Access Journals (Sweden)

    Jordaan Frances

    2004-04-01

    Full Text Available Abstract Background Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. Results With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA. Conclusion The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.

  12. Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism.

    Science.gov (United States)

    Krupka, C; Kufer, P; Kischel, R; Zugmaier, G; Lichtenegger, F S; Köhnke, T; Vick, B; Jeremias, I; Metzeler, K H; Altmann, T; Schneider, S; Fiegl, M; Spiekermann, K; Bauerle, P A; Hiddemann, W; Riethmüller, G; Subklewe, M

    2016-02-01

    Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, Pcheckpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity. PMID:26239198

  13. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

    OpenAIRE

    Wesolowski, Janusz; Alzogaray, Vanina; Reyelt, Jan; Unger, Mandy; Juarez, Karla; Urrutia, Mariela; Cauerhff, Ana; Danquah, Welbeck; Rissiek, Björn; Scheuplein, Felix; Schwarz, Nicole; ADRIOUCH, Sahil; Boyer, Olivier; Seman, Michel; Licea, Alexei

    2009-01-01

    Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in cam...

  14. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed. PMID:25264572

  15. An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

    OpenAIRE

    Masoumeh Hajirezaei; Mojtaba Darbouy; Manoochehr Rasouli; Bahram Kazemi

    2015-01-01

    Background: The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without ...

  16. An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

    Directory of Open Access Journals (Sweden)

    Masoumeh Hajirezaei

    2015-10-01

    Full Text Available Background: The homologous recombination (HR is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+ plasmid.Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method. 

  17. Immunoglobulin G4: an odd antibody

    NARCIS (Netherlands)

    R.C. Aalberse; S.O. Stapel; J. Schuurman; T. Rispens

    2009-01-01

    Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also

  18. Anti-sulfotyrosine antibodies

    Science.gov (United States)

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  19. Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

    Institute of Scientific and Technical Information of China (English)

    Wei-Yun ZHANG; Tak-Chun YIP; Cheuk-Sang KWOK

    2004-01-01

    Human embryonic kidney 293 cells were transfected with plasmid pcDNA-H520C9scFv-rhIL2 containing a chimeric cDNA encoding the humanized 520C9 scFv/recombinant human IL-2 fusion protein (H520C9scFv-rhIL-2). The transfected cells in plateau growing phase were cultured in serum-free medium for three days. The supernatant was collected, concentrated and purified using an affinity column packed with CNBr-activated Sepharose 4B coupled with anti-rhIL-2 mouse monoclonal antibody. The purified fusion protein was analyzed by ELISA, SDS-PAGE and Western blot. The fusion protein showed only one band in both silver stained electrophoresis gel and Western blot developed by ECL chemiluminescence system.Its molecular weight was confirmed to be about 45 kD. This fusion protein possessed binding specificity against p 185 positive SKOV3 and B 16/neu cells, and it might stimulate IL-2-dependent CTLL-2 cell proliferation as well.

  20. 全价值链成本管控体系建设实践研究%Construction Practice Study of the System of Whole Value Chain Cost Management and Control

    Institute of Scientific and Technical Information of China (English)

    崔洁元; 韦英

    2015-01-01

    全价值链成本管控体系建设是以价值链管理和战略成本管理理念为指导,以作业成本管理为基础,全面收集、整理、分析和利用价值链上各环节的成本信息,以支持价值链成本管控体系的构建和优化。本文通过对全价值链成本管控进行理论阐述,在具体实践中运用精益管理理念,创新管理思维,理论联系实际,建设性地提出了推进全价值链成本管控体系建设的思路和具体实施方法,对构建企业全价值链成本管控体系有一定的借鉴意义。%Guided by value chain management and the theory of strategic cost management, and based on activity-based costing management, the whole value chain management system collets, sorts out, analyzes and makes use of the information on every value chain link to support the construction and optimization of value chain cost management system. The paper expounds the theory of whole value chain management and control by employing lean management concept, innovating management thinking, combining theory and practice, putting forward ideas and concrete measures to promote the construction of whole value chain management system, which may be reference to constructing whole value chain management system for enterprises.

  1. Analysis of hapten binding and catalytic determinants in a family of catalytic antibodies.

    Science.gov (United States)

    Ulrich, H D; Schultz, P G

    1998-01-01

    We report here the cloning and kinetic analysis of a family of catalytic antibodies raised against a common transition state (TS) analog hapten, which accelerate a unimolecular oxy-Cope rearrangement. Sequence analysis revealed close homologies among the heavy chains of the catalytically active members of this set of antibodies, which derive mainly from a single germline gene, whereas the light chains can be traced back to several different, but related germline genes. The requirements for hapten binding and catalytic activity were determined by the construction of hybrid antibodies. Characterization of the latter antibodies again indicates a strong conservation of binding site structure among the catalytically active clones. The heavy chain was found to be the determining factor for catalytic efficiency, while the light chain exerted a smaller modulating effect that depended on light chain gene usage and somatic mutations. Within the heavy chain, the catalytic activity of a clone, but not hapten binding affinity, depended on the sequence of the third complementarity determining region (CDR). No correlation between high affinity for the hapten and high rate enhancement was found in the oxy-Cope system, a result that stands in contrast to the expectations from transition state theory. A mechanistic explanation for this observation is provided based on the three-dimensional crystal structure of the most active antibody, AZ-28, in complex with the hapten. This study demonstrates the utility of catalytic antibodies in examining the relationship between binding energy and catalysis in the evolution of biological catalysis, as well as expanding our understanding of the molecular basis of an immune response. PMID:9451442

  2. Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6

    Science.gov (United States)

    Stadler, Christiane R.; Bähr-Mahmud, Hayat; Plum, Laura M.; Schmoldt, Kathrin; Kölsch, Anne C.; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    abstract The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype. These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity. In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted. PMID:27141353

  3. Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction.

    Science.gov (United States)

    Capote, Nieves; Gorris, M Teresa; Martínez, M Carmen; Asensio, Margarita; Olmos, Antonio; Cambra, Mariano

    2006-03-01

    ABSTRACT The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3' minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected. PMID:18944448

  4. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  5. Antithyroid microsomal antibody

    Science.gov (United States)

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked to an increased risk ...

  6. Round table on the Supply Chain for NPPs construction: Localization - Daya Bay Experience; REEL Handling and Lifting Systems, More than 60 years expertise in lifting and handling equipment in production process

    International Nuclear Information System (INIS)

    The second day afternoon began with the round table on the Supply Chain for NPPs construction with Philippe Frantz, President of REEL, Marc Lachaise, Head of procurement of NNB at EDF Energy, and Steven Lau, First Deputy General Manager of DNMC. Philippe Frantz started to present the activities and the contribution of REEL in the construction of NPPs as a main supplier of handling system. Then, Marc Lachaise took the lead to present Hinkley Point C Project, the Values of NNB and the key role of the supply chain in this Project. Steven Lau went on to describe the link of the supply chain with the operating of NPPs and explained the cooperation between EDF and CGNPC in order to secure the supply of equipment. Following their presentation, they started the open discussion with the audience by explaining their strategy to make or to buy and the link of this strategy to their core business. They also highlighted the new relations and the new partnership between supplier and customer. They insisted on the necessity to invest on supply chain and to have a strong Nuclear Safety Culture in the supply chain

  7. 基于电子商务技术的供应链系统构建分析%The Analysis of the Construction of Supply Chain System Based on E-commerce Technology

    Institute of Scientific and Technical Information of China (English)

    张慧

    2013-01-01

    Based on the research on the relationship between supply chain system and e-commerce the necessity of constructing the supply chain for e-commerce is analyzed and summered up. Consequently, the strategies for constructing a supply chain for e-commerce are proposed starting from the characteristics of the supply chain system for e-commerce environment, so as to further improve the suitability of supply chain system in the development of e-commerce.%本文在研究供应链系统和电子商务相互关系的基础上,分析得出构建电子商务供应链的必要性,从而从电子商务供应链的特点出发得出如何构建一个电子商务供应链的策略,从而进一步提高供应链系统在电子商务发展中的适应性。

  8. Construction of copper-based coordination polymers with 1D chain, 2D plane and wavy networks: Syntheses, structures, thermal behaviors and photoluminescence properties

    Indian Academy of Sciences (India)

    Jianghua Li; Chi Zhang

    2015-11-01

    Three Cu-based coordination polymers (CPs), including [CuII ( -1 -NCS)2 (O-1 -DMF)2 (2 -3,3’-bptz)] (1), [CuI (1,3-2-NCS)(2-3,3’-bptz)] (2) and [(CuI (1,3-μ2- NCS))(2-4,4’-bptz)] (3) (DMF = , -dimethyl formamide, 3,3’-bptz = 3,6-bis(3-pyridyl)tetrazine and 4,4’-bptz = 3,6-bis(4-pyridyl)tetrazine) have been successfully constructed by solution diffusion reactions by using Cu(NO3)2 ·3H2O or CuNCS and KNCS with 3,3’-bptz / 4,4’-bptz ligands, respectively. The resulting crystalline materials have been characterized by the single-crystal X-ray diffraction analyses, elemental analyses, FT-IR spectra, thermogravimetric analyses and powder X-ray diffraction (PXRD). Single crystal X-ray analyses revealed that CP 1 is organized in one-dimensional (1D) chain in which the Cu(II) ions are coordinated by 1 -NCS− anions and 1-DMF molecules, and linked by 2-3,3’-bptz bridging ligands. CPs ,2 and 3 are structural isomers. CP 2 exhibits two-dimensional (2D) (4,4)-plane-like network in which Cu(I) ions are linked by 2-NCS − and 2-3,3’-bptz ligands. In CP 3, Cu(I) ions are connected by 2 -NCS − and 2-4,4’-bptz ligands to form 2D saw-tooth wavy network. In addition, the photoluminescence properties of CPs 1-3 were also investigated in the solid state at room temperature.

  9. The antibody repertoire in evolution: chance, selection, and continuity.

    Science.gov (United States)

    Marchalonis, John J; Adelman, Miranda K; Schluter, Samuel F; Ramsland, Paul A

    2006-01-01

    All jawed vertebrates contain the genetic elements essential for the function of the adaptive/combinatorial immune response, have diverse sets of natural antibodies resulting from segmental gene recombination, express comparable functional repertoires and can produce specific antibodies following appropriate immunization. Profound variability occurs in the third hypervariable (CDR3) segments of light and heavy chains even within antibodies of the same ostensible specificity. Germline VH and VL elements, as well as the joining (J) segments are highly conserved among the distinct vertebrate species. Conservation is particularly noted among the VH3-like sequences of all jawed vertebrates in the FR2 and FR3 segments, as well as in the FGXGT(R or K)L J-segment characteristic of light chains and TCRs and the WGXGT(uncharged)VT JH segments. Human VH3-53 and Vlambda6 family orthologs may be present over the entire range of vertebrates. Models of the three-dimensional structures of shark VH/VL combining sites indicate similarity in framework structure and comparable CDR usage to those of man. Although carcharhine shark VH regions show greater than 50% identity to the human VH germline prototype, searches of lower deuterostome and invertebrate databases fail to detect molecules with significant relatedness. Overall, antibodies of jawed vertebrates show tremendous individual diversity, but are constructed incorporating design features that arose with the evolutionary emergence of the jawed vertebrates and have been conserved through at least 450 million years of evolutionary time. PMID:16083959

  10. Selection of apoptotic cell specific human antibodies from adult bone marrow.

    Directory of Open Access Journals (Sweden)

    Caroline Grönwall

    Full Text Available Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

  11. On the Application of Value Chain Theory in Construction Engineering Enterprise Cost Management%浅析价值链理论在建筑工程企业成本管理的应用

    Institute of Scientific and Technical Information of China (English)

    陆乃炎; 韩吉鹏

    2013-01-01

    On the basis of value chain theory, this paper points out the common defects in the cost management of construction engineering in China and then analyzes the relationship between value chain and cost control. Finally, the outstanding contribution of value chain theory to constriction engineering cost management is analyzed.%本文在介绍价值链理论的基础上,提出了我国建筑工程成本管理普遍存在的缺陷,接着对价值链与成本控制的关系进行分析,最后分析价值链在建筑工程成本管理中的突出贡献。

  12. Molecular-specific urokinase antibodies

    Science.gov (United States)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  13. Antibody fragments: Hope and hype

    OpenAIRE

    Nelson, Aaron L

    2010-01-01

    The antibody molecule is modular and separate domains can be extracted through biochemical or genetic means. It is clear from review of the literature that a wave of novel, antigen-specific molecular forms may soon enter clinical evaluation. This report examines the developmental histories of therapeutics derived from antigen-specific fragments of antibodies produced by recombinant processes. Three general types of fragments were observed, antigen-binding fragments (Fab), single chain variabl...

  14. Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pyloriisolates and its antibody in sera of patients

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA+ H pylori) isolates cause diseases.METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed,and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively.Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients′ sera, and the correlations between infection with CagA+ H pylori and gastritis as well as peptic ulcer were analyzed.RESULTS: Of all the clinical specimens obtained, 80.8%(126/156) were found to have H pylori isolates and 97.2%of the isolates (106/109) were positive for caaA gene. In comparison with the reported data, the cloned cagA1fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences,respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein, rCagA1was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109)expressed CagA and 88.1% of the patients′ serum samples (96/109) were CagA antibody-positive. The percentage of

  15. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do not...... scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  16. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    OpenAIRE

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable...

  17. Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Zhong; Yongping Li; Shuqi Huang; Bo Ning; Chunyan Zhang; Jianliang Zheng; Guanguang Feng

    2002-01-01

    Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

  18. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†.

    Science.gov (United States)

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-02-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  19. Expression and DNA sequence analysis of a human embryonic skeletal muscle myosin heavy chain gene.

    OpenAIRE

    Karsch-Mizrachi, I; M. Travis; Blau, H; Leinwand, L A

    1989-01-01

    Vertebrate myosin heavy chains (MHC) are represented by multiple genes that are expressed in a spatially and temporally distinct pattern during development. In order to obtain molecular probes for developmentally regulated human MHC isoforms, we used monoclonal antibodies to screen an expression cDNA library constructed from primary human myotube cultures. A 3.4 kb cDNA was isolated that encodes one of the first MHCs to be transcribed in human skeletal muscle development. A portion of the cor...

  20. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    International Nuclear Information System (INIS)

    The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO22+ was used as a source of RNA for the development of a recombinant (Fab)2 fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire κ light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab)2 protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab)2 fragments that bound to the UO22+-DCP complex with an affinity indistinguishable from that of a (Fab)2 fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea et al

  1. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    Energy Technology Data Exchange (ETDEWEB)

    X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake

    2005-04-18

    The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO{sub 2}{sup 2+} was used as a source of RNA for the development of a recombinant (Fab){sub 2} fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire {kappa} light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab){sub 2} protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab){sub 2} fragments that bound to the UO{sub 2}{sup 2+}-DCP complex with an affinity indistinguishable from that of a (Fab){sub 2} fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the

  2. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

  3. A multi-Fc-species system for recombinant antibody production

    OpenAIRE

    Nizak Clément; Vielemeyer Ole; El Marjou Ahmed; Moutel Sandrine; Benaroch Philippe; Dübel Stefan; Perez Franck

    2009-01-01

    Abstract Background Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural anti...

  4. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

    DEFF Research Database (Denmark)

    Álvarez-Cienfuegos, Ana; Alanes, Natalia Nuñez del Prado; Compte, Marta;

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we...... three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody...... of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas....

  5. Relacionamento interorganizacional na cadeia de suprimentos: um estudo de caso na indústria da construção civil The interorganizational relationship in the supply chain: a case study in the civil construction industry

    Directory of Open Access Journals (Sweden)

    Renata Albergaria de Mello Bandeira

    2009-01-01

    Full Text Available O escopo deste trabalho consiste em promover uma discussão sobre os mecanismos de coordenação das relações interorganizacionais - poder e cooperação - na cadeia de suprimentos da indústria da construção civil. O poder, como um construto de relações interorganizacionais, tem recebido um tratamento irregular e conflitante por parte dos analistas. No entanto, esta abordagem ignora relações existentes que são muito apropriadas para certos contextos do relacionamento interfirmas. Logo, foi desenvolvido um estudo de caso em um elo da indústria da construção civil com o intuito de identificar suas relações como sendo baseadas em dominação ou cooperação. São analisadas, ainda, as implicações dos resultados da pesquisa na gestão da cadeia de suprimentos da construção civil.The article discusses the relationship in the supplier chain in the civil construction industry. The main objective is to define if it is a cooperative relation among the intervenients or if the most powerful dictates the rules. Power as an interorganizational construct has been observed by analysts with an irregular and conflictive point of view. However, these analysts ignore completely existing relationships that are very appropriate in some existing relationships among enterprises. Due to that, it was verified het type of relationship developed by two intervenients in one step of the supply chain of a civil construction company to determine if it is cooperative or if, otherwise, the main actor uses its power to lead the chain. The researchers also analyze what are the implications of this study in the civil construction industry's supply management.

  6. Putting Markov Chains Back into Markov Chain Monte Carlo

    OpenAIRE

    Barker, Richard J.; Schofield, Matthew R.

    2007-01-01

    Markov chain theory plays an important role in statistical inference both in the formulation of models for data and in the construction of efficient algorithms for inference. The use of Markov chains in modeling data has a long history, however the use of Markov chain theory in developing algorithms for statistical inference has only become popular recently. Using mark-recapture models as an illustration, we show how Markov chains can be used for developing demographic models and also ...

  7. Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

    Science.gov (United States)

    Bender, E; Woof, J M; Atkin, J D; Barker, M D; Bebbington, C R; Burton, D R

    1993-04-01

    The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries. PMID:8518367

  8. Negative tail fusions can improve ruggedness of single domain antibodies.

    Science.gov (United States)

    Goldman, Ellen R; Brozozog-Lee, P Audrey; Zabetakis, Dan; Turner, Kendrick B; Walper, Scott A; Liu, Jinny L; Anderson, George P

    2014-03-01

    Single-domain antibodies (sdAbs), the recombinantly expressed binding domains derived from the heavy-chain-only antibodies found in camelids and sharks, are valued for their ability to refold after heat denaturation. However, some sdAbs are prone to aggregation on extended heating at high concentration. Additionally, sdAbs prepared cytoplasmically often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold. Genetic fusions of sdAbs with the acid tail of α-synuclein (ATS) resulted in constructs that had enhanced ability to resist aggregation. In addition, almost complete refolding was observed even in the absence of the disulfide bond. These sdAb-ATS fusions expand the utility of sdAbs. They provide sdAbs that are resistant to aggregation, and enable the production of re-foldable sdAbs in the reducing environment of the cytoplasm. PMID:24440507

  9. Immunogenicity of an engineered internal image antibody.

    OpenAIRE

    Billetta, R; Hollingdale, M. R.; Zanetti, M

    1991-01-01

    We engineered an antibody expressing in the third complementarity-determining region of its heavy chain variable region a "foreign" epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP) of the circumsporozoite protein of Plasmodium falciparum parasite, one of the etiologic agents of malaria in humans. A monoclonal antibody to P. falciparum specific for the (NANP)n amino acid sequence bound to the engineered antibody, and a synthetic (NANP)3 peptide blocked this interaction. Immunization...

  10. 建筑施工企业供应链管理现状及存在的问题%Supply-chain management status and existing problems of building construction enterprise

    Institute of Scientific and Technical Information of China (English)

    史国红

    2011-01-01

    概括了我国建筑施工企业供应链管理现状,根据项目流程总结了目前建筑供应链管理亟待解决的问题,并剖析了其原因,最后提出先进管理手段,以实现企业与供应商之间的互通。%This thesis outlines supply-chain management status of building construction enterprise,summarizes urgent problems needing solving in temporary building supply-chain management according to project procedure,and analyzes its causes.In the end,it proposes advanced management means,with a view to realize mutual connection between enterprise and supplier.

  11. Supply chain management in product development.

    OpenAIRE

    Tzortzopoulos, Patricia; Kagioglou, Mike; Cooper, Rachel; Dyson, E

    2009-01-01

    Mounting emphasis on construction supply chain management (CSCM) is due to both global sourcing of materials and a shortage of labor. These factors force increasing amounts of value-added work to be conducted off-site deep in the supply chain. Construction Supply Chain Management Handbookcompiles in one comprehensive source an overview of the diverse research and examples of construction supply chain practice around the world.Reflecting the emergence of CSCM as an important area of multi-nati...

  12. Engineering of FRT-lacZ fusion constructs: induction of the Pseudomonas aeruginosa fadAB1 operon by medium and long chain-length fatty acids

    OpenAIRE

    Son, Mike S.; Nguyen, David T.; Kang, Yun (Kenneth); Hoang, Tung T.

    2008-01-01

    Without prior knowledge of the promoters of various genes in bacteria, it can be difficult to study gene regulation using reporter-gene fusions. Regulation studies of promoters are ideal at their native locus, which do not require prior knowledge of promoter regions. Based on a previous study with FRT-lacZ-KmR constructs, we constructed two novel FRT-lacZ-GmR plasmids. This allows easy engineering of P. aeruginosa reporter-gene fusions, post-mutant construction with the Flp-FRT system. We dem...

  13. 影视旅游价值链构建及发展策略研究%Construction of Film and TV Tourism Value Chain and Development Strategy

    Institute of Scientific and Technical Information of China (English)

    曹菲

    2013-01-01

      This paper uses the value chain theory of professor Michael E. Porter, sets up the value chain of film and TV tourism, designs the activities content of each link from main body activities and auxiliary activities, and analyzes the development of film and TV tourism from the angle of node activity strategy.%  本文运用Michael E. Porter教授提出的价值链理论,建立影视旅游价值链,从主体活动和辅助活动两个角度设计各个环节的活动内容,并从节点活动角度分析了发展影视旅游的策略。

  14. Quantity Flexibility Contract Based on Construction Supply Chain Management%基于建筑供应链管理的工程采购数量弹性契约研究

    Institute of Scientific and Technical Information of China (English)

    査京民; 黄雷

    2013-01-01

    Adoption of integrated supply chain management is the key to reduce cost in construction industry. Aiming at the disadvantages of low degree cooperation,inefficient purchasing processes,and poor adaptability to demand uncertainty,the traditional purchasing model and the purchasing model under construction supply chain management environment were compared. The quantity flexibility purchasing contract model was set up based on contract theory,which proves that the contract model can effectively cope with demand uncertainty,enable contractor to flexibly carry out certain purchasing activity and further coordinate the construction supply chain.%建筑业采用集成式的供应链管理是降低建设工程成本的重要方法。为解决传统工程采购模式存在的合作度低、采购效率低下、对不确定性的应变能力差等缺陷。对比了建筑供应链管理环境下与传统工程采购模式的特点,基于供应链契约理论,构建了承包商与供应商之间的数量弹性采购契约模型,并证明了该契约模式可以有效应对工程采购中需求的不确定性。结论表明,建筑供应链管理下的数量弹性采购契约模式可以使承包商灵活地进行特定的采购活动,进而使建筑供应链上下游企业在一定程度上达到协调。

  15. 基于价值链管理的内部控制构建思想%Constructing Thoughts of Internal Control Based on Value Chain Management

    Institute of Scientific and Technical Information of China (English)

    张其镇

    2011-01-01

    Development and application of information technology and value chain theory provide a good opportunity to improve the management level of enterprise and gain a competitive advantage. This paper, combining with the value chain theory, analyzes the key steps to build the internal control which can adapt to the management of value chain under the guidance of the internal control structure. This paper .argues that internal control should be value-added activities which take getting a competitive advantage as the strategic goal, take theall kinds of "flow" in business as the main line of control and constantly self-perfect.%信息技术与价值链理论的发展与应用,为提高企业管理水平,获得竞争优势提供了一个良好的契机.本文结合价值链理论,分析了在内部控制结构指导下构建一个能适应价值链管理的内部控制的关键步骤.本文认为,内部控制应是一个以获取竞争优势为战略目标,以企业内各种"流"为控制主线并能不断自我完善的增值活动.

  16. Clinical application of a new antimyosin antibody

    International Nuclear Information System (INIS)

    A mouse monoclonal antibody, 3-48 (Rougier Bio-Tech Ltd, Montreal) which recognizes the alpha and beta heavy chains of human atrial and ventricular myosin, and the beta heavy chain of human slow skeletal muscle, has recently been developed. In the rat isoproterenol-induced infarction model and the canine model of selective obstruction of a coronary artery, the antibody was shown to be specifically localized to the necrotic myocardium. A selected group of patients with known infarction was imaged with the 111indium labeled F(ab')2 protion of this antibody in a pre-clinical feasibility study, and the results therefrom are reported in this communication. (orig.)

  17. Based on JMI Model the Study in Implementation Strategy of Construction Supply Chain%基于JMI模式的建筑供应链实施策略研究

    Institute of Scientific and Technical Information of China (English)

    刘新欣; 王红春

    2014-01-01

    工程建设项目消耗的物资数量大、品种多,能否合理安排物资储备,直接关系到建设项目的总进度和总成本。随着建筑供应链思想的不断深化,结合工程建设自身的特点,文中提出联合库存管理模式,明确应用条件及实施策略,可以有效地降低施工企业的总库存成本,加快企业的资金周转速率,实现供应商与建筑企业之间的战略共赢,最终提高建筑企业的综合竞争力。%The number of construction projects goods consumed in large and variety,set up arrangements for material reserves sensibly,directly related to the overall progress and the total cost of the construction project. With the continue deepening of the construction supply chain ideology,combined with the characteristics of construction,the paper proposes the joint management inventory,nail down application conditions and implementation strategy,reduce the total inventory cost of construction enterprises effectively,accelerate the capital turnover rate of enterprises,realize win-win strategy between providers and construction companies,at last,improve the overall competitiveness of construction enterprises.

  18. Value Chain Theory in Agent Construction Project Investment Control:From Construction Agency Perspective%价值链理论在代建项目投资控制中的应用研究--基于代建企业视角

    Institute of Scientific and Technical Information of China (English)

    刘逸凡; 刘万琳

    2013-01-01

    From the view of the Construction Agency,this paper used the value chain analysis model to study investment control within the whole process of the Agent Construction Project,proposed to establish a internal value chain which involves two key segments (the optimization of the design choices in the design phase and the control of design changes in construction phase) of the Construction Agency, thus successfully control the project investment. Through the value chain support systems,sort out all the value activities within theses two key segments,draw conclusions of effective measures that control the project investment,thereby improve the management performance of Construction Agency and enable them to gain a competitive advantage during the bidding process that held by government.%  本文从代建企业的角度出发,利用价值链理论分析模型对代建项目全过程的投资控制进行研究,提出以设计阶段设计方案的优化选择和施工阶段设计变更的控制作为企业内部价值链两大关键环节,从而有效地对代建项目进行投资控制。通过价值链中的支持系统,梳理关键环节中的价值活动,得出代建项目投资控制的有效措施,进而提高代建企业的管理绩效,在政府委托招标过程中获得竞争优势。

  19. Distinct antibody species: structural differences creating therapeutic opportunities.

    Science.gov (United States)

    Muyldermans, Serge; Smider, Vaughn V

    2016-06-01

    Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

  20. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

    Science.gov (United States)

    Fuchs, P; Breitling, F; Little, M; Dübel, S

    1997-06-01

    The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed. PMID:9219032

  1. Insight into earthquake sequencing: analysis and interpretation of time-series constructed from the directed graph of the Markov chain model

    Science.gov (United States)

    Cavers, M. S.; Vasudevan, K.

    2015-02-01

    Directed graph representation of a Markov chain model to study global earthquake sequencing leads to a time-series of state-to-state transition probabilities that includes the spatio-temporally linked recurrent events in the record-breaking sense. A state refers to a configuration comprised of zones with either the occurrence or non-occurrence of an earthquake in each zone in a pre-determined time interval. Since the time-series is derived from non-linear and non-stationary earthquake sequencing, we use known analysis methods to glean new information. We apply decomposition procedures such as ensemble empirical mode decomposition (EEMD) to study the state-to-state fluctuations in each of the intrinsic mode functions. We subject the intrinsic mode functions, the orthogonal basis set derived from the time-series using the EEMD, to a detailed analysis to draw information-content of the time-series. Also, we investigate the influence of random-noise on the data-driven state-to-state transition probabilities. We consider a second aspect of earthquake sequencing that is closely tied to its time-correlative behavior. Here, we extend the Fano factor and Allan factor analysis to the time-series of state-to state transition frequencies of a Markov chain. Our results support not only the usefulness the intrinsic mode functions in understanding the time-series but also the presence of power-law behaviour exemplified by the Fano factor and the Allan factor.

  2. A Novel Three-dimensional Inorganic Open-framework Constructed from [Mo4O16]n8n- Chains Linked through Ag+

    Institute of Scientific and Technical Information of China (English)

    GUO Hong-Xu; LIU Shi-Xiong

    2005-01-01

    A novel 3-D inorganic bimetallic compound Ag2Mo2O7 has been hydrothermally synthesized and structurally characterized by X-ray diffraction. The compound crystallizes in mono- clinic, space group P21/c with a = 6.1274(3), b = 13.1836(6), c = 7.8780(3)(A), β = 110.673(3)(A), V = 595.42(5)(A)3, Z = 4, Mr = 519.62, Dc = 5.797 g/cm3, F(000) = 936, μ(MoKα) = 10.579 mm-1, F(000) = 936, the final R = 0.0204 and wR = 0.0477 for 1268 observed reflections with I > 2σ(I). In the title compound, the [Mo4O16]n8n- chains are linked through O-Ag-O bridging bonds to form a 3D network. It represents an unprecedented linking mode between the isopolyoxomolybdate chain and the transition metal oxide cluster. IR, TGA and fluorescence properties of the title compound are also studied.

  3. 生产性服务业:构建中国制造业国家价值链的关键%Producer Services: the Key to Constructing the National Value Chain of Chinese Manufacturing

    Institute of Scientific and Technical Information of China (English)

    贾根良; 刘书瀚

    2012-01-01

    生产性服务业在全球价值链的系统整合中处于关键性地位,拥有强大的研发能力并掌控整个价值链是生产性服务业的核心功能。依托巨大的国内市场,以生产性服务业为龙头,构建独立自主的制造业国家价值链,是中国制造业转型升级的根本措施。在中国制造业国家价值链的构建中,生产性服务业主要通过四种途径发挥着核心作用:中国制造业的领导型企业作为系统整合者主要执行的是包括研发在内的生产性服务功能,这是成长为中国跨国公司的基本途径;专业化市场交易平台承担着国内市场高度组织化并获取国际定价权的重要职能;国家价值链的国外布局是构建中国制造业全球价值链的初步尝试;电子商务革命和连锁专卖体系等新型生产性服务业的兴起,不仅是中国制造业企业打破营销商垄断国内终端销售渠道的有力途径,也是其突破跨国公司垄断发达国家终端销售市场的历史机遇。%Producer services, whose core functions are possessing strong R&D capabilities and controlling the whole value chain, are in a key position in the system integration of global value chain. The fundamental measure for the transforming and upgrading of Chinese manufacturing is to construct independent national manufacturing value chain with producer services as the leader, and relies on huge domestic market. During the construction of the national manufacturing value chain, producer services play a central role mainly in four ways: As system integrators, China' s leading manufacturing enterprises mainly perform the productive service functions which include research and development, and this is the basic way for these enterprises to grow into multinational corporations; Specialized market trading platform undertakes vital responsibilities to build a highly organized domestic market and gain international pricing power; The layout of

  4. Bispecific antibodies.

    Science.gov (United States)

    Kontermann, Roland E; Brinkmann, Ulrich

    2015-07-01

    Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with 'two-target' functionality can interfere with multiple surface receptors or ligands associated, for example with cancer, proliferation or inflammatory processes. BsAbs can also place targets into close proximity, either to support protein complex formation on one cell, or to trigger contacts between cells. Examples of 'forced-connection' functionalities are bsAbs that support protein complexation in the clotting cascade, or tumor-targeted immune cell recruiters and/or activators. Following years of research and development (R&D), the first bsAb was approved in 2009. Another bsAb entered the market in December 2014 and several more are in clinical trials. Here, we describe the potentials of bsAbs to become the next wave of antibody-based therapies, focusing on molecules in clinical development. PMID:25728220

  5. Value Analysis of Construction Projects under the Lean Supply Chain%基于精益供应链的工程建设项目价值分析

    Institute of Scientific and Technical Information of China (English)

    谢秋玲; 叶青

    2012-01-01

    Combining with the lean supply chain theory and taking the value as the key of solution and decision-making, value analysis of construction project is studied, and the application process of value analysis is also illustrated by case study. The value of each solution is comprehensively evaluated by fuzzy quality function deployment, and compared with each other to find the optimal one. The value analysis of construction projects under the lean supply chain is an effective decision-making method.%结合精益供应链理论,以价值作为方案决策要点,对工程建设项目价值分析进行研究,通过案例说明价值分析在工程建设项目中的应用流程.采用模糊质量功能展开法综合评价各方案的价值,并进行对比分析,找出价值最优方案.基于精益供应链的工程建设项目价值分析是一种行之有效的方案决策方法.

  6. 基于精益价值链理论的建筑业企业精益建造能力评价研究%Evaluation of Construction Enterprise’s Lean Construction Ability Based on the Theory of Lean Value Chain

    Institute of Scientific and Technical Information of China (English)

    陈礼靖; 佘健俊; 李梅

    2013-01-01

    精益建造是一种先进的生产管理模式,对新时期建筑业企业精益建造能力的评价研究有助于建筑业企业改进施工生产模式,真正做到精益采购、精益施工以及精益交付。根据精益价值链的特点和要求建立了一套建筑业企业精益建造能力评价指标体系,其中重点对采购流程中的准时采购、按需采购和施工流程中的质量管理以及消除浪费等指标进行了分析,利用模糊层次分析法对建筑业企业的精益建造能力进行综合评价,并进行实证分析,以期为建筑业企业精益建造能力的评价与提升提供一种借鉴方法。%Lean construction is an advanced production management mode. The research of the evaluation of construction enterprise’s lean construction ability in the new period can help construction enterprises to improve production mode,and to realize lean procurement,lean construction and lean delivery. The paper firstly introduces the concept and characteristics of lean value chain,and then creates a set of index system of construction enterprise’s lean construction ability according to the requirements of the lean value chain. Specifically,the paper focuses on analyzing some index,such as just in time purchasing,on-demand procurement in procurement process,as well as quality management and waste elimination in the construction process,then conducting a comprehensive evaluation of construction enterprise’s lean ability by fuzzy analytic hierarchy process,and finally the paper carries on an empirical analysis,which provides a reference method for the evaluation and improvement of construction enterprise’s lean construction ability.

  7. Single-domain antibodies for biomedical applications.

    Science.gov (United States)

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-02-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development. PMID:26551147

  8. Antibody remodeling: a general solution to the design of a metal-coordination site in an antibody binding pocket.

    OpenAIRE

    Roberts, V A; Iverson, B L; Iverson, S A; Benkovic, S J; Lerner, R A; Getzoff, E D; Tainer, J A

    1990-01-01

    To develop a general approach to designing cofactor-binding sites for catalytic antibodies, we characterized structural patterns in the binding sites of antibodies and zinc enzymes. Superposition of eight sets of antibody light- and heavy-chain variable domains identified structurally conserved sites within the sequence-variable complementarity determining regions. The pattern for catalytic zinc sites included two ligands close in sequence, a sequence-distant ligand, and a main-chain hydrogen...

  9. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library.

    Science.gov (United States)

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon

    2015-10-01

    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis. PMID:26277276

  10. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  11. Overcoming low yields of plant-made antibodies by a protein engineering approach.

    Science.gov (United States)

    Zischewski, Julia; Sack, Markus; Fischer, Rainer

    2016-01-01

    The commercial development of plant-based antibody production platforms is often limited by low and variable yields, but little is known about the factors that affect antibody accumulation during and after translation. Here, we present a strategy to identify yield-limiting regions in the transcript and protein. We exchanged variable heavy chain (VH) domain sequences between two human antibodies at structurally conserved positions, thus creating ten chimeric VH domains containing sequences from M12 (∼1000 μg/g leaf fresh weight [FW]) and 4E10 (∼100 μg/g FW). After transient expression in Nicotiana benthamiana leaves, we measured mRNA and protein levels by quantitative real-time PCR and surface plasmon resonance spectroscopy, respectively. Transcript levels were similar for all constructs, but antibody levels ranged from ∼250 μg/g to over 2000 μg/g FW. Analysis of the expression levels showed that: i) 4E10 yields were only marginally increased by suppression of post-transcriptional gene silencing; ii) the CDR3 of 4E10 contains a protease site; and iii) a bipartite, yield-limiting region exists in the CDR2/CDR3. Our findings highlight the strong impact of cotranslational and posttranslational events on antibody yields and show that protein engineering is a powerful tool that can be used to overcome the remaining limitations affecting antibody production in plants. PMID:26632507

  12. Research on Enterprise Social Responsibility Construction in Supply Chain Based on the Perspectives of Network Governance%基于网络治理视角的供应链企业社会责任研究

    Institute of Scientific and Technical Information of China (English)

    周翼翔

    2015-01-01

    At present, corporate social responsibility is no longer confined to the scope of an enterprise , but extended to different links of supply chain.In supply chain, the position, profit distribution, and corporate social responsibility performance of each enterprise are not identical, which makes both core and non-core enterprises lack of responsibility.According to the situation of"prisoner's dilemma", we construct the model to pro-mote corporate social responsibility in supply chain .Only from point to chain and then to network governance can we gradually change the current situation of deficient corporate social responsibility on the basis of stimulus , contract constraint, mutual competition combined with external govern-ance in our country.%企业社会责任的范围已不再局限于某个企业,已扩展到供应链的不同环节。在供应链中,由于各企业的地位、利益分配以及企业社会责任表现各不相同,使得无论核心企业还是非核心企业都缺乏履行责任的动力。针对这种“囚徒困境”的局面,构建供应链企业社会责任推进模式,在经济刺激、协议约束、相互竞争的基础上结合外部治理,由点到链,再由链到网络治理逐步推进来解决我国目前企业社会责任缺失的现状。

  13. A chain of FPU cells

    OpenAIRE

    Verhulst, F.

    2015-01-01

    In contrast to the classical Fermi-Pasta-Ulam (FPU) chain, the inhomogeneous FPU chain shows nearly all the principal resonances. Using this fact, we can construct a periodic FPU chain of low dimension, for instance a FPU cell of four degrees-of-freedom, that can be used as a building block for a chain of FPU cells. Differences between chains in nearest-neighbour interaction and those in overall interaction are caused by symmetry. We will also show some results on the dynamics of a particular...

  14. Dynamics of corporate strategy from a value chain perspective : A study of the Swedish telecom and construction industries during the 90’s

    OpenAIRE

    de Paula, Andes

    2006-01-01

    Changes in sectors and industries have brought new challenges to corporations as well as been important driving forces for the dynamics in strategy at the corporate level. With the dramatic developments of the 1990’s in mind, such as multilateral free-trade agreements, liberalization, privatization, sharp industry growth/decline, increased competition and globalization, in particular within the telecom and the construction industry, this study contributes to describing and understanding strat...

  15. Insight into earthquake sequencing: analysis and interpretation of time-series constructed from the directed graph of the Markov chain model

    Directory of Open Access Journals (Sweden)

    M. S. Cavers

    2015-02-01

    Full Text Available Directed graph representation of a Markov chain model to study global earthquake sequencing leads to a time-series of state-to-state transition probabilities that includes the spatio-temporally linked recurrent events in the record-breaking sense. A state refers to a configuration comprised of zones with either the occurrence or non-occurrence of an earthquake in each zone in a pre-determined time interval. Since the time-series is derived from non-linear and non-stationary earthquake sequencing, we use known analysis methods to glean new information. We apply decomposition procedures such as ensemble empirical mode decomposition (EEMD to study the state-to-state fluctuations in each of the intrinsic mode functions. We subject the intrinsic mode functions, the orthogonal basis set derived from the time-series using the EEMD, to a detailed analysis to draw information-content of the time-series. Also, we investigate the influence of random-noise on the data-driven state-to-state transition probabilities. We consider a second aspect of earthquake sequencing that is closely tied to its time-correlative behavior. Here, we extend the Fano factor and Allan factor analysis to the time-series of state-to state transition frequencies of a Markov chain. Our results support not only the usefulness the intrinsic mode functions in understanding the time-series but also the presence of power-law behaviour exemplified by the Fano factor and the Allan factor.

  16. Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum.

    Science.gov (United States)

    Shkoporov, A N; Khokhlova, E V; Savochkin, K A; Kafarskaia, L I; Efimov, B A

    2015-06-01

    Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and infants and present a promising platform for construction of genetically modified probiotic agents for treatment of gastrointestinal disorders. In this study, three strains of Bifidobacterium longum were constructed that express and secrete biologically active single-chain antibodies against human TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the level of 25 μg L(-1) in broth culture and was mostly retained in the cytoplasm, while VHH-type antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3-1 mg L(-1) and secreted very efficiently. The biological activity of both antibody types was demonstrated in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after intragastric administration of transformed B. longum strains to (C57/BL6 × DBA/2)F1 mice. The obtained B. longum strains may serve as prototypes for construction of novel probiotic medications against inflammatory bowel disease and C. difficile-associated disease. PMID:25994292

  17. THE CONSTRUCTION AND PRELIMINARY APPRAISEMENT OF HSV-2gD GENE DNA VACCINE

    Institute of Scientific and Technical Information of China (English)

    王军阳; 范桂香; 胜利; 袁育康

    2002-01-01

    Objective To prevent infection from herpes simplex virus type 2(HSV-2),and make the foundation for the construction of multi-valent DNA vaccine. Methods The complete DNA sequence,which encoded the amino acid sequence of the viral glycoprotein D(gD),was obtained from the HSV-2 genome by polymerase chain reaction(PCR).The fragment was inserted into the lower stream of Cytomegalovirus(CMV) promoter in the eukaryotic expression plasmid pcDNA3.1(+), then immunized mice by bilateral intramuscular injection into the rear legs with this recombinant plasmid and tested the specific antibodies against glycoprotein D by ELISA. Results Animal experiment have demonstrated that the recombinant plasmid(pcDNA-gD2)inoculated into mice could induce the production of specific antibodies against glycoprotein D. Conclusion The eukaryotic plasmid pcDNA-gD2 constructed by us could correctly express gD gene and induce the production of specific antibodies.

  18. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    Directory of Open Access Journals (Sweden)

    Kaku Yoshihiro

    2012-09-01

    Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

  19. Cell-Free Synthesis Meets Antibody Production: A Review

    OpenAIRE

    Marlitt Stech; Stefan Kubick

    2015-01-01

    Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufactu...

  20. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  1. Model protocells from single-chain lipids.

    Science.gov (United States)

    Mansy, Sheref S

    2009-03-01

    Significant progress has been made in the construction of laboratory models of protocells. Most frequently the developed vesicle systems utilize single-chain lipids rather than the double-chain lipids typically found in biological membranes. Although single-chain lipids yield less robust vesicles, their dynamic characteristics are highly exploitable for protocellular functions. Herein the advantages of using single-chain lipids in the construction of protocells are discussed. PMID:19399223

  2. Model Protocells from Single-Chain Lipids

    OpenAIRE

    Mansy, Sheref S.

    2009-01-01

    Significant progress has been made in the construction of laboratory models of protocells. Most frequently the developed vesicle systems utilize single-chain lipids rather than the double-chain lipids typically found in biological membranes. Although single-chain lipids yield less robust vesicles, their dynamic characteristics are highly exploitable for protocellular functions. Herein the advantages of using single-chain lipids in the construction of protocells are discussed.

  3. Model Protocells from Single-Chain Lipids

    Directory of Open Access Journals (Sweden)

    Sheref S. Mansy

    2009-03-01

    Full Text Available Significant progress has been made in the construction of laboratory models of protocells. Most frequently the developed vesicle systems utilize single-chain lipids rather than the double-chain lipids typically found in biological membranes. Although single-chain lipids yield less robust vesicles, their dynamic characteristics are highly exploitable for protocellular functions. Herein the advantages of using single-chain lipids in the construction of protocells are discussed.

  4. Development and utilization of camelid VHH antibodies from alpaca for 2,2',4,4'-tetrabrominated diphenyl ether detection.

    Science.gov (United States)

    Bever, Candace R S; Majkova, Zuzana; Radhakrishnan, Rajeswaran; Suni, Ian; McCoy, Mark; Wang, Yanru; Dechant, Julie; Gee, Shirley; Hammock, Bruce D

    2014-08-01

    An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies. PMID:25005746

  5. RA8, A human anti-CD25 antibody against human treg cells

    Energy Technology Data Exchange (ETDEWEB)

    Arias, Robyn; Flanagan, Meg; Miller, Keith D.; Nien, Yu-Chih; Hu, Peisheng; Gray, Dixon; Khawli, Leslie A.; Epstein, Alan L.

    2007-06-01

    Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 109 single chain variable fragments (scFv). Two unique clones with low KD values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25+ cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.

  6. Research on Integrated Green Supply Chain Management

    Institute of Scientific and Technical Information of China (English)

    DENG Lei; WANG Xu

    2006-01-01

    On the basis of the analyzing product life cycle and value chain management in green supply chain, integrated green supply chain is put forward and constructed which involve lean production, agile manufacturing and green supply chain. This integrated structure provides an effective method for resolving some questions such as cost, market, environment, etc. in enterprise. A case study is presented at the end of paper to demonstrate how integrated supply chain implemented successfully in enterprise.

  7. 基于建筑供应链的不允许缺货联合库存模型研究%Study on Joint Managed Inventory Model Under No-Shortage Based on Construction Materials Supply Chain

    Institute of Scientific and Technical Information of China (English)

    钟德强; 郭薇

    2014-01-01

    研究由一个总承包商与多个分包商组成的建筑材料供应链联合库存问题,在不允许缺货的情况下,建立了库存成本模型。传统独立库存与供应链联合库存的成本比较证明,供应链联合库存总成本低于传统独立库存总成本。并以一个总承包商、2个分包商为例,研究了联合库存下的成本分配问题,证明用Shapley值法分配成本,各参与方在供应链联合库存下分担的成本均低于传统独立库存下的成本。%Investigates the joint managed inventory(JMI) problems of construction materials supply chain consist-ing of one general contractor and multiple sub-contractors. On the condition that short supply is not allowed,establishes the inventory cost model. Comparing the traditional independent inventory cost and that of supply chain JMI, proves that the total cost of the JMI is lower. Taking one general contractor and two sub-contractors for an example, studies the cost allocation of joint managed inventory, and verifies that using Shapley value method to allocate the cost, the shared cost of each participant in supply chain JMI is lower than that of traditional independent inventory.

  8. Synthesis, crystal structure and properties of two 1D nano-chain coordination polymers constructed by lanthanide with pyridine-3,4-dicarboxylic acid and 1,10-phenanthroline

    International Nuclear Information System (INIS)

    The hydrothermal reactions of LnCl3.6H2O (Ln=Eu, Tb), pyridine-3,4-dicarboxylic acid (3,4-pydaH2), 1,10-phenthroline (phen) and NaOH in aqueous medium yield two metal-organic hybrid materials, [Eu2(3,4-pyda)3(phen)(H2O).H2O]n (1) and [Tb2(3,4-pyda)3(phen)(H2O).H2O]n (2), respectively. Both compounds have similar topology structure containing one-dimensional nano-chain, which is further assembled into a three-dimensional supramolecular network via π-π stacking interactions and hydrogen bonds. To the best of our knowledge, they represent the first example of nano-chain coordination polymers constructed by 3,4-pydaH2 and chelate heterocylic ligand. Interestingly, the 3,4-pyda anion exhibits three kinds of coordination modes in these complexes. The coordination modes of 3,4-pyda in complexes 1 and 2 have not been observed in other coordination polymers containing 3,4-pyda ligands. Compounds 1 and 2 exhibit strong fluorescent emission bands in the solid state at room temperature. Their magnetic analyses show that they exhibit different magnetic interactions. - Graphical abstract: Two novel lanthanide coordination polymers [M2(pydc)3(phen)(H2O).H2O]n (M=Eu(1) and Tb(2), pydc=pyridine-3,4-dicarboxylate, phen=1,10-phenthroline) have been synthesized and characterized. Both compounds reveal a one-dimensional nano-chain, which is further assembled into a three-dimensional supramolecular network via π-π stacking interactions and hydrogen bonds. Their luminescent and magnetic properties have been investigated

  9. 物联网环境下建筑供应链库存协同管理的研究%Study on Construction Supply Chain Collaborative Inventory Management in IOT Environment

    Institute of Scientific and Technical Information of China (English)

    王连月

    2014-01-01

    In this paper, through illustrating the connotation of the collaborative inventory management of the construction supply chain, we analyzed the problems therein and pointed out that in order to realize the collaborative inventory management for the supply chain, the IOT network and inventory management information platform of the nodal enterprises should be set up first.%为了实现建筑供应链库存协同管理,需要各节点企业数据准确传输和信息充分共享。通过对建筑供应链库存协同管理内涵的论述,分析了供应链库存协同管理的问题,提出实现供应链库存协同管理需要构建节点企业物联网和库存管理信息平台。

  10. Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

    Science.gov (United States)

    Shaw, D M; Embleton, M J; Westwater, C; Ryan, M G; Myers, K A; Kingsman, S M; Carroll, M W; Stern, P L

    2000-12-15

    The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. PMID:11113573

  11. Dimerisation strategies for shark IgNAR single domain antibody fragments.

    Science.gov (United States)

    Simmons, David P; Abregu, Fiona A; Krishnan, Usha V; Proll, David F; Streltsov, Victor A; Doughty, Larissa; Hattarki, Meghan K; Nuttall, Stewart D

    2006-08-31

    Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (approximately 13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix-turn-helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (approximately 26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats. PMID:16962608

  12. 基于供应链的应急物流虚拟联合体的构建%The Construction of Emergency Logistics Virtual Union Based on Supply Chain

    Institute of Scientific and Technical Information of China (English)

    陈伟珂; 花翠

    2014-01-01

    This paper based on supply chain management theory,follows the point -line -surface construction principle, proposed to construct a framework of emergency logistics virtual consortium,in order to give full play to the leading role of the government,the army vanguard role,the market supply,the practical realization of military logistics and logistics,the organic combination of market mechanism and administration mechanism.The purpose is to effectively integrate social and professional resources,improve the overall response speed and supply efficiency of emergency logistics.%文中基于供应链管理理论,遵循点-线-面构建原则,提出应急物流虚拟联合体的构建框架,以充分发挥政府主导作用,军队先锋作用,市场供给作用,切实实现军队物流和地方物流、市场机制和行政机制的有机结合。目的是有效集成社会专业资源,提高应急物流的整体响应速度和供应效率。

  13. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  14. 抗阿尔茨海默病Aβ人源单链抗体基因的筛选和表达%Cloning and expression of human single-chain Fv antibody against Aβ1-42 peptide involved in Alzheimer disease

    Institute of Scientific and Technical Information of China (English)

    贾静; 刘喆; 赵雪梅; 梁平

    2008-01-01

    目的 从人源噬菌体单链抗体库中筛选与阿尔茨海默病发病中起关键作用的β淀粉样多肽(Aβ)1-42特异性结合的单链可变区抗体(scFv)基因,利用原核生物大肠杆菌进行可溶性表达,以获得抗AB1-42人源抗体.方法 利用噬菌体展示技术对人源噬菌体单链抗体库进行多轮富集,以Aβ1-42为抗原经酶联免疫吸附(ELISA)检测,筛选与Aβ1-42特异性结合的阳性噬菌体克隆,再用阳性噬菌体感染E.coli HB2151进行可溶性表达,经SDS-PAGE、Western blot及ELISA法检测scFv单抗的可溶性表达水平及抗原结合活性.并对阳性scFv抗体基因进行基因测序鉴定.结果 获得了7个特异性的抗Aβ1-42 scFv阳性噬菌体克隆,其中4个克隆ELISA检测吸光度值(A值)高于阴性对照5倍以上;其中1株(A 10)获得可溶性单链抗体的成功表达,表达产物主要位于菌体中,ELISA效价显示在全菌蛋白中A值高于对照5倍以上.其相对分子质量约为33 000.DNA测序表明所获抗体的可变区基因属于scFv抗体基因,推导得到的氨基酸序列具有典型的抗体可变区结构.结论 用人源噬菌体单链抗体库筛选出抗Aβ1-42的人源抗体单链可变区基因,并成功表达了具有抗原结合活性的可溶性单链抗体,为进一步研究阿尔茨海默病的发病机制和治疗奠定了基础.%Objective Screening of antibody clones specific for β amyloid peptide 1-42 from human phage-display single-chain Fv(scFv)antibody library,and to clone the antibody gene and to express it in a bacterial system,with an ultimate intention to obtain human anti-Aβ1-42 antibody for Alzheimer disease(AD) therapy.Methods β amyloid peptide 1-42 was bound on the solid surface of 96 wells plate as the antigen for the binding antibody clones from a human phage-display scFv antibody library.After four rounds of biopanning,random,well-separated colonies were identified by ELISA test.The specific positive phage clones were

  15. Recombinant shark natural antibodies to thyroglobulin.

    Science.gov (United States)

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL. PMID:15954089

  16. 替代失活法构建变形链球菌LuxS基因缺陷株%Construction of Streptococcus mutans LuxS gene deletion mutants by long flanking homology polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    李月恒; 陈惠珍; 周智; 陈娇; 李锐; 尹一兵; 张雪梅

    2011-01-01

    目的 LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,构建该基因的缺陷菌.方法 采用长臂同源多聚酶链反应(LFH-PCR)方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷菌株,并采用PCR鉴定.结果 对变形链球菌LuxS基因缺陷菌株进行PCR和DNA序列测定分析证实构建成功.结论 成功构建出变形链球菌LuxS基因的缺陷菌株,为后期针对变形链球菌LuxS基因的相关研究奠定基础.%Objective To construct LuxS deletion mutant of Streptococcus mutans and get the capsule deficient strain. Method Long flanking homology polymerase chain reaction(LFH-PCR) was introduced to generate a gene disruption construct consisting of Emr cassette with long flanking homology regions to the target gene. The electroporation competence of Streptococcus mutans was then transformed with this PCR product. Then the positive transformants were counted on selective agar containing erythromycin and identified by PCR. Result Identification by PCR and sequencing confirmed the validity of the LuxS deletion mutant of Streptococcus mutans. Conclusion The successful construction of the LuxS deletion mutant can be used in further functional genome research.

  17. Monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs) are antibodies having single specificity for a given antigen site (epitope). The development of hybridoma technology and the relative ease by which MAbs can be prepared has revolutionized many aspects of serological applications in diagnosis and differentiation of disease producing agents. The property of monospecificity offers advantages in diagnostic applications over polyclonal sera in that tests can be defined exactly with regard to the antigen detected and the affinity of reaction between the given antigenic site and the monoclonal reagent. In addition, MAbs offer better possibilities for test standardization, because the same reagent can be used in different laboratories. Such an MAb can be supplied by a central laboratory or 'grown' from hybridoma cells, ensuring that the resultant product is identical from laboratory to laboratory and that the part of the test involving the MAb reaction is the same. The methodologies for inoculation regimes, mice, cloning methods, selection of fusion partners, etc., have been validated extensively in developed country laboratories. The decision to establish a MAb production facility must be examined on a strict cost-benefit basis, since it is still expensive to produce a product. There are many MAbs available that should be sought to allow exploitation in developing tests. If a production facility is envisaged, it should produce reagents for national needs, i.e. there should be a clear problem oriented approach whereby exact needs are defined. In the field of veterinary applications, MAbs are the central reagent in many immunoassays based on the enzyme linked immunosorbent assay (ELISA). The development of specific tests for diagnosing diseases is dominated by MAbs and has been fuelled by a strong research base, mainly in developed countries allied to developing countries through the study of related diseases. Thus, there are very many assays dependent on MAbs, some of which form the basis of

  18. 建筑供应链库存控制模式与优化方法研究--基于CPFR理论%On the Control Models and Optimization Methods of Construction Supply Chain:Based on the Theory of CPFR

    Institute of Scientific and Technical Information of China (English)

    杜泽超; 王俊

    2015-01-01

    For a long time, inventory management is the difficult point of the supply chain management of construction. Based on the theory of CPFR, this paper studies the inventory control models and optimization methods of construction supply chain and gets the final conclusion.%一直以来,库存管理就是建筑供应链管理的重难点问题。本文基于CPFR理论研究了建筑供应链库存控制模式与优化方法,并得出了最终结论。

  19. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  20. CONSTRUCTION AND CHARACTERIZATION OF MUTANT PROINSULIN WITH SHIFTED INTRA-A CHAIN DISULFIDE-BOND%(A11,A12)胰岛素原移位突变体的表征研究

    Institute of Scientific and Technical Information of China (English)

    井健

    2009-01-01

    With PCR technique a mutant proinsulin gene with CysA11Ser and SerA12Cys was constructed to investigate intra A-chain disulfide effect on insulin biological activity. After expression, unfolding and refolding, mutant proinsulin was purified with (A6, A12) intra-A chain disulfide-bond. The mutant proinsulin showed 55% radioimmunoactivity (RIA) and 11% of receptor binding activity of native proinsulin. Mass spectrometry analysis, IEF and amino acid composition assay were carried out to determine mutant proinsulin. Data showed that the A6-A12 intra-A chain disulfide bond was formed, having some negative effect on native proinsulin structure, especially in regard to the stability of insulin receptor binding region.%将(CysA11Ser,SerA12Cys)人胰岛素原移位突变体基因克隆至高效表达质粒pET22b多克隆位点区,构建重组表达质粒pET-A112,并在大肠杆菌E.coli BL21(DE3)pLys中进行了表达.表达产物存在于包涵体中,通过包涵体的分离及二硫键变复性,获得A6-A12二硫键突变型的胰岛素原突变体.SDS-PAGE电泳显示突变型胰岛素原的电泳迁移率与野生型胰岛素原基本相同.质谱检测表明,突变性胰岛素原的分子结构均一、纯度良好,氨基酸组成与理论值基本一致.以野生型人胰岛素原为对照进行的胰岛素受体试验及胰岛素放射免疫试验表明,A6-A12二硫键错接型胰岛素原突变体的受体结合活性和放免活性是野生型胰岛素原的11%与55%.

  1. Host defence against C. albicans infections in IgH transgenic mice with V(H) derived from a natural anti-keratin antibody.

    Science.gov (United States)

    Li, Wei; Fu, Meng; An, Jin-Gang; Xing, Ying; Zhang, Ping; Zhang, Xin; Wang, Yao-Chun; Li, Cheng-Xin; Tian, Rong; Su, Wen-Jing; Guan, Hai-Hong; Wang, Gang; Gao, Tian-Wen; Han, Hua; Liu, Yu-Feng

    2007-02-01

    Fungal infections have been increasing and life-threatening in recent years, but host immune responses, especially the humoral immunity, to fungi have not been fully understood. In the present study, we report that natural antibodies from unimmunized mice bind to Candida albicans. We established a monoclonal natural antibody, 3B4, which recognized a surface antigen located at germ tubes of C. albicans. The 3B4 antibody protected mice from C. albicans-induced death in passive immunization, by mechanisms involving suppressing germ tube formation and modulating phagocytosis. Interestingly, 3B4 also bound to a self-antigen keratin. To further study the generation and anti-C. albicans activities of natural antibodies in vivo, we constructed a mu chain transgenic mouse (TgV(H)3B4) using the V(H) gene from 3B4. TgV(H)3B4 had elevated serum anti-keratin/C. albicans IgM, and were resistant to C. albicans infections. Analyses of B cell development showed that in TgV(H)3B4, B cells secreting the anti-keratin/C. albicans antibodies were enriched in the B1 B cell compartment. Our findings reveal an important role of keratin-reactive natural antibodies in anti-C. albicans immune responses, and suggest that keratin may function in selecting B cells into the B1 B cell compartment, where natural antibodies are made to fight fungal infections. PMID:16925788

  2. Chain Teleportation

    OpenAIRE

    Lee, Chien-er

    2004-01-01

    By means of the idea of measurements on the crossed space-time nonlocal observables, we extend the mechanism for the two-way quantum teleportation to the chain teleportation among N spatially separated spin-1/2 systems. Since in the process only the local interactions are used, the microcausality is automatically satisfied.

  3. MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

    Directory of Open Access Journals (Sweden)

    Duarte Keila M.R.

    2001-01-01

    Full Text Available Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV isolated in Brazil. One antibody (8G7G2 isotyped as IgG2b (kappa light chain showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV. It can be used in identification of tomato mosaic virus (ToMV.

  4. What is a Responsible Supply Chain?

    Directory of Open Access Journals (Sweden)

    Terje Ingebrigt Vaaland

    2012-02-01

    Full Text Available The paper introduces the concept of responsible supply chain based on two dimensions, the core processes of a supply chain and the concept of corporate social responsibility (CSR. It is suggested that a responsible supply chain is achieved through manifested core values of the supply chain actors, strategies, and tactics. The paper further discusses the individual supply chain actors’ responsibility in securing a responsible supply chain beyond the actors’ direct control. A conceptual model and a definition of a responsible supply chain are offered. Our model not only provides structure to the extant research but also develops new constructs and relationships that improve the conceptualization of the responsible supply chain. The paper is based on a review of 81 research articles published between 2000 and 2010 in which elements of CSR and supply chain processes are included.

  5. The Involvement of the Intermediate Chain of Cytoplasmic Dynein in Binding the Motor Complex to Membranous Organelles of Xenopus Oocytes

    OpenAIRE

    Steffen, Walter; Karki, Sher; Vaughan, Kevin T.; Vallee, Richard B.; Holzbaur, Erika L F; Weiss, Dieter G.; Kuznetsov, Sergei A.

    1997-01-01

    Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could...

  6. Catch Chain

    OpenAIRE

    Talbert, Robert

    2010-01-01

    Catch Chain is a book of poems that traces the journey of a Corrections Officer who attempts to combat issues of isolation, inhumane treatment of inmates and societal rejection in jails by embarking upon a cross-country road trip. However, the same issues the officer initially wrestled with begin cropping up in different cities, on various highways and in a multitude of states. The excitement and adventure of the open road runs parallel to the recurring imprisonment of the guard's mind.

  7. Polynucleotides encoding anti-sulfotyrosine antibodies

    Science.gov (United States)

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  8. Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

    Science.gov (United States)

    Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

    2015-11-13

    Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. PMID:26396190

  9. Towards Intelligent Supply Chains

    DEFF Research Database (Denmark)

    Siurdyban, Artur; Møller, Charles

    2012-01-01

    applied to the context of organizational processes can increase the success rate of business operations. The framework is created using a set of theoretical based constructs grounded in a discussion across several streams of research including psychology, pedagogy, artificial intelligence, learning...... of deploying inapt operations leading to deterioration of profits. To address this problem, we propose a unified business process design framework based on the paradigm of intelligence. Intelligence allows humans and human-designed systems cope with environmental volatility, and we argue that its principles......, business process management and supply chain management. It outlines a number of system tasks combined in four integrated management perspectives: build, execute, grow and innovate, put forward as business process design propositions for Intelligent Supply Chains....

  10. Extended linear chain compounds

    CERN Document Server

    Linear chain substances span a large cross section of contemporary chemistry ranging from covalent polymers, to organic charge transfer com­ plexes to nonstoichiometric transition metal coordination complexes. Their commonality, which coalesced intense interest in the theoretical and exper­ imental solid state physics/chemistry communities, was based on the obser­ vation that these inorganic and organic polymeric substrates exhibit striking metal-like electrical and optical properties. Exploitation and extension of these systems has led to the systematic study of both the chemistry and physics of highly and poorly conducting linear chain substances. To gain a salient understanding of these complex materials rich in anomalous aniso­ tropic electrical, optical, magnetic, and mechanical properties, the conver­ gence of diverse skills and talents was required. The constructive blending of traditionally segregated disciplines such as synthetic and physical organic, inorganic, and polymer chemistry, crystallog...

  11. Antibody engineering & therapeutics, the annual meeting of the antibody society December 7–10, 2015, San Diego, CA, USA

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M.; Lund-Johansen, Fridtjof; Bradbury, Andrew R.M.; Carter, Paul J.; Melis, Joost P.M.

    2016-01-01

    ABSTRACT The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  12. The multiple readings of 'metaphor' in the classroom: co-construction of inferential chains As múltiplas leituras da 'metáfora' em sala de aula: co-construção de cadeias inferenciais

    Directory of Open Access Journals (Sweden)

    Mara Sophia Zanotto

    2010-01-01

    Full Text Available This paper is part of a research project whose central aim is to empirically investigate the multiple readings of 'metaphors' in literary texts. The methodology employed is interpretive and the main research technique is the 'Group-Think Aloud'. In this paper, the data discussed were generated by a group of readers engaged in the reading of 'The Pulverized Mountain', a poem by Drummond de Andrade. The analysis focuses on the interpretations of the final verses that, due to incongruities presented, constitute enigmas for the reader to decipher. Analysis has shown that the apparent data chaos and complexity has, in fact, an organization in inferential chains co-constructed by metonymic and metaphoric processes.Este trabalho faz parte do projeto de pesquisa que tem como objetivo central investigar empiricamente as múltiplas leituras de metáforas em textos literários. A metodologia é a interpretativista e a técnica principal de pesquisa é o 'Pensar Alto em Grupo'. Neste trabalho são discutidos os dados de um grupo de leitores lendo o poema 'A Montanha Pulverizada', de Drummond de Andrade. O foco da análise são as interpretações dos versos finais, que, devido às incongruências que apresentam, constituem enigmas para o leitor decifrar. A análise mostrou que o aparente caos e complexidade dos dados têm, de fato, uma organização em cadeias inferenciais co-construídas por processos metonímicos e metafóricos.

  13. Stationary Probability Vectors of Higher-order Markov Chains

    OpenAIRE

    Li, Chi-Kwong; Zhang, Shixiao

    2013-01-01

    We consider the higher-order Markov Chain, and characterize the second order Markov chains admitting every probability distribution vector as a stationary vector. The result is used to construct Markov chains of higher-order with the same property. We also study conditions under which the set of stationary vectors of the Markov chain has a certain affine dimension.

  14. Markov Chain Approximations to Singular Stable-like Processes

    OpenAIRE

    Xu, Fangjun

    2012-01-01

    We consider the Markov chain approximations for singular stable-like processes. First we obtain properties of some Markov chains. Then we construct the approximating Markov chains and give a necessary condition for weak convergence of these chains to singular stable-like processes.

  15. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    2016-01-01

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology. PMID:27236550

  16. Antibodies that bind complex glycosaminoglycans accumulate in the Golgi

    OpenAIRE

    Radic, Marko; Weigert, Martin G.; Khan, Salar N.; Han, Jiong; Kalinina, Olga; Luning Prak, Eline T.

    2013-01-01

    Light (L) chains that edit anti-DNA heavy (H) chains rescue B-cell development by suppressing DNA binding. However, exceptional editor L chains allow B cells to reach splenic compartments even though their B-cell receptors remain autoreactive. Such incompletely edited B cells express multireactive antibodies that accumulate in the Golgi and are released as insoluble, amyloid-like immune complexes. Here, we examine examples of incomplete editing from the analysis of variable to joining (VJ) ge...

  17. Recombinant approaches to IgG-like bispecific antibodies

    Institute of Scientific and Technical Information of China (English)

    Jonathan S MARVIN; Zhenping ZHU

    2005-01-01

    One of the major obstacles in the development of bispecific antibodies (BsAb)has been the difficulty of producing the materials in sufficient quality and quantity by traditional technologies, such as the hybrid hybridoma and chemical conjugation methods. In contrast to the rapid and significant progress in the development of recombinant BsAb fragments (such as diabody and tandem single chain Fv), the successful design and production of full length IgG-like BsAb has been limited. Compared to smaller fragments, IgG-like BsAb have long serum halflife and are capable of supporting secondary immune functions, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. The development of IgG-like BsAb as therapeutic agents will depend heavily on our research progress in the design of recombinant BsAb constructs (or formats) and production efficiency. This review will focus on recent advances in various recombinant approaches to the engineering and production of IgG-like BsAb.

  18. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  19. Effects of an amyloid-beta 1-42 oligomers antibody screened from a phage display library in APP/PS1 transgenic mice

    Science.gov (United States)

    Wang, Jianping; Li, Nan; Ma, Jun; Gu, Zhiqiang; Yu, Lie; Fu, Xiaojie; Liu, Xi; Wang, Jian

    2016-01-01

    We screened anti-Aβ1-42 antibodies from a human Alzheimer’s disease (AD) specific single chain variable fragment (scFv) phage display library and assessed their effects in APP/PS1 transgenic mice. Reverse transcription-PCR was used to construct the scFv phage display library, and screening identified 11A5 as an anti-Aβ1-42 antibody. We mixed 11A5 and the monoclonal antibody 6E10 with Aβ1-42 and administered the mixture to Sprague-Dawley rats via intracerebroventricular injection. After 30 days, rats injected with the antibody/ Aβ1-42 mixture and those injected with Aβ1-42 alone were tested on the Morris water maze. We also injected 11A5 and 6E10 into APP/PS1 transgenic mice and assessed the concentrations of Aβ in brain and peripheral blood by ELISA at 1-month intervals for 3 months. Finally we evaluated behavior changes in the Morris water maze. Rats injected with Aβ1-42 and mixed antibodies showed better performance in the Morris water maze than did rats injected with Aβ1-42 alone. In APP/PS1 transgenic mice, Aβ concentration was lower in the brains of the antibody-treated group than in the control group, but higher in the peripheral blood. The antibody-treated mice also exhibited improved behavioral performance in the Morris water maze. In conclusion, anti-Aβ1-42 antibodies (11A5) screened from the human scFv antibody phage display library promoted the efflux or clearance of Aβ1-42 and effectively decreased the cerebral Aβ burden in an AD mouse model. PMID:26820640

  20. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    Science.gov (United States)

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries. PMID:24423623

  1. A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

    OpenAIRE

    Trilling, Anke K.; Hans de Ronde; Linda Noteboom; Adèle van Houwelingen; Margriet Roelse; Saurabh K Srivastava; Willem Haasnoot; Maarten A Jongsma; Arend Kolk; Han Zuilhof; Jules Beekwilder

    2011-01-01

    BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis an...

  2. Heavy chain–only antibodies are spontaneously produced in light chain–deficient mice

    OpenAIRE

    Zou, Xiangang; Osborn, Michael J.; Bolland, Daniel J.; Smith, Jennifer A.; Corcos, Daniel; Hamon, Maureen; Oxley, David; Hutchings, Amanda; Morgan, Geoff; Santos, Fatima; Kilshaw, Peter J.; Michael J. Taussig; Corcoran, Anne E; Brüggemann, Marianne

    2007-01-01

    In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain–only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily...

  3. Detection of seral p53 antibody in patients with breast carcinoma by immuno-polymerase chain reation%PCR法检测乳腺癌患者血清抗p53蛋白抗体的研究

    Institute of Scientific and Technical Information of China (English)

    李洁

    2006-01-01

    目的为乳腺癌诊断提供血清学新方法.方法用免疫-聚合酶链反应(immuno-polymerase chain reanon,PCR)法检测血清抗p53蛋白(一种抑癌基因)抗体,酶免疫组化方法检测组织p53蛋白表达.结果乳腺癌患者血清抗p53蛋白抗体阳性率为39.5%,而非癌患者和正常人血清抗p53蛋白抗体均为阴性,乳腺癌患者血清中抗p53蛋白抗体显著高于非癌患者和正常人(P<0.01).p53蛋白阳性表达的乳腺癌患者抗p53蛋白抗体阳性率为64.2%,明显高于p53蛋白阴性表达组,血清p53抗体测定与p53蛋白表达密切相关(P<0.01).结论检测血清抗p53蛋白抗体是检测组织p53蛋白理想的替代工具,抗p53蛋白抗体可以作为乳腺癌血清学诊断新标志,用于乳腺癌的普查和早期诊断.

  4. Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases

    OpenAIRE

    Erster, Oran; Thomas, Jerry M; Hamzah, Juliana; Jabaiah, Abeer M.; Getz, Jennifer A.; Schoep, Tobias; Hall, Sejal S.; Ruoslahti, Erkki; Daugherty, Patrick S.

    2012-01-01

    As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded...

  5. Specificities of monoclonal antibodies against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis.

    OpenAIRE

    Huber-Lukac, M; Lüthy, P; Braun, D G

    1983-01-01

    Eight hybrid cell lines secreting monoclonal antibodies directed against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis were grown in BALB/c mice. Ascites fluids were collected, and the antibodies were purified by antigen-affinity chromatography. The specificity of each monoclonal antibody for the toxin and protoxin was established by the indirect enzyme-linked immunosorbent assay. All the antibodies consisted of gamma 1 heavy and kappa light chains. They were reac...

  6. Antiphospholipid Antibody Syndrome

    Science.gov (United States)

    ... page from the NHLBI on Twitter. What Is Antiphospholipid Antibody Syndrome? Antiphospholipid (AN-te-fos-fo-LIP-id) antibody ... weeks or months. This condition is called catastrophic antiphospholipid syndrome (CAPS). People who have APS also are at ...

  7. Antibodies Against Three Forms of Urokinase

    Science.gov (United States)

    Morrison, Dennis R.; Atassi, M. Zouhair

    2007-01-01

    Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

  8. Production and Characterization of Recombinant Light Chain and Carboxyterminal Heavy Chain Fragments of Tetanus Toxin

    OpenAIRE

    Yousefi, Mehdi; Khosravi-Eghbal, Roya; Hemmati, Azam; Shokri, Fazel

    2013-01-01

    Background Light chain (LC) and heavy chain carboxyterminal subdomain (HCC) fragments are the most important parts of tetanus neurotoxin (TeNT) which play key roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a prokaryotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies. Methods LC and HCC gene segments were amplified from Clostridium tetani genomic DNA by PCR, clone...

  9. The antibody mining toolbox

    OpenAIRE

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba

    2013-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

  10. RECOMBINANT ANTI-TENASCIN ANTIBODY CONTRUCTS

    International Nuclear Information System (INIS)

    The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr ?-particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

  11. 基于港口服务供应链的高职港口物流专业群建设实践%Practice of Construction of Higher Vocational Port Logistics Specialty Group Based on Port Service Supply Chain

    Institute of Scientific and Technical Information of China (English)

    靳荣利; 王贵斌; 陈艳玲

    2014-01-01

    阐述了基于港口服务供应链构建港口物流专业群的基本内涵,并对基于港口服务供应链的高职港口物流专业群建设的实践进行了探讨。%In this paper, we proposed the basic connotation of the construction of the port logistics specialty group based on the port service supply chain and introduced the practice of the construction of the higher vocational port logistics specialty group.

  12. Hepatitis A virus antibody

    International Nuclear Information System (INIS)

    A description is presented of a radioimmunoassay designed to prove the presence of the antibody against the hepatitis A virus (HA Ab, anti-Ha) using an Abbott HAVAB set. This proof as well as the proof of the antibody against the nucleus of the hepatitis B virus is based on competition between a normal antibody against hepatitis A virus and a 125I-labelled antibody for the binding sites of a specific antigen spread all over the surface of a tiny ball; this is then indirect proof of the antibody under investigation. The method is described of reading the results from the number of impulses per 60 seconds: the higher the titre of the antibody against the hepatitis A virus in the serum examined, the lower the activity of the specimen concerned. The rate is reported of incidence of the antibody against the hepatitis A virus in a total of 68 convalescents after hepatitis A; the antibody was found in 94.1%. The immunoglobulin made from the convalescents' plasma showed the presence of antibodies in dilutions as high as 1:250 000 while the comparable ratio for normal immunoglobulin Norga was only 1:2500. Differences are discussed in the time incidence of the antibodies against the hepatitis A virus, the antibodies against the surface antigen of hepatitis B, and the antibody against the nucleus of the hepatitis V virus. (author)

  13. Antecedents and Dimensions of Supply Chain Robustness

    OpenAIRE

    Durach, Christian F.; Wieland, Andreas; Machuca, Jose A.D.

    2015-01-01

    Purpose – The purpose of this paper is to provide groundwork for an emerging theory of supply chain robustness – which has been conceptualized as a dimension of supply chain resilience – through reviewing and synthesizing related yet disconnected studies. The paper develops a formal definition of supply chain robustness to build a framework that captures the dimensions, antecedents and moderators of the construct as discussed in the literature. Design/methodology/approach – The...

  14. Construction of Human ScFv Phage Display Library against Ovarian Tumor

    Institute of Scientific and Technical Information of China (English)

    XIA Jinsong; BI Hao; YAO Qin; QU Shen; ZONG Yiqiang

    2006-01-01

    In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

  15. Chain reaction

    International Nuclear Information System (INIS)

    Chain Reaction is a work of recent American political history. It seeks to explain how and why America came to depend so heavily on its experts after World War II, how those experts translated that authority into political clout, and why that authority and political discretion declined in the 1970s. The author's research into the internal memoranda of the Atomic Energy Commission substantiates his argument in historical detail. It was not the ravages of American anti-intellectualism, as so many scholars have argued, that brought the experts back down to earth. Rather, their decline can be traced to the very roots of their success after World War II. The need to over-state anticipated results in order to garner public support, incessant professional and bureaucratic specialization, and the sheer proliferation of expertise pushed arcane and insulated debates between experts into public forums at the same time that a broad cross section of political participants found it easier to gain access to their own expertise. These tendencies ultimately undermined the political influence of all experts. (author)

  16. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111In, 67Ga and 131I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  17. Construction of Green Supply Chain Management System and Implementation Steps of China's Phytochemistry Enterprise%中国植提企业绿色供应链管理体系构建与实施步骤研究

    Institute of Scientific and Technical Information of China (English)

    胡雪松; 杜娅

    2011-01-01

    分析了中国植提企业实施绿色供应链管理的必要性,构建了绿色供应链管理职能体系,提出了实施绿色供应链管理的步骤.%The necessity of implementing green supply chain management in China's phytochemistry enterprises is analyzed and a functions system of green supply chain management is built, and the implementation steps are proposed.

  18. Expression and assembly of a fully active antibody in algae

    Science.gov (United States)

    Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

    2003-01-01

    Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

  19. The Invariance Principle for p-(→θ) Chain

    Institute of Scientific and Technical Information of China (English)

    Di He HU; Zheng Yan XIAO

    2007-01-01

    There are two parts in this paper. In the first part we construct the Maxkov chain in random environment(MCRE), the skew product Markov chain and p-(→θ) chain from a random transition matrix and a two-dimensional probability distribution, and in the second part we prove that the invariance principle for p-(→θ) chain, a more complex non-homogeneons Markov chain, is true under some reasonable conditions. This result is more powerful.

  20. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J;

    2016-01-01

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform...... well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby...... reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples...

  1. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    Directory of Open Access Journals (Sweden)

    Kristensen Peter

    2009-01-01

    Full Text Available Abstract Background Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.

  2. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

    Science.gov (United States)

    Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet, Jaume; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva, Baldo; Sanz, Laura; Blanco, Francisco J; Alvarez-Vallina, Luis

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIE(XVIII) modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas. PMID:27345490

  3. Crater Chains

    Science.gov (United States)

    2003-01-01

    [figure removed for brevity, see original site] The large crater at the top of this THEMIS visible image has several other craters inside of it. Most noticeable are the craters that form a 'chain' on the southern wall of the large crater. These craters are a wonderful example of secondary impacts. They were formed when large blocks of ejecta from an impact crashed back down onto the surface of Mars. Secondaries often form radial patterns around the impact crater that generated them, allowing researchers to trace them back to their origin.Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.Image information: VIS instrument. Latitude 19.3, Longitude 347.5 East (12.5 West). 19 meter/pixel resolution.

  4. Antibody phage display applications for nuclear medicine imaging and therapy

    International Nuclear Information System (INIS)

    Antibody-based constructs genetically engineered from genes of diverse origin provide a remarkable opportunity to develop functional molecular imaging techniques and specific molecular targeted radionuclide therapies. Phage display libraries of antibody fragment genes can be used to select antibody-based constructs that bind any chosen epitope. A large naive human antibody-based library was used to illustrate binding of antibody constructs to a variety of common and unique antigens. Antibody-based libraries from hybridoma cells, lymphocytes from immunized humans or from mice and human antibody repertoires produced in transgenic mice have also been described. Several orders of magnitude of affinity enhancement can be achieved by random or site specific mutations of the selected binding peptide domains of the scFv. Affinities (Kd) as high as 10-11 M (10 pM) for affinity-matured scFv have been documented. Such gene libraries thus offer an almost limitless variety of antibody-based molecular binding peptide modules that can be used in creative ways for the construction of new targeting agents for functional or molecular imaging and therapy

  5. Antibody phage display applications for nuclear medicine imaging and therapy

    Energy Technology Data Exchange (ETDEWEB)

    Winthrop, M.D.; Denardo, G.L.; Denardo, S.J. [Sacramento Univ. of California Davis Medical Center, Sacramento, CA (United States). Dept. of Internal Medicine, Div. of Radiodiagnosis and Terapy

    2000-09-01

    Antibody-based constructs genetically engineered from genes of diverse origin provide a remarkable opportunity to develop functional molecular imaging techniques and specific molecular targeted radionuclide therapies. Phage display libraries of antibody fragment genes can be used to select antibody-based constructs that bind any chosen epitope. A large naive human antibody-based library was used to illustrate binding of antibody constructs to a variety of common and unique antigens. Antibody-based libraries from hybridoma cells, lymphocytes from immunized humans or from mice and human antibody repertoires produced in transgenic mice have also been described. Several orders of magnitude of affinity enhancement can be achieved by random or site specific mutations of the selected binding peptide domains of the scFv. Affinities (K{sub d}) as high as 10{sup -}11 M (10 pM) for affinity-matured scFv have been documented. Such gene libraries thus offer an almost limitless variety of antibody-based molecular binding peptide modules that can be used in creative ways for the construction of new targeting agents for functional or molecular imaging and therapy.

  6. Cysteinylation of a monoclonal antibody leads to its inactivation.

    Science.gov (United States)

    McSherry, Troy; McSherry, Jennifer; Ozaeta, Panfilo; Longenecker, Kenton; Ramsay, Carol; Fishpaugh, Jeffrey; Allen, Steven

    2016-01-01

    Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism. PMID:27050640

  7. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    International Nuclear Information System (INIS)

    CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance

  8. A simple vector system to improve performance and utilisation of recombinant antibodies

    Directory of Open Access Journals (Sweden)

    Vincent Karen J

    2006-12-01

    Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

  9. Graphs: Associated Markov Chains

    OpenAIRE

    Murthy, Garimella Rama

    2012-01-01

    In this research paper, weighted / unweighted, directed / undirected graphs are associated with interesting Discrete Time Markov Chains (DTMCs) as well as Continuous Time Markov Chains (CTMCs). The equilibrium / transient behaviour of such Markov chains is studied. Also entropy dynamics (Shannon entropy) of certain structured Markov chains is investigated. Finally certain structured graphs and the associated Markov chains are studied.

  10. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans; Dyrberg, T

    1990-01-01

    affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the...... beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact......, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides....

  11. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  12. Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

    Directory of Open Access Journals (Sweden)

    Subhash G. Vasudevan

    2012-02-01

    Full Text Available Domain III of the dengue virus envelope protein (EDIII, aa295-395 has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library ( > 10 billion independent clones. Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9 that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.

  13. Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

    Institute of Scientific and Technical Information of China (English)

    Heng-ChuanXIA; Wei-GuoHU; Xin-XiuYANG; FengLI; Zu-ChuanZHANG

    2004-01-01

    Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have beenwidely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIPrecently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin thatcan selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6,6F8, 4H10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have beensuccessfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies areIgG1, IgG1, IgG1, IgG1, IgG2a and IgM. Their affinity constants were determined to be 1.42×108, 2.71×108,8.72×07, 2.06×l08, 1.36×108 and 1.51×108 M-1 in a sequent order, measured by non-competitive ELISA.The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel,which consisted ofa monoclonal antibody 4H 10 and Sepharose 4B, was prepared and used to purify Moschatinfrom pumpkin seeds crude extract.

  14. Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

    Institute of Scientific and Technical Information of China (English)

    Heng-Chuan XIA; Wei-Guo HU; Xin-Xiu YANG; Feng LI; Zu-Chuan ZHANG

    2004-01-01

    Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have beenwidely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIPrecently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin thatcan selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6,6F8, 4H 10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have beensuccessfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies areIgG1, IgG1, IgG1, IgG1, IgG2a and IgM. Their affinity constants were determined to be 1.42×108, 2.71×108,8.72×107, 2.06×108, 1.36×108 and 1.51×108M-1 in a sequent order, measured by non-competitive ELISA.The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel,which consisted ofa monoclonal antibody 4H10 and Sepharose 4B, was prepared and used to purify Moschatinfrom pumpkin seeds crude extract.

  15. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies*

    Science.gov (United States)

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-01-01

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies. PMID:25737448

  16. Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

    Science.gov (United States)

    Acero Sánchez, Josep Ll; Fragoso, Alex; Joda, Hamdi; Suárez, Guillaume; McNeil, Calum J; O'Sullivan, Ciara K

    2016-07-01

    The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot. PMID:27220524

  17. Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies*

    OpenAIRE

    Kalantari-Dehaghi, Mina; Chen, Yumay; Deng, Wu; Chernyavsky, Alex; Marchenko, Steve; Wang, Ping H.; Grando, Sergei A.

    2013-01-01

    Background: Previous studies suggested that mitochondrial antibodies contribute to pemphigus vulgaris (PV).Results: PV sera elicited mitochondrial damage, and mitochondria-protecting drugs exhibited protective effect in cell culture and mouse skin.Conclusion: PV antibodies altered O2 respiration, disrupted electron transfer chain, and increased reactive oxygen species.Significance: Results provide the mechanism of therapeutic action and justify the use of mitochondria-protecting drugs in PV.T...

  18. Expression and assembly of a fully active antibody in algae

    OpenAIRE

    Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

    2003-01-01

    Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene...

  19. Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus.

    Science.gov (United States)

    Wang, Man; Zhang, Yan; Li, Benqiang; Zhu, Jianguo

    2015-06-01

    Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis. PMID:25910693

  20. Engineering antibody therapeutics.

    Science.gov (United States)

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  1. Radiology's value chain.

    Science.gov (United States)

    Enzmann, Dieter R

    2012-04-01

    A diagnostic radiology value chain is constructed to define its main components, all of which are vulnerable to change, because digitization has caused disaggregation of the chain. Some components afford opportunities to improve productivity, some add value, while some face outsourcing to lower labor cost and to information technology substitutes, raising commoditization risks. Digital image information, because it can be competitive at smaller economies of scale, allows faster, differential rates of technological innovation of components, initiating a centralization-to-decentralization technology trend. Digitization, having triggered disaggregation of radiology's professional service model, may soon usher in an information business model. This means moving from a mind-set of "reading images" to an orientation of creating and organizing information for greater accuracy, faster speed, and lower cost in medical decision making. Information businesses view value chain investments differently than do small professional services. In the former model, producing a better business product will extend image interpretation beyond a radiologist's personal fund of knowledge to encompass expanding external imaging databases. A follow-on expansion with integration of image and molecular information into a report will offer new value in medical decision making. Improved interpretation plus new integration will enrich and diversify radiology's key service products, the report and consultation. A more robust, information-rich report derived from a "systems" and "computational" radiology approach will be facilitated by a transition from a professional service to an information business. Under health care reform, radiology will transition its emphasis from volume to greater value. Radiology's future brightens with the adoption of a philosophy of offering information rather than "reads" for decision making. Staunchly defending the status quo via turf wars is unlikely to constitute a

  2. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  3. Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis.

    Science.gov (United States)

    Xu, Chuang; Chen, Yuanyuan; Chang, Qiaocheng; Xia, Cheng; Yang, Wei; Zhang, Hongyou

    2015-08-01

    MAdCAM-1 plays an important role in mediating immune response and inflammation. This study aimed to express and purify a fusion protein of MAdCAM-1 in prokaryotic cells and to prepare rat anti-bovine MAdCAM-1 polyclonal antibodies. Prokaryotic expression vector pGEX-4T-1-MAdCAM-1 and pET-28a-MAdCAM-1 were constructed, respectively. The above plasmids were transformed into BL21 Escherichia coli strain. These recombinant strains were induced by IPTG and identified by Western blot analysis and SDS-PAGE. Wistar rats were immunized with recombinant protein (pET-28a-MAdCAM-1) emulsified with Freund's adjuvant, and antibody titers were measured by indirect ELISA. Antibody titers reached the highest value (1:128,000) after the third immunization. Western blot showed that rat anti-bovine MAdCAM-1 polyclonal antibody can not only recognize recombinant MAdCAM-1 protein expressed in E. coli but also recognizes natural MAdCAM-1 protein extracted from bovine tissues. However, commercial anti-mouse MAdCAM-1 monoclonal antibodies did not recognize the recombinant MAdCAM-1 protein or natural protein, which indicated no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis showed that MAdCAM-1 expression was limited in mammary lymphoid nodes of subclinical mastitis in dairy cows. We speculate that MAdCAM-1 expression is inconsistent in different periods of the dairy cows. The successful preparation of rat anti-bovine MAdCAM-1 polyclonal antibody and its preliminary application in dairy cows provide the foundation for further study of the mechanism of anti-inflammation of MAdCAM-1 in dairy cows with subclinical mastitis. PMID:26301930

  4. Construction of Customization Marketing Service Model for Agricultural Product Cold Chain Logistics Enterprises%农产品冷链物流企业的定制营销服务模型构建

    Institute of Scientific and Technical Information of China (English)

    原惠群; 舒文定

    2012-01-01

    在借鉴第三方物流企业定制营销模式的基础上,通过介绍农产品冷链物流企业实现定制化营销服务的依据与模式,对农产品冷链物流企业定制化营销中的各个模块进行了具体的说明,为我国农产品冷链物流企业开展满足客户个性化需求的定制营销提供一定的借鉴。%China agricultural cold chain has a vast market,a trend of diversification and personalization for the customer demand.According to the personalized demand of customers,the customization marleting is increasingy respected and widely recognized by enterprises and customers.Therefore,it is particularly important to further analyse customization behavior of agricultural cold chain logistics enterprise.Based on the reference to the customization marketing model of the third party logistics enterprise,this paper describes each module of customization marketing in details,through introducing the basis and mode of achieving customized marketing services for the agricultural product cold chain logistics enterprises.It is hoped that a certain reference can be provided to our country agricultural cold chain logistics enterprises for implementing the customization marketing to meet customer demand for individual requirements.

  5. Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries

    Science.gov (United States)

    Tung, Chao-Ping; Chen, Ing-Chien; Yu, Chung-Ming; Peng, Hung-Pin; Jian, Jhih-Wei; Ma, Shiou-Hwa; Lee, Yu-Ching; Jan, Jia-Tsrong; Yang, An-Suei

    2015-01-01

    Broadly neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information. PMID:26456860

  6. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    Science.gov (United States)

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  7. [Construction and panning of scFv phage display library against recombinant interleukin 4 receptor].

    Science.gov (United States)

    Yang, Guangyong; Guo, Haitao; Liu, Ximing; He, Guangzhi; Tian, Weiyi; Cai, Kun; Wang, Ping; Wang, Wenjia

    2016-06-01

    Objective To construct the recombinant human interleukin 4 receptor (rhIL-4R) single-chain Fv (scFv) antibody library by phage display technique to obtain the anti-IL-4R scFv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rhIL-4R. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv by splice overlap extension PCR (SOE-PCR). Both scFv and the pCANTAB5E vector were respectively double-digested with restriction endonuclease Sfi I and Not I, connected with T4 ligase, and then transformed into the competent cells E.coli TG1; it was cultured in medium to obtain the phage scFv antibody library; after three rounds of enrichment and panning, the specific antigen scFv with high affinity was selected for the sequencing. Results After three rounds of panning, we obtained a diversity of approximately 2×10(8) anti-rhIL-4R scFv antibody library. Sequencing analysis of one positive clone showed that the anti-rhIL-4R scFv was 741 bp and coded 247 amino acids. The analysis of VBASE2 database indicated that VH and VL gene sequences of anti-rhIL-4R protein all had three complementarity determining regions and four backbone areas.Conclusion The anti-rhIL-4R scFv was obtained from the scFv antibody library. PMID:27371853

  8. Genetically delivered antibody protects against West Nile virus

    OpenAIRE

    Pereboev, Alexander; Borisevich, Viktoriya; Tsuladze, George; Shakhmatov, Mikhail; Hudman, Deborah; Kazachinskaia, Elena; Razumov, Ivan; Svyatchenko, Viktor; Loktev, Valery; Yamshchikov, Vladimir

    2007-01-01

    Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capa...

  9. On Markov Chains and Filtrations

    OpenAIRE

    Spreij, Peter

    1997-01-01

    In this paper we rederive some well known results for continuous time Markov processes that live on a finite state space.Martingale techniques are used throughout the paper. Special attention is paid to the construction of a continuous timeMarkov process, when we start from a discrete time Markov chain. The Markov property here holds with respect tofiltrations that need not be minimal.

  10. Preparation of single chain variable fragment of MG7 mAb by phage display technology

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Yong-Zhan Nie; Dai-Ming Fan; Xue-Yong Zhang

    2001-01-01

    AIM To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS mRNA was isolated from MG7-producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOⅢ of highly expressing MG7binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigenbinding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE. RESULTS The VH, VL and ScFv DNAs were about 340bp,320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATO Ⅲ cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATO Ⅲ cells.SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32. CONCLUSION The MG7 ScFv was successfully produced by phage antibody technology, which may

  11. Rational design of humanized dual-agonist antibodies.

    Science.gov (United States)

    Zhang, Yong; Liu, Yan; Wang, Ying; Schultz, Peter G; Wang, Feng

    2015-01-14

    The ultralong heavy chain complementarity determining region 3 (CDR3H) of bovine antibody BLV1H12 folds into a novel "stalk-knob" structural motif and has been exploited to generate novel agonist antibodies through replacement of the "knob" domain with cytokines and growth factors. By translating this unique "stalk-knob" architecture to the humanized antibody trastuzumab (referred to hereafter by its trade name, Herceptin, Genentech USA), we have developed a versatile approach to the generation of human antibody agonists. Human erythropoietin (hEPO) or granulocyte colony-stimulating factor (hGCSF) was independently fused into CDR3H, CDR2H, or CDR3L of Herceptin using an engineered "stalk" motif. The fusion proteins express in mammalian cells in good yields and have similar in vitro biological activities compared to hEPO and hGCSF. On the basis of these results we then generated a bi-functional Herceptin-CDR fusion protein in which both hEPO and hGCSF were grafted into the heavy- and light-chain CDR3 loops, respectively. This bi-functional antibody fusion exhibited potent EPO and GCSF agonist activities. This work demonstrates the versatility of the CDR-fusion strategy for generating functional human antibody chimeras and provides a novel approach to the development of multi-functional antibody-based therapeutics. PMID:25494484

  12. Linking Single Domain Antibodies that Recognize Different Epitopes on the Same Target.

    Science.gov (United States)

    Glaven, Richard H; Anderson, George P; Zabetakis, Dan; Liu, Jinny L; Long, Nina C; Goldman, Ellen R

    2012-01-01

    Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb. PMID:25585631

  13. Affinity purification of antibodies

    Science.gov (United States)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  14. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  15. Production Of Human Antibodies

    Science.gov (United States)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  16. RBC Antibody Screen

    Science.gov (United States)

    ... the baby is Rh-positive and the mother's antibody status is negative for anti-D, the mother is given additional RhIG. This test also may be used to help diagnose autoimmune-related hemolytic anemia ... when a person produces antibodies against his or her own RBC antigens. This ...

  17. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...

  18. Contributions of Conventional and Heavy-Chain IgG to Immunity in Fetal, Neonatal, and Adult Alpacas▿

    OpenAIRE

    Daley-Bauer, L. P.; Purdy, S. R.; Smith, M. C.; Gagliardo, L. F.; Davis, W.C.; Appleton, J.A.

    2010-01-01

    In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes t...

  19. Health supply chain management.

    Science.gov (United States)

    Zimmerman, Rolf; Gallagher, Pat

    2010-01-01

    This chapter gives an educational overview of: * The actual application of supply chain practice and disciplines required for service delivery improvement within the current health environment. * A rationale for the application of Supply Chain Management (SCM) approaches to the Health sector. * The tools and methods available for supply chain analysis and benchmarking. * Key supply chain success factors. PMID:20407173

  20. Silicone chain extender

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a silicone chain extender, more particularly a chain extender for silicone polymers and copolymers, to a chain extended silicone polymer or copolymer and to a functionalized chain extended silicone polymer or copolymer, to a method for the preparation thereof and...

  1. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  2. 5. technical conference 'car body chain': modern concepts in car body engineering - design, construction, materials, projecting, fabrication, quality, recycling; 5. Fachtagung 'Prozesskette Karosserie': Konzepte der Neuzeit im Karosseriebau - Design, Konstruktion, Werkstoffeinsatz, Planung, Fertigung, Qualitaet

    Energy Technology Data Exchange (ETDEWEB)

    Westkaemper, E.; Schraft, R.D. (eds.)

    2000-07-01

    The conference discussed the optimisation of the car body process chain and the integration of new technologies. Cost reduction and lightweight construction with new materials were the key issues. This proceedings volume comprises 19 papers, all of which can be found in this database. [German] Fuer die Tagung 'Konzepte der Neuzeit im Karosseriebau' steht die Aufgabe bevor, die Prozesskette zu optimieren und zu beschleunigen sowie neue Technologien einzubinden. Die wichtigsten Ziele, die Senkung der Herstellungskosten und die Reduzierung der Fahrzeugmasse durch den Einsatz von innovativen Werkstoffen, werden an diesen Objekten dargestellt. In dieser Tagung sind 19 Referate abgehalten und befinden sich in dem vorliegenden Band. (orig.)

  3. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. PMID:27214604

  4. Risk Migration In Supply Chain Inventory Financing Service

    OpenAIRE

    Zheng Qin; Xiaochao Ding

    2011-01-01

    Inventory financing affects the risks of both for banks and supply chain companies. Traditionally, supply chain research focus more on material flow than financial. We construct a supply chain financing risk-information migration model (RMM). In this model, we discussed the preconditions to adopt inventory financing when the enterprises are facing cash constraints. And we simulated the whole operate of supply chain and bank behavior with Matlab. The simulation result shows if loan conditions ...

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

    Science.gov (United States)

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

  6. The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

    OpenAIRE

    Feige, Matthias J.; Gräwert, Melissa A.; Marcinowski, Moritz; Hennig, Janosch; Behnke, Julia; Ausländer, David; Herold, Eva M.; Peschek, Jirka; Castro, Caitlin D.; Flajnik, Martin; Hendershot, Linda M.; Sattler, Michael; Groll, Michael; Buchner, Johannes

    2014-01-01

    Sharks are among the evolutionary oldest living organisms with an immune system that possesses a number of elements similar to ours, including antibodies. In this article, we present structural insights into one of the most ancient antibodies, shedding light on the molecular evolution of the immune system and the structural features of heavy chain-only antibodies. Sharks enrich urea in their blood to prevent osmotic loss of water in the marine environment. Urea, however, denatures proteins if...

  7. Radiolabelled antibodies in imaging

    International Nuclear Information System (INIS)

    Recent technological advances make it possible to produce pure (monoclonal) antibodies in unlimited quantities without the need for continuous immunization of animals and to label these antibodies with a variety of radionuclides which can be traced by single-photon computed tomography. An outline review of the state of the art is presented, with particular reference to the imaging of myocardial infarcts and to tumour imaging studies using labelled monoclonal antibodies (sup(99m)Tc and 125I). Lengthy bibliography. (U.K.)

  8. 农产品电子商务供应链体系构建研究%Study on Construction of Farm Produce E-commerce Supply Chain System

    Institute of Scientific and Technical Information of China (English)

    刘丽华

    2012-01-01

    In this paper, we established an advanced and high-efficiency supply chain system and innovated the mode of the production and sales of agricultural products in a bid to satisfy the demand of the consumers for fresh green food.%电子商务时代,企业应立足农产品物流,应用电子商务,专注供应链管理,构建先进高效的供应链体系,创新农产品产销模式,以满足消费者对新鲜绿色食品的需求.

  9. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  10. An abstract approach to Loewner's chains

    CERN Document Server

    Arosio, Leandro; Hamada, Hidetaka; Kohr, Gabriela

    2010-01-01

    We present a new abstract construction of Loewner chains in one and several variables which holds also on hyperbolic complex manifolds and prove that there is essentially a one-to-one correspondence between evolution families of order $d$ and Loewner chains of the same order. As a consequence we obtain a Loewner-Kufarev generalized PDE on complex manifolds. We apply such results to the study of biholomorphic mappings from the unit ball of $\\C^n$.

  11. HIV Antibody Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  12. Antinuclear antibody panel

    Science.gov (United States)

    ... blood may be due to: Chronic liver disease Collagen vascular disease Drug-induced lupus erythematosus Myositis (inflammatory muscle disease) ... Saunders; 2011:chap 51. Read More Antibody Arthritis Collagen vascular disease Drug-induced lupus erythematosus Liver disease Scleroderma Systemic ...

  13. PRODUCTION OF MONOCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    TOLKOVA E.S.

    2015-01-01

    Full Text Available The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  14. PRODUCTION OF MONOCLONAL ANTIBODIES

    OpenAIRE

    TOLKOVA E.S.

    2015-01-01

    The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  15. Immunoglobulins, antibody repertoire and B cell development.

    Science.gov (United States)

    Butler, J E; Zhao, Y; Sinkora, M; Wertz, N; Kacskovics, I

    2009-03-01

    Swine share with most placental mammals the same five antibody isotypes and same two light chain types. Loci encoding lambda, kappa and Ig heavy chains appear to be organized as they are in other mammals. Swine differ from rodents and primates, but are similar to rabbits in using a single VH family (VH3) to encode their variable heavy chain domain, but not the family used by cattle, another artiodactyl. Distinct from other hoofed mammals and rodents, Ckappa:Clambda usage resembles the 1:1 ratio seen in primates. Since IgG subclasses diversified after speciation, same name subclass homologs do not exist among swine and other mammals unless very closely related. Swine possess six putative IgG subclasses that appear to have diversified by gene duplication and exon shuffle while retaining motifs that can bind to FcgammaRs, FcRn, C1q, protein A and protein G. The epithelial chorial placenta of swine and the precosial nature of their offspring have made piglets excellent models for studies on fetal antibody repertoire development and on the postnatal role of gut colonization, maternal colostrum and neonatal infection on the development of adaptive immunity during the "critical window" of immunological development. This chapter traces the study of the humoral immune system of this species through its various eras of discovery and compiles the results in tables and figures that should be a useful reference for educators and investigators. PMID:18804488

  16. Thermoresponsive Agarose Based Microparticles for Antibody Separation.

    Science.gov (United States)

    Ooi, Huey Wen; Ketterer, Benedikt; Trouillet, Vanessa; Franzreb, Matthias; Barner-Kowollik, Christopher

    2016-01-11

    We report the development of thermoresponsive 4-mercaptoethylpyridine (MEP)-based chromatographic microsphere based resins for antibody separation that show switchable release abilities by adsorbing immunoglobulins at 40 °C and releasing the proteins at 5 °C. The thermoswitchable release properties were introduced to the porous resins by the grafting of linear poly(N-isopropylacrylamide) (PNIPAM) chains synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, which were modified to possess MEP end functionalities. Adsorption of γ-globulins as a model antibody on the shortest PNIPAM-MEP (3 kDa) grafted microparticles display binding capacities of up to 20 g L(-1) at 40 °C and a significant decrease in binding capacity to less than 2.5 g L(-1) at 5 °C. By switching the temperature to 5 °C, the release of bound γ-globulins is shown to be as high as 90%. The effects of polymer chain length on the binding capacity are studied in detail and found to be critical as they influence the density of MEP functionalities on the particle surfaces. PMID:26626821

  17. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.

    Science.gov (United States)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J; Helmsing, Saskia; Meier, Doris; Hust, Michael; Schröder, Christoph; Bertinetti, Daniela; Winter, Gerhard; Pardes, Khalid; Funk, Mia; Vala, Andrea; Giese, Nathalia; Herberg, Friedrich W; Dübel, Stefan; Hoheisel, Jörg D

    2016-09-25

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis. PMID:26709003

  18. Conformational Isomerism Can Limit Antibody Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Debler, E.W.; Muller, R.; Hilvert, D.; Wilson, I.A.

    2009-05-14

    Ligand binding to enzymes and antibodies is often accompanied by protein conformational changes. Although such structural adjustments may be conducive to enzyme catalysis, much less is known about their effect on reactions promoted by engineered catalytic antibodies. Crystallographic and pre-steady state kinetic analyses of antibody 34E4, which efficiently promotes the conversion of benzisoxazoles to salicylonitriles, show that the resting catalyst adopts two interconverting active-site conformations, only one of which is competent to bind substrate. In the predominant isomer, the indole side chain of Trp{sup L91} occupies the binding site and blocks ligand access. Slow conformational isomerization of this residue, on the same time scale as catalytic turnover, creates a deep and narrow binding site that can accommodate substrate and promote proton transfer using Glu{sup H50} as a carboxylate base. Although 34E4 is among the best catalysts for the deprotonation of benzisoxazoles, its efficiency appears to be significantly limited by this conformational plasticity of its active site. Future efforts to improve this antibody might profitably focus on stabilizing the active conformation of the catalyst. Analogous strategies may also be relevant to other engineered proteins that are limited by an unfavorable conformational pre-equilibrium.

  19. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  20. Construction management

    CERN Document Server

    Pellicer, Eugenio; Teixeira, José C; Moura, Helder P; Catalá, Joaquín

    2014-01-01

    The management of construction projects is a wide ranging and challenging discipline in an increasingly international industry, facing continual challenges and demands for improvements in safety, in quality and cost control, and in the avoidance of contractual disputes. Construction Management grew out of a Leonardo da Vinci project to develop a series of Common Learning Outcomes for European Managers in Construction. Financed by the European Union, the project aimed to develop a library of basic materials for developing construction management skills for use in a pan-European context. Focused exclusively on the management of the construction phase of a building project from the contractor's point of view, Construction Management covers the complete range of topics of which mastery is required by the construction management professional for the effective delivery of new construction projects. With the continued internationalisation of the construction industry, Construction Management will be required rea...

  1. Spin chain sigma models with fermions

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez, Rafael [Institut de Physique, Universite de Neuchatel, Breguet 1, CH-2000 Neuchatel (Switzerland)]. E-mail: rafael.hernandez@cern.ch; Lopez, [Departamento de Fisica Teorica C-XI and Instituto de Fisica Teorica C-XVI, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

    2004-11-01

    The complete one-loop planar dilatation operator of N=4 supersymmetric Yang-Mills is isomorphic to the hamiltonian of an integrable PSU(2,2 vertical bar 4) quantum spin chain. We construct the non-linear sigma models describing the continuum limit of the SU(1|3) and SU(2|3) sectors of the complete N=4 chain. We explicitly identify the spin chain sigma model with the one for a superstring moving in AdS{sub 5} x S{sup 5} with large angular momentum along the five-sphere. (author)

  2. Decay Chain Deduction of Uranium Fission Products.

    Science.gov (United States)

    Guo, Huiping; Tian, Chenyang; Wang, Xiaotian; Lv, Ning; Ma, Meng; Wei, Yingguang

    2016-07-01

    Delayed gamma spectrum is the fingerprint of uranium materials in arms control verification technology. The decay chain is simplified into basic state linear chain and excitation state linear chain to calculate and analyze the delayed gamma spectra of fission products. Formulas of the changing rule for nuclide number before and after zero-time are deduced. The C program for calculating the delayed gamma ray spectra data is constructed, and related experiments are conducted to verify this theory. Through analysis of the delayed gamma counts of several nuclides, the calculated results are found to be consistent with experimental values. PMID:27218290

  3. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2011-02-15

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  4. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    International Nuclear Information System (INIS)

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2±1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  5. 武汉软件服务外包产业价值链构建与价值提升策略%Industry Value Chain Construction and Value Promotion Strategy for Wuhan Software and Service Outsourcing

    Institute of Scientific and Technical Information of China (English)

    王梅源; 郑双怡; 熊兰兰

    2015-01-01

    产业价值链同时包含产业链和价值增值两个因素,有助于寻找与探索产业价值提升路径和策略。从软件生命周期、软件产品与服务、企业业务特征3个视角构建了软件服务外包产业价值链,针对武汉市产业发展现状和存在的问题,提出现阶段武汉软件服务外包产业应走一条“以技术升级和能力升级为主,创新研发与全球运营并重,产品升级与服务升级并重”的价值提升路径,并从区位优势、产业转移、在岸外包、龙头企业、创新发展5个方面提出产业价值提升的对策和建议。%Industry value chain includes two factors of the industrial chain and the value added at the same time, helps us find and explore the path and strategy of industry value upgrade. We build the software and service outsourcing industry value chain from three perspectives of the software life cycle, software products and services and enterprise business char‐acteristics. In view of the present situation and the existed question of Wuhan industry development, put forward a sug‐gestion that at present stage Wuhan software and service outsourcing industry must go a path of upgrade industries, that is "to upgrade technology and capability is given priority to, innovation development and global operations, product upgra‐ding and service upgrading pay equal attention to". We put forward the corresponding countermeasures and suggestions for industry value enhancement from five aspects of location advantage, industrial transfer, onshore outsourcing, leading en‐terprise, innovation and development.

  6. Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.

    Directory of Open Access Journals (Sweden)

    Shradha Bagaria

    Full Text Available Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

  7. Cloning murine antibody V-genes with non-degenerate primers and conversion to a recombinant antibody format.

    Science.gov (United States)

    Bialon, Magdalena; Schellenberg, Ludmila; Herzog, Nicolas; Kraus, Stefan; Jörißen, Hannah; Fischer, Rainer; Stein, Christoph; Nähring, Jörg; Barth, Stefan; Püttmann, Christiane

    2014-12-01

    Monoclonal antibodies are produced in cultured hybridoma cell lines, but these cells tend to be unstable; it is therefore necessary to rescue the corresponding genetic information. Here we describe an improved method for the amplification of antibody variable gene (V-gene) information from murine hybridoma cells using a panel of specific, non-degenerate primers. This primer set allows sequences to be rescued from all murine V-genes, except the lambda light chain genes, which rarely contribute to murine immune diversity. We tested the primers against a range of antibodies and recovered specific amplification products in all cases. The heavy and light chain variable regions were subsequently joined by a two-step cloning strategy or by splice overlap extension PCR. PMID:25545205

  8. 湖北省农副产品冷链物流网络构建研究%Construction of Farm and Sideline Products Cold Chain Logistics Network in Hubei

    Institute of Scientific and Technical Information of China (English)

    旷玉玲; 海峰; 傅俊; 水璐; 方威

    2012-01-01

    将冷链物流网络从纵向上划分为冷链物流需求网络、冷链物流功能网络和冷链物流目标客户网络三个子网络.根据湖北省农副产品的发展现状,结合湖北省“两圈一带”的战略思路,提出了湖北省构建农副产品冷链物流网络的具体步骤、方法和建议.%This paper divides the cold chain logistics network vertically into three sub-systems, namely, demand network, function network and objective customer network. In view of the development of the farm and sideline products industry in Hubei and the strategic guideline of "two circles and one belt", it proposes some specfic procedures, measures and suggestions.

  9. COFFEE COMMODITY CHAIN

    OpenAIRE

    Tine Olsen; Brett Inder

    2008-01-01

    To explain the value added along the coffee commodity chain we propose and estimate a theoretical model of the coffee commodity chain. The theoretical model consists of four markets and five agents in the coffee commodity chain and predicts that prices in the coffee commodity chain move together but are also influenced by income, technology and production. A vector error correction model is used to test the theoretical predictions. In addition to the theoretical conclusions the empirical mode...

  10. The Global Value Chain

    DEFF Research Database (Denmark)

    Sørensen, Olav Jull

    The conference paper aims to develop the global value chain concept by including corporate internal value adding activities and competition to the basic framework in order to turn the global value chain into a strategic management tool......The conference paper aims to develop the global value chain concept by including corporate internal value adding activities and competition to the basic framework in order to turn the global value chain into a strategic management tool...

  11. PRODUCTION OF A HUMAN RECOMBINANT ANTIBODY AGAINST SEROTYPE A CANDIDA ALBICANS

    Directory of Open Access Journals (Sweden)

    A A. Jafari

    2005-07-01

    Full Text Available After using 3 different generations of antibodies including human and non-human hyperimmune sera, monoclonal antibodies and chimeric antibodies, more recently a newer approach has been developed in which the antibody genes are cloned directly from a patient peripheral B-lymphocytes and expressed in a host like E. coli. In this study the Candida albicans serotype A (NCTC 3153 mannan was purified using a modified Fehling method and used for selection of human recombinant antibody from a C. albicans phage antibody library. After four rounds of affinity selecting (panning, 2 predominant clones were chosen by DNA fingerprinting and ELISA. A 248 amino acid DNA fragment coding for anti-C. albicans mannan scFv was sequenced and cloned in a pBAD-TOPO cloning vector to produce a soluble and phage free antibody. The analysis of antibody sequences by V base Index (DNAPLOT confirmed the human antibody origin with the VH4 family in V segment of heavy variable chain and VL3 (Lambda 3 in J segment of the light variable chain. This antibody fragment was purified using immobilized metal affinity chromatography and inmmunoblotted as a 31kDa recombinant protein.

  12. Antibody structural modeling with prediction of immunoglobulin structure (PIGS)

    DEFF Research Database (Denmark)

    Marcatili, Paolo; Olimpieri, Pier Paolo; Chailyan, Anna; Tramontano, Anna

    2014-01-01

    Antibodies (or immunoglobulins) are crucial for defending organisms from pathogens, but they are also key players in many medical, diagnostic and biotechnological applications. The ability to predict their structure and the specific residues involved in antigen recognition has several useful...... applications in all of these areas. Over the years, we have developed or collaborated in developing a strategy that enables researchers to predict the 3D structure of antibodies with a very satisfactory accuracy. The strategy is completely automated and extremely fast, requiring only a few minutes (∼10 min on...... average) to build a structural model of an antibody. It is based on the concept of canonical structures of antibody loops and on our understanding of the way light and heavy chains pack together....

  13. Antibody structural modeling with prediction of immunoglobulin structure (PIGS)

    KAUST Repository

    Marcatili, Paolo

    2014-11-06

    © 2014 Nature America, Inc. All rights reserved. Antibodies (or immunoglobulins) are crucial for defending organisms from pathogens, but they are also key players in many medical, diagnostic and biotechnological applications. The ability to predict their structure and the specific residues involved in antigen recognition has several useful applications in all of these areas. Over the years, we have developed or collaborated in developing a strategy that enables researchers to predict the 3D structure of antibodies with a very satisfactory accuracy. The strategy is completely automated and extremely fast, requiring only a few minutes (~10 min on average) to build a structural model of an antibody. It is based on the concept of canonical structures of antibody loops and on our understanding of the way light and heavy chains pack together.

  14. Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

    Institute of Scientific and Technical Information of China (English)

    Mei-Yun LIU; Wei HAN; Yan-Li DING; Tian-Hong ZHOU; Rui-Yang TIAN; Sheng-Li YANG; Hui LIU; Yi GONG

    2005-01-01

    B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

  15. Chain-Chain Based Routing Protocol

    Directory of Open Access Journals (Sweden)

    Samia A Ali

    2011-05-01

    Full Text Available Wireless sensor network (WSN is an emerging technology for monitoring physical world. WSNs consist of large numbers of sensor nodes operated by battery mostly in harsh environment. Thus energy conservation is a primary issue for organization of these sensor nodes. Another crucial issue is the data delivery time by sensor nodes to the sink node, especially in Military, medical fields, and security monitoring systems where minimum delay is desirable. Number of protocols has been proposed in the literature for routing. One of such protocols is the cluster based routing protocol LEACH (low energy adaptive clustering hierarchy. LEACH protocol organizes WSN into a set of clusters and a periodic voting for cluster head is performed in order to be evenly distributed among all the sensors of the WSN. This periodical cluster head voting in LEACH, however, consumes an amount of non-negligible energy and other resources. For energy conservation, PEGASIS (power efficient gathering in sensor information systems a near optimal chain-based protocol has been proposed, however, it is faced with the challenge of long delay for the transmitted data. Another routing protocol called CCM (Chain-Cluster based Mixed routing, which is mainly a hybrid of LEACH and PEGASIS is proposed, the consumed energy increases as network size increases. In this paper, we propose an efficient routing protocol called CCBRP (Chain-Chain based routing protocol, it achieves both minimum energy consumption and minimum delay. The CCBRP protocol mainly divides a WSN into a number of chains (Greedy algorithm is used to form each chain as in PEGSIS protocol and runs in two phases. In the first phase, sensor nodes in each chain transmit data to their chain leader nodes in parallel. In the second phase, all chain leader nodes form a chain (also, using Greedy algorithm and choose randomly a leader node then all chain leader nodes send their data to this chosen leader node. This chosen leader node

  16. Tumor cell-selective apoptosis induction through targeting of KV10.1 via bifunctional TRAIL antibody

    Directory of Open Access Journals (Sweden)

    Pardo Luis A

    2011-09-01

    Full Text Available Abstract Background The search for strategies to target ion channels for therapeutic applications has become of increasing interest. Especially, the potassium channel KV10.1 (Ether-á-go-go is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins. Methods We designed a single-chain antibody against an extracellular region of KV10.1 (scFv62 and fused it to the human soluble TRAIL. The KV10.1-specific scFv62 antibody -TRAIL fusion protein was expressed in CHO-K1 cells, purified by chromatography and tested for biological activity. Results Prostate cancer cells, either positive or negative for KV10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in KV10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking KV10.1 expression. In co-cultures with KV10.1-positive cancer cells the fusion protein also induced apoptosis in bystander KV10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander. Conclusions KV10.1 represents a novel therapeutic target for cancer. We could design a strategy that selectively kills tumor cells based on a KV10.1-specific antibody.

  17. Discrete Quantum Markov Chains

    CERN Document Server

    Faigle, Ulrich

    2010-01-01

    A framework for finite-dimensional quantum Markov chains on Hilbert spaces is introduced. Quantum Markov chains generalize both classical Markov chains with possibly hidden states and existing models of quantum walks on finite graphs. Quantum Markov chains are based on Markov operations that may be applied to quantum systems and include quantum measurements, for example. It is proved that quantum Markov chains are asymptotically stationary and hence possess ergodic and entropic properties. With a quantum Markov chain one may associate a quantum Markov process, which is a stochastic process in the classical sense. Generalized Markov chains allow a representation with respect to a generalized Markov source model with definite (but possibly hidden) states relative to which observables give rise to classical stochastic processes. It is demonstrated that this model allows for observables to violate Bell's inequality.

  18. Gushing metal chain

    Science.gov (United States)

    Belyaev, Alexander; Sukhanov, Alexander; Tsvetkov, Alexander

    2016-03-01

    This article addresses the problem in which a chain falls from a glass from some height. This phenomenon demonstrates a paradoxical rise of the chain over the glass. To explain this effect, an initial hypothesis and an appropriate theory are proposed for calculating the steady fall parameters of the chain. For this purpose, the modified Cayley's problem of falling chain given its rise due to the centrifugal force of upward inertia is solved. Results show that the lift caused by an increase in linear density at the part of chain where it is being bent (the upper part) is due to the convergence of the chain balls to one another. The experiments confirm the obtained estimates of the lifting chain.

  19. Usability Constructs

    DEFF Research Database (Denmark)

    Hertzum, Morten; Clemmesen, Torkil; Hornbæk, Kasper Anders Søren;

    2007-01-01

    Whereas research on usability predominantly employs universal definitions of the aspects that comprise usability, people experience their use of information systems through personal constructs. Based on 48 repertory-grid interviews, this study investigates how such personal constructs are affecte...

  20. Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin.

    Science.gov (United States)

    Sakamoto, Seiichi; Taura, Futoshi; Pongkitwitoon, Benyakan; Putalun, Waraporn; Tsuchihashi, Ryota; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-04-01

    A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv. PMID:20217398