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Sample records for chain antibody constructs

  1. 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

  2. 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

  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. Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

    Institute of Scientific and Technical Information of China (English)

    Dao-Feng Yang; Hui-Fen Zhu; Zhi-Hua Wang; Guan-Xin Shen; De-Ying Tian

    2005-01-01

    AIM: To construct fusion protein of a single-chain antibody(scFv) against transferrin receptor (TfR) with alkalinephosphatase (AP).METHODS: The VH-linker-VL, namely scFv gene, wasprepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by SfiⅠ and NotⅠ, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E. coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and NotⅠ restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E. coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity.CONCLUSION: We have successfully prepared the antihuman TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

  7. Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

    Institute of Scientific and Technical Information of China (English)

    刘喜富; 萧飒; 顾征; 王勇; 张卫国; 陈艾; 林晴; 黄华梁; 孙健; 陈润生; 沈倍奋; 陈兴

    1997-01-01

    Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

  8. 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

  9. 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-...... for combating HER2+ breast cancer. © 2013 by Tabriz University of Medical Sciences.......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......-binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems...

  10. Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human beta-glucuronidase

    NARCIS (Netherlands)

    Haisma, HJ; Brakenhoff, RH; Van der Meulen-Muileman, I.H.; Pinedo, HM; Boven, E

    1998-01-01

    We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human beta-glucuronidase by recombinant DNA technology. The sequences encoding the murine monoclonal antibody 323/A3 light- and heavy-chain variable genes were joined by a

  11. 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

  12. Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Li Wang; Yu-Ling Zheng; Ru Ma; Bao-Li Wang; Ai-Guang Guo; Yong-Qiang Jiang

    2005-01-01

    AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

  13. Construction and Analysis of Three-dimensional Graphic Model of Single-chain Fv Derived from an Anti-human Placental Acidic Isoferritin Monoclonal Antibody by Computer

    Institute of Scientific and Technical Information of China (English)

    ZHOU Chun; SHEN Guanxin; ZHU Huifen; YANG Jing; ZHANG Yue; FENG Jiannan; SHEN Beifen

    2000-01-01

    A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

  14. 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

  15. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging and radioimmumotheraphy. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, N.K.V.; Larson, S.M.

    1993-11-01

    For the past several years, we have studied the anti-G{sub D2} murine monoclonal antibody, 3F8, in radiolabeled form, for diagnosis and therapy of neuroblastoma. The targeting properties of this antibody/antigen system are exceptional, with uptakes consistently in the highest range of reported results for in vivo human studies. The radioiodinated antibody 3F8 is now used by us as our criteria for diagnosis and staging of advanced neuroblastoma. This antibody is showing considerable promise also in our Phase I trials in Stage 4 neuroblastoma, and major responses are being seen at current dose level, with manageable marrow toxicity, but no limiting organ toxicity.

  16. 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

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

    NARCIS (Netherlands)

    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 a

  18. 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...

  19. 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.

  20. 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.

  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. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  3. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  4. 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...

  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. Quality in Construction- A Supply Chain Perspective

    DEFF Research Database (Denmark)

    Koch, Christian; Larsen, Casper Schultz

    2006-01-01

    in the delivery network, using operation management approaches. The paper presents case study work done in Danish construction. The method was observation of work at the construction site followed by interviews with actors backwards upstream the supply chain to the origin of the failure. The building project...... followed generated 160 failures over a three month observation period. The economic consequences are calculated to 8% of the production costs. The analysis of relations in the supply network showed that most of the failures were generated in the knowledge stream and then occasionally transformed...

  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. 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.

  9. 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

  10. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    1999-01-01

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  11. 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

  12. Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

    Science.gov (United States)

    He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

    2015-03-01

    Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

  13. 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.

  14. 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.

  15. 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.

  16. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  17. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    Science.gov (United States)

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  18. 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

  19. Formalizing the Process of Constructing Chains of Lexical Units

    Directory of Open Access Journals (Sweden)

    Grigorij Chetverikov

    2015-06-01

    Full Text Available Formalizing the Process of Constructing Chains of Lexical Units The paper investigates mathematical aspects of describing the construction of chains of lexical units on the basis of finite-predicate algebra. Analyzing the construction peculiarities is carried out and application of the method of finding the power of linear logical transformation for removing characteristic words of a dictionary entry is given. Analysis and perspectives of the results of the study are provided.

  20. 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.

  1. Roles of supply chain management in construction

    NARCIS (Netherlands)

    Vrijhoef, R.; Koskela, L.

    1999-01-01

    Supply chain management (SCM) is a concept that has flourished in manufacturing, originating from Just-In-Time (JIT) production and logistics. Today, SCM represents an autonomous managerial concept, although still largely dominated by logistics. SCM endeavors to observe the entire scope of the suppl

  2. 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

  3. 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.

  4. 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.

  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. Construction and screening of phage display single chain antibody library against histidine-rich protein Ⅱ of Plasmodium falciparum%抗恶性疟原虫富含组氨酸蛋白Ⅱ单链抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    侯云霞; 董文其; 徐伟文; 王萍; 陈白虹; 李明

    2001-01-01

    Objective To construct phage display single-chain antibody fragments (scFvs) library against histidine-rich protein Ⅱ (HRP-Ⅱ) of Plasmodium falciparum and select specific scFvs of anti- HRP-Ⅱ for the purpose of malaria diagnosis. Method The genes of variable fragments of heavy chain (VH) and light chain (VL) were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein. The VH and VL genes were then assembled by the method of splicing overlapping extension and cloned into phagemid vector pCANTAB 5E. The scFv phage antibodies were expressed at the surface of the phage after the rescue by helper phage M13K07. HRP- Ⅱ protein was used as antigenic reagent for panning and screening. Results The total RNA from the spleen cells was isolated, and cDNA obtained and VH and VL gene regions amplified using PCR. The VH and VL gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1 Escherichia coli. The repertoire of the phage antibody was about 106. After panning and screening, 8 positive clones expressed scFv antibodies which were specific for HPR-Ⅱ as demonstrated by ELISA. Conclusion Phage display technology can be used as a powerful tool in making scFv antibodies which have the potential to be used as reagents in the diagnosis and therapy of malaria.%目的构建抗恶性疟原虫富含组氨酸蛋白Ⅱ(Histidine- rich protein II, HRP-II)单链抗体库,并筛选出阳性克隆。方法用噬菌体抗体库技术构建抗恶性疟原虫HRP-Ⅱ单链抗体库,并以HRP-Ⅱ为靶抗原对该库进行了三轮"亲和吸附-援救-感染扩增"的富集,挑取单菌落筛选并鉴定阳性克隆。结果获得目的基因并成功构建抗恶性疟原虫HRP-Ⅱ的单链抗体库,库容为106,并从中筛选出8株阳性克隆。结论噬菌体抗体库技术具有高效的筛选性能,抗 HRP-Ⅱ单链抗体的制备为其在恶性疟的免疫快速诊断方法中的应用奠定了基础。

  7. γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitrophenyl 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 kcat = 198 minγ1 and the rate enhancement kcat/kuncat = 122 419. When 0.16 mg·mLγ1 EP6 was preincubated in vitro with 0.132 mmol·Lγ1 (220 g·kgγ1 = 1.1×LD95) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP6 could prolong the latent time of spasm and death when mice were passively immunized with EP6 intravenously 15 min before 1×LD95 of soman challenge. These results demonstrate that EP6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

  8. 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

  9. 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...

  10. Effectiveness of Practicing Supply Chain Management in Construction Site

    Directory of Open Access Journals (Sweden)

    Mamter S.

    2014-01-01

    Full Text Available Construction Supply chain management comprised of the network of organization involved in the different processes and activities which produce the material, components and services that come together to design, procurement and deliver a building. It also consists of different organizations involved in the construction process including client/owner, designer, contractor, subcontractor and suppliers. This paper shall present on the implementation of supply chain management in construction and the effectiveness of practicing SCM in construction site. A field study is done from the viewpoint of contractor and consultant then analysed by using average index methods and presented in a statistical analysis. From the analysis, it reveals that effectiveness of practicing the SCM give a lot of good performances and granted benefits to contractor. The statistical analysis produced first ranking effectiveness of SCM is can minimize waste of material and labor for construction project.

  11. Antibody elbow angles are influenced by their light chain class

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R; Zemla, A; Wilson, I; Rupp, B

    2006-01-12

    We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa-chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195{sup o}) elbow angles. This apparent hyperflexibility of lambda-chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is also described.

  12. 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...

  13. Production of a human single-chain variable fragment antibody against esophageal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ming-Yan Xu; Xiao-Hu Xu; Geng-Zhen Chen; Xiao-Ling Deng; Jonathan Li; Xiao-Jun Yu; Mei-Zhen Chen

    2004-01-01

    AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×106 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup Ⅳ of immunoglobulin, and variable light (VL) gene from the κchain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  19. Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

    Science.gov (United States)

    Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

    2011-11-15

    Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

  20. Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

    Science.gov (United States)

    Prechl, J; Tchorbanov, A; Horváth, A; Baiu, D C; Hazenbos, W; Rajnavölgyi, E; Kurucz, I; Capel, P J; Erdei, A

    1999-05-01

    Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines. PMID:10408376

  1. Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

    Science.gov (United States)

    Prechl, J; Tchorbanov, A; Horváth, A; Baiu, D C; Hazenbos, W; Rajnavölgyi, E; Kurucz, I; Capel, P J; Erdei, A

    1999-05-01

    Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

  2. 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.

  3. 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.

  4. Heavy Chain Only Antibodies: A New Paradigm in Personalized HER2+ Breast Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Seyed Moein Moghimi

    2013-01-01

    Full Text Available 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-binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems for combating HER2+ breast cancer.

  5. 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

  6. 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.

  7. 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

  8. 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.

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

    Science.gov (United States)

    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

    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 VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > 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

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

    Science.gov (United States)

    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

    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

  11. 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

  12. 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

  13. 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

  14. Improving properties of llama heavy chain antibodies using in silico analysis

    NARCIS (Netherlands)

    Lutje Hulsik, D.

    2009-01-01

    Microbicides offer a promising way to prevent HIV infection in developing countries. Llama heavy chain antibody fragments (VHHs) that neutralize HIV are an ideal candidate for usage as active microbicide component. A VHH is the smallest intact antigen binding domain which allows it to reach for anti

  15. Improved expression of single-chain antibodies in Ustilago maydis.

    Science.gov (United States)

    Sarkari, Parveen; Reindl, Michèle; Stock, Janpeter; Müller, Olaf; Kahmann, Regine; Feldbrügge, Michael; Schipper, Kerstin

    2014-12-10

    To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

  16. 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.

  17. Generation and characterization of a single-chain anti-EphA2 antibody.

    Science.gov (United States)

    Goldgur, Yehuda; Susi, Petri; Karelehto, Eveliina; Sanmark, Hanna; Lamminmäki, Urpo; Oricchio, Elisa; Wendel, Hans-Guido; Nikolov, Dimitar B; Himanen, Juha P

    2014-12-01

    Recombinant antibody phage library technology provides multiple advantages, including that human antibodies can be generated against proteins that are highly conserved between species. We used this technology to isolate and characterize an anti-EphA2 single-chain antibody. We show that the antibody binds the antigen with 1:1 stoichiometry and has high specificity for EphA2. The crystal structure of the complex reveals that the antibody targets the same receptor surface cavity as the ephrin ligand. Specifically, a lengthy CDR-H3 loop protrudes deep into the ligand-binding cavity, with several hydrophobic residues at its tip forming an anchor-like structure buried within the hydrophobic Eph pocket, in a way similar to the ephrin receptor-binding loop in the Eph/ephrin structures. Consequently, the antibody blocks ephrin binding to EphA2. Furthermore, it induces apoptosis and reduces cell proliferation in lymphoma cells lines. Since Ephs are important mediators of tumorigenesis, such antibodies could have applications both in research and therapy.

  18. 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

  19. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  20. 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)

  1. 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 risk consequences of this finding as 42 % of deliverers in Soroti District reported drinking raw milk, compared with 15 % in Gulu District (P 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

  2. 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.

  3. 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...

  4. Construction of a Single-chain Fv Antibody against Epidermal Growth Factor Receptor and Its Antitumor Activity%抗表皮生长因子受体的单链抗体ER(Fv)的构建及其抗肿瘤活性研究

    Institute of Scientific and Technical Information of China (English)

    盛唯瑾; 尚伯杨; 苗庆芳; 甄永苏

    2011-01-01

    OBJECTIVE To express single-chain Fv (scFv) antibody against epidermal growth factor receptor (EGFR) with Escherichia coli, and study its antitumor activity. METHODS Recombinant plasmids containing scFv gene fragment were constructed and transformed into competent E. Coli BL21(DE3)star?. The scFv antibody, ER(Fv) , was produced after IPT.G induction, and then purified with Ni ion affinity chromatography. ELISA was used for measuring the binding efficiency of ER(Fv) to EGFR, and flow cy-tometry was applied to assay the binding activity between ER(Fv-LDP) and tumor cells. The cytotoxicity of ER(Fv) against tumor cells in vitro was assessed by MTT assay, and the tumor inhibitory effect in vivo was observed in nude mice bearing human epidermoid carcinoma A431 xenograft. RESULTS ER(Fv) with His-tag was produced in the form of inclusion bodies, and was purified with Ni ion affinity chromatography. Finally 10 mg of purified protein were obtained from 1 L of culture medium. ER( Fv) possessed powerfulbinding affinity for EGFR with an affinity constant (Ka)of 4. 0 × 107 L · Mol-1. ER( Fv) protein bound specifically to the membrane of EGFR-overexpressing tumor cells with an dissociation constant (Kd) of 10-7 mol · L-1. ER( Fv) showed potent cytotoxicity against tumor cells. In A431 xenografts, ER( Fv) presented antitumor efficacy. The tumor growth inhibition rates by ER( Fv) at dose of 5, 10 mg · Kg-1, and 20 mg · Kg-1 were 43. 9% , 46. 7% and 54. 8% , respectively. CONCLUSION The preparation of ER( Fv) with EGFR binding affinity and antitumor activity provides the basis for the development of EGFR-targeted antibody-based drugs.%目的 利用大肠杆菌表达抗表皮生长因子受体(EGFR)的单链抗体并对其抗肿瘤活性进行研究.方法 构建含有抗EGFR单链抗体基因片段的重组质粒,IPTG诱导大肠杆菌表达单链抗体ER(Fv),用Ni离子亲和柱纯化单链抗体.ELISA测定ER(Fv)与纯EGFR抗原的结合能力;流式细胞术分析ER(Fv)

  5. Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating.

    Science.gov (United States)

    Baek, Du-San; Kim, Yong-Sung

    2014-03-28

    Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

  6. 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.

  7. 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

  8. 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.

  9. 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 ...

  10. 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.

  11. 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

  12. Cloning,sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

    Institute of Scientific and Technical Information of China (English)

    ZHANGJINSONG; MINGYEH

    1994-01-01

    The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Constructing Chains of Enablers for Alternative Economic Futures

    DEFF Research Database (Denmark)

    Hull Kristensen, Peer

    2016-01-01

    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...

  19. 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

  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. 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)

  2. 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

  3. 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

  4. 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 ...

  5. 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...

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody.

    Directory of Open Access Journals (Sweden)

    Timo Heidt

    Full Text Available BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111Indium ((111In via bifunctional DTPA ( = (111In-LIBS/(111In-control. Autoradiography after incubation with (111In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2, 4010 ± 630 DLU/mm(2 and 4520 ± 293 DLU/mm(2 produced a significantly higher ligand uptake compared to (111In-control (2101 ± 76 DLU/mm(2, 1181 ± 96 DLU/mm(2 and 1866 ± 246 DLU/mm(2 indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2 vs. 17390 ± 7470 DLU/mm(2; P<0.05. These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01. CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of

  11. 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.

  12. Evaluating the modus operandi of construction supply chains using organisation control theory

    Directory of Open Access Journals (Sweden)

    Milind Jagtap

    2015-07-01

    Full Text Available Supply chains are omnipresent. However, the modus operandi of the construction supply chainis not clearly established in the literature. This might be attributable to the character of construction projects and the structure of the construction industry. Formal and informal control mechanisms are well established in retail and manufacturing supply chains which is evident in improved product performance. However, there is a paucity of research on the construction supply chain especially at identifying the interplay of control mechanisms and their relationship with project performance. In the case of large and complex construction projects, the client-contractor relationship requires input control, behaviour control and output control for successful project delivery. In the light of organisation control theory and the existing literature on construction supply chains, this study evaluates the modus operandi of the client-contractor relationship based on three control mechanisms: input control (project risk and reward power, and intra-project communication, behaviour control (opportunism and output control (project performance using a structural equation model. A survey data of 258 construction professionals working on construction projects in India was collected. The study findings reveal that input control, in terms of project risk and reward power, and intraproject communication, largely influence behaviour control in terms of opportunism. However, behaviour controls do not directly affect output control in terms of project performance; rather, a direct effect of the input control mechanism of output control is particularly evident.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. AN EMERGENT FORM OF CLIENT-LED SUPPLY CHAIN GOVERNANCE IN UK CONSTRUCTION: CLANS

    Directory of Open Access Journals (Sweden)

    Tennant, Stuart

    2012-04-01

    Full Text Available Drawing inspiration and legitimacy from the traditions of organisational theory and in particular alternative mechanisms of organisational governance, the research explores an emergent, clan form of client-led supply chain governance in UK construction. Clan mechanisms of organisational governance are described as hybrid structures of exchange, neither pro-market nor organisational hierarchy. Not to be mistaken with alternative mechanisms of exchange such as networks, clan forms of client-led supply chain management are readily distinguishable by their highly socialised marketplace, enduring relationships and community of practice. A qualitative research strategy is adopted for this exploration of clan forms of client-led supply chain governance. Data collection uses semi-structured interviews, recorded, coded and analyzed. Participants include senior industry figures from a cross-section of construction stakeholder organisations, including client bodies, first tier service providers and construction contractors. In contrast to much of the prevailing work in construction supply chain management research, the findings draw specific attention to a hybrid form of organisational governance rarely discussed: namely clans. In light of challenging economic conditions, the recognition and potential contribution of clans as an alternative mechanism of governance is a timely and valuable contribution to the ongoing construction supply chain management debate.

  18. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

    Science.gov (United States)

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-01-01

    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  19. Antiproliferative and Apoptotic Effects of a Specific Antiprostate Stem Cell Single Chain Antibody on Human Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Foroogh Nejatollahi

    2013-01-01

    Full Text Available Prostate stem cell antigen (PSCA is a highly glycosylated cell surface protein which is overexpressed in several malignancies including prostate, pancreas, and urinary bladder cancers. Tumor suppression has been reported by anti-PSCA antibody. Small and high affinity single chain antibodies (scFv have been introduced as effective agents for cancer immunotargeting approaches. In the present study, we used a phage antibody display library of scFv and selected two antibodies against two immunodominant epitopes of PSCA by panning process. The reactivity of the scFvs for the corresponding epitopes was determined by phage ELISA. The binding specificity of antibodies to PSCA-expressing prostate cancer cell line, DU-145, was analyzed by flow cytometry. The antiproliferative and apoptotic induction effects were evaluated by MTT and Annexin-V assays, respectively. Results represented functional scFv C5-II which could bind specifically to DU-145 cells and significantly inhibited the proliferation of these cells (61% with no effect on PSCA-negative cells. The antibody also induced apoptosis in the PSCA expressing cells. The percentage of the apoptotic cells after 24 hrs of exposure to 500 scFv/cell was 33.80%. These results demonstrate that the functional anti-PSCA scFv C5-II has the potential to be considered as a new agent for targeted therapy of prostate cancer.

  20. Microinjection of antibodies to the calpactin I light chain in MDBK cells causes precipition of the cytoskeletal calpactin I complex without affecting the distribution of related proteins.

    Science.gov (United States)

    Glenney, J R

    1990-01-01

    The calpactin I complex is composed of two heavy chain (39K) and two light chain (11K) subunits. The heavy chain is a member of a protein family that includes lipocortins, endonexin and chromobindins while the light chain is a member of the S100 family (7 distinct members are known). We have found that the kidney epithelial cell line MDBK expresses four members of the heavy chain family and two members of the light chain protein family. Antibodies to the light chain of calpactin I were found to cause the precipitation of injected antibody together with the associated heavy chain without apparent effect on the distribution of related proteins. This suggests a differential targeting of various members of the calpactin heavy and light chain families even within the same cell.

  1. 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.

  2. Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

    Science.gov (United States)

    Torán, J L; Sánchez-Pulido, L; Kremer, L; del Real, G; Valencia, A; Martínez-A, C

    2001-01-01

    We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

  3. 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.

  4. Construction of human phage antibody library and screening for human monoclonal antibodies of amylin%人源性噬菌体抗体库的构建及抗amylin抗体的初步筛选

    Institute of Scientific and Technical Information of China (English)

    公倩; 李常颖; 畅继武; 朱铁虹

    2012-01-01

    AIM- To screen monoclonal antibodies to amyiin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. METHODS: The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I , Xho I and Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive donas were determined by Phage-ELISA analysis. RESULTS; A Fab phage antibody library with 0.8x 108 members was constructed with the efficacy of about 70%. DNA sequence analysis indicated VH gene belonged to VH3 gene family and Vλ gene belonged to the Vλ gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. CONCLUSION; The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.%目的:构建人源性噬菌体抗体库,从中筛选胰淀素(amylin)单克隆抗体(mAb),测定其特异性及抗原结合活性.方法:从正常健康人的外周血淋巴细胞中提取总RNA,用RT-PcR方法扩增人免疫球蛋白Fd段和轻链基因,构建噬菌体抗体库.酶切和PcR鉴定后,阳性克隆进行DNA测序分析.用人amylin抗原对抗体库进行筛选富集,将得到的阳性克隆进行Phage-ELISA鉴定,结果进行统计学分析.结果:最终构建的抗体库库容约为0.8×108,酶切鉴定显示有插入片段,抗体库重组率为70%.阳性克

  5. Improved Binding Activity of Antibodies against Major Histocompatibility Complex Class I Chain-Related Gene A by Phage Display Technology for Cancer-Targeted Therapy

    Directory of Open Access Journals (Sweden)

    Achara Phumyen

    2012-01-01

    Full Text Available Major histocompatibility complex class I chain-related gene A (MICA is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8. The 12 amino acid residues in the complementarity determining regions (CDRs on the V domain of the heavy chain CDR3 (HCDR3 of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3–7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21 were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.

  6. 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.

  7. Targeting Antibodies to Carbon Nanotube Field Effect Transistors by Pyrene Hydrazide Modification of Heavy Chain Carbohydrates

    Directory of Open Access Journals (Sweden)

    Steingrimur Stefansson

    2012-01-01

    Full Text Available Many carbon nanotube field-effect transistor (CNT-FET studies have used immobilized antibodies as the ligand binding moiety. However, antibodies are not optimal for CNT-FET detection due to their large size and charge. Their size can prevent ligands from reaching within the Debye length of the CNTs and a layer of charged antibodies on the circuits can drown out any ligand signal. In an attempt to minimize the antibody footprint on CNT-FETs, we examined whether pyrene hydrazide modification of antibody carbohydrates could reduce the concentration required to functionalize CNT circuits. The carbohydrates are almost exclusively on the antibody Fc region and this site-specific modification could mediate uniform antibody orientation on the CNTs. We compared the hydrazide modification of anti-E. coli O157:H7 polyclonal antibodies to pyrenebutanoic acid succinimidyl ester-coated CNTs and carbodiimide-mediated antibody CNT attachment. Our results show that the pyrene hydrazide modification was superior to those methods with respect to bacteria detection and less than 1 nM labeled antibody was required to functionalize the circuits.

  8. 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.

  9. 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

    , 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...... window chains already or about to having a commercial impact. Currently, it seems the greening of markets is beginning to affect the roles different companies play in the chain. We see a marked shift towards more sys-temic, smart eco-innovative solutions which fit well with nanotech opportunities......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...

  10. 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

    , 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...... window chains already or about to having a commercial impact. Currently, it seems the greening of markets is beginning to affect the roles different companies play in the chain. We see a marked shift towards more sys-temic, smart eco-innovative solutions which fit well with nanotech opportunities......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...

  11. 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

  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. 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

  14. 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.

  15. 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.

  16. 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

  17. 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 aci...

  18. 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.

  19. 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.

  20. 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

  1. Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

    Institute of Scientific and Technical Information of China (English)

    Zuanning Yuan; Minge Du; Yiwen Chen; Fei Dou

    2013-01-01

    Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli-gomers and constructed a naïve human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe-cifical y recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked immunosorbent assay demonstrated this antibody bound specifical y to human amyloid-beta 42 te-tramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc-cessful y constructed a human phage display library and screened a single-domain antibody that specifical y recognized amyloid-beta 42 oligomers.

  2. 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

  3. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1

    Science.gov (United States)

    Ghadge, Ghanashyam D.; Pavlovic, John; Koduvayur, Sujatha P.; Kay, Brian K.; Roos, Raymond P.

    2013-01-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. PMID:23607939

  4. 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.

  5. 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

  6. A bilateral and multi-issue negotiation framework to support a supply chain of construction industry

    Directory of Open Access Journals (Sweden)

    Fernando Schramm

    2013-12-01

    Full Text Available Any interaction involving individuals, whose objectives are conflicting with each other, may establish a negotiation process. In a negotiation, each party should develop his/her own strategy and, normally, a win-lose vision is frequently adopted. The main consequence of this behavior is a result, in which both parties lose, especially when the negotiation involves more than one aspect, such as negotiations resulting from purchases of material for construction industry, where aspects like price, quality and lead-time should be considered. Most of the negotiation involving construction industry adopts a win-lose vision; and, commonly, only the issue price is considered. The goal of this paper is to propose a framework to support negotiations between two parties (buyer and seller in the supply chain of construction industry. The combination of a win-win strategy with a multicriteria analysis produces a best compromise solution for both parties. A simulation of negotiation using realistic data is presented.

  7. Construction of antibody-like nanoparticles for selective protein sequestration in living cells

    Science.gov (United States)

    Liu, Yibin; Fang, Simin; Zhai, Junqiu; Zhao, Meiping

    2015-04-01

    We demonstrate the successful construction of fluorescently labeled magnetic antibody-like nanoparticles (ANPs) via a facile one-step surface-initiated in situ molecular imprinting approach over silica coated magnetite (Fe3O4@SiO2) core-shell nanocomposites. The as-prepared ANPs had a highly compact structure with an overall size of 83 +/- 5 nm in diameter and showed excellent aqueous dispersion stability. With the predetermined high specificity to the target protein and high biocompatibility, the ANPs enabled rapid, efficient, selective and optically trackable sequestration of target proteins within living cells. This work represents the first example of fully artificially engineered multifunctional ANPs for the intracellular protein-sequestration without disruption of the cells. The established approach may be further extended to generate ANPs for various proteins of interest and provide useful tools for related biological research and biomedical applications.We demonstrate the successful construction of fluorescently labeled magnetic antibody-like nanoparticles (ANPs) via a facile one-step surface-initiated in situ molecular imprinting approach over silica coated magnetite (Fe3O4@SiO2) core-shell nanocomposites. The as-prepared ANPs had a highly compact structure with an overall size of 83 +/- 5 nm in diameter and showed excellent aqueous dispersion stability. With the predetermined high specificity to the target protein and high biocompatibility, the ANPs enabled rapid, efficient, selective and optically trackable sequestration of target proteins within living cells. This work represents the first example of fully artificially engineered multifunctional ANPs for the intracellular protein-sequestration without disruption of the cells. The established approach may be further extended to generate ANPs for various proteins of interest and provide useful tools for related biological research and biomedical applications. Electronic supplementary information (ESI

  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. Serum major-histocompatibility-complex class Ⅰ-related chain A antibody detection for the evaluation of graft dysfunction in renal allograft recipients

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ming; LU Fu-ming; QU Lian-xi; HE Jun; YUAN Xiao-niao; GU Yong

    2011-01-01

    Background In addition to the well-known antibodies against human leukocyte antigens (HLA)-induced kidney-graft rejection, polymorphic major-histocompatibility-complex (MHC) class Ⅰ-related chain A (MICA) antigens can elicit antibodies and have been suggested to play a role in the antibody-mediated allograft rejection (AMR). We carded out a prospective study of MICA antibodies in post-renal transplant patients to determine the association between MICA antibodies, C4d staining, histological features, and graft outcome.Methods We tested 52 patients who had biopsy results due to graft dysfunction. The MICA antibodies in concurrent sera were determined by Luminex. All patients were followed up for one year after renal biopsy. The influence of antibody production on the function of graft was analyzed.Results Antibodies against MICA were positive in 15 out of the 52 patients (28.9%). The presence of MICA antibodies was associated with renal-allograft deterioration. During one-year follow-up, the estimated glomerular filtration rate (eGFR) decreased (24.0±3.4)% among recipients with anti-MICA antibodies. However, among recipients without anti-MICA antibodies, the eGFR has declined only (8.4+3.0)% (P=0.017). The association between C4d staining,histological features and MICA antibody production was found no significant difference.Conclusion Besides anti-HLA antibodies, the presence of post-transplant MICA antibody is associated with poor graft outcome and increases the risk of graft failure.

  10. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    Science.gov (United States)

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera. PMID:24001307

  11. ANTECEDENTS OF SUPPLIER RELATION QUALITY IN THE GHANAIAN CONSTRUCTION SUPPLY CHAIN

    Directory of Open Access Journals (Sweden)

    Bondinuba

    2016-06-01

    Full Text Available Effective and efficient management of suppliers within a supply chain is an essential requirement for improving organisational performance within construction companies. However, factors inherent within the supply chain of supplier-buyer exchanges such as culture, politics, dependence and trust may influence supplier relationship quality (SRQ. This research therefore seeks to identify the influence that these factors have upon SRQ in the Ghanaian construction industry and develop a conceptual framework that explains the interconnectivity between them. A literature review is used to develop a conceptual framework of the antecedents influencing supplier relationship quality. Primary ‘perception’ data obtained from 152 building material suppliers is used to test the proposed model using Partial Least Squares (PLS. Findings reveal that culture, politics, dependence and trust have a significant influence on relationship quality in supply chain collaborations amongst purchasers and suppliers of building materials. While politics has a strong influence on dependence, it also generates a negative influence on SRQ and trust. The research confirms the positive effect of trust and dependence in SRQ management and extends understanding of the influence of culture and politics. Practical implications suggest that managers of building material suppliers should focus upon building trust and dependence and be discouraged from over-reliance upon politics and political affiliations as a basis for long-term relationship building.

  12. 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.

  13. 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...

  14. Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

    Science.gov (United States)

    Pakdeepak, Kanet; Pata, Supansa; Chiampanichayakul, Sawitree; Kasinrerk, Watchara; Tatu, Thanusak

    2016-01-01

    Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced. PMID:27050832

  15. New Construction of Eigenstates and Separation of Variables for SU(N) Quantum Spin Chains

    CERN Document Server

    Gromov, Nikolay; Sizov, Grigory

    2016-01-01

    We conjecture a new way to construct eigenstates of integrable XXX quantum spin chains with SU(N) symmetry. The states are built by repeatedly acting on the vacuum with a single operator B^{good}(u) evaluated at the Bethe roots. Our proposal serves as a compact alternative to the usual nested algebraic Bethe ansatz. Furthermore, the roots of the operator B^{good}(u) give the separated variables of the model, explicitly generalizing Sklyanin's approach to the SU(N) case. We present many tests of the conjecture and prove it in several special cases. We focus on rational spin chains with fundamental representation at each site, but expect many of the results to be valid more generally.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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/ .

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

    Directory of Open Access Journals (Sweden)

    George O Badescu

    Full Text Available 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.

  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. 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

  4. 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

  5. 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

  6. 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)

  7. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pr

  8. 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...

  9. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

    Science.gov (United States)

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill

    2015-04-01

    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  10. Novel one-dimensional lanthanide acrylic acid complexes: an alternative chain constructed by hydrogen bonding

    Science.gov (United States)

    Li, Hui; Hu, Chang Wen

    2004-12-01

    Novel one-dimensional (1D) chains of three lanthanide complexes La(L 1) 3(CH 3OH)]·CH 3OH (L 1=(E)-3-(2-hydroxyl-phenyl)-acrylic acid) 1, La(L 2) 3(H 2O) 2]·2.75H 2O (L 2=(E)-3-(3-hydroxyl-phenyl)-acrylic acid) 2, and La(L 3) 3(CH 3OH) 2(H 2O)]·CH 3OH (L 3=(E)-3-(4-hydroxyl-phenyl)-acrylic acid) 3 are reported. The crystal structure data are as follows for 1: C 29H 29LaO 11, monoclinic, P2 1/ n, a=15.4289(12) Å, b=7.9585(6) Å, c=23.041(2) Å, β=99.657(2)°, Z=4, R1=0.0637, w R2=0.0919; for 2: C 27H 30.50LaO 13.75, triclinic, P-1, a=8.4719(17) Å, b=13.719(3) Å, c=14.570(3) Å, α=62.19(3)°, β=99.657(2)°, γ=78.22(3)°, Z=2, R1=0.0384, w R2=0.0820; and for 3: C 30H 35LaO 13, monoclinic, P2(1)/ c, a=9.5667(6) Å, b=24.3911(15) Å, c=14.0448(9) Å, β=109.245(2)°, Z=4, R1=0.0374, w R2=0.0630. All the three structure data were collected using graphite monochromated molybdenum Kα radiation and refined using full-matrix least-squares techniques on F 2. These structures show that four kinds of the carboxylato bridge modes are included in these chains to link the La(III) ions. It is the first time that it has been found that the intra-chain hydrogen bonding can construct an alternative chain even, when the coordination bridge mode is the same along the chain (complex 2). There are 2D and 3D hydrogen bonding in the crystal lattices of complexes 1- 3.

  11. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  12. A New Combination of Mutated loxPs in a Vector for Construction of Phage Antibody Libraries

    Institute of Scientific and Technical Information of China (English)

    Yu GAN; Xin-Tai ZHAO

    2005-01-01

    In the construction of large antibody libraries by in vivo recombination, two non-homogeneous loxP sites are required for the exchange of Vgenes between phagemids to create many new VH-VL combinations.The mutated loxP511 was designed not to recombine with the wild-type loxP (loxPwt) in early studies and a combination of the two has been used to construct antibody libraries. But recent reports have shown that recombination occurs between loxPwt and loxP511. This suggests that the combinational use of loxP511 and loxPwt might lead to the loss of the V gene diversity of antibody libraries. Therefore, it is necessary to find a new combination of loxPs to avoid the excision recombination in the antibody library. In this study,we found that the excision recombination between loxP511 and loxP2272, another mutated loxP sequence,was undetectable within one phagemid, while the excision recombination between loxP511 and loxPwt occurred at a frequency of 40%, higher than that reported previously. Furthermore, the in vivo recombination of different phagemids with loxP511 and loxP2272 showed that the V gene exchange was efficiently mediated to produce new VH-VL combinations. It was concluded that the loxP511 and loxP2272 combination was more favorable for reducing the excision recombination and constructing large phage antibody libraries with high diversity.

  13. Construction of chimeric antibodies: cloning of immunoglobulin genes including their promoter regions by PCR.

    Science.gov (United States)

    Mocikat, R; Kütemeier, G; Harloff, C

    1992-03-01

    In the production of recombinant antibodies, it is necessary to have an immunoglobulin gene promoter for driving the expression of the antibody genes. Here we describe a simple PCR method that allows cloning of the immunoglobulin genes together with their own promoters despite the fact that the sequence of the upstream part of the gene is unknown.

  14. 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

  15. Berlin Brandenburg International (BER: planning and implementation of a concrete supply chain for the airport construction site

    Directory of Open Access Journals (Sweden)

    Guido Riedel

    2012-12-01

    Full Text Available Background: With the decision to extend the airport Berlin-Schönefeld to the new airport Berlin Brandenburg International (BER in 2006, a construction of superlatives has emerged. One of the biggest challenges was the supply of around 2.5 million cubic meters of high quality concrete that had to be produced for the construction of the airport. Due to the scale of this enterprise as well as its environment, the logistic solution of raw material supply has to be found.       Method: The planning of the concrete supply chain for the airport construction site BER had to be carried out with two major goals: the stability of the supply chain to assure that the demands of the construction site are met and delays are prevented, as well as assurance of the high quality standards of the concrete production and to avoid an alkali silica reaction and the resulting unavoidable disaggregation of the concrete. External effects, such as the carbon dioxide emission and the effect of the supply chain on adjoining residents were key factors that had to be integrated in a holistic supply chain concept.  The principle underlying method is an analysis of limiting conditions for two approaches: a centralized supply chain with on-site concrete factory and upstream transport of raw materials versus a decentralized supply chain with off-site factories and downstream transport of ready-mixed concrete. Results: The analysis of constraints and the effects on key requirements of the concrete supply chain for the BER airport construction site lead to the installation of the most modern concrete plant in Europe. The benefits of a centralized supply chain are significant. On one hand, the high quality standards can be met with the on-site mixture of the concrete and centralized quality assurance, on the other hand, the majority of the supply traffic for the construction site was moved from the road to train-bound logistics, meeting the emission requirements of the

  16. CD28嵌合抗体及与抗原分子的3D空间结构模建%Construction of 3D model of CD28 chimeric antibody with its antigen docked

    Institute of Scientific and Technical Information of China (English)

    程钢; 秦媛媛; 程迪; 陈曦; 张学光; 邱玉华

    2012-01-01

    AIM: To construct a 3D model of the chimeric antibodies (AntiCD28: ch-2F5) with corresponding antigen molecuie docked to theoretically verify the rationality of the binding of antibody with its antigen and to provide a method of 3D identification between antigen and antibody and spatial structure analysis. METHODS; We analyzed the sequence by submitting it to http://www. ncbi. nlm. nih. gov/ and made a comparison using integratly the 3 databases of Gen-Bank, Protein data bank and GENO-3D. The 3D model was constructed by Swiss-model homology modeling server and molecular docking online was performed by GRAMM-X Protein Docking Web Server. Chimeric heavy chain, light chain, heavy-light chain complex, heavy-light chain and antigen complex were displayed and photographed by the Chimera Software. Meanwhile, the spatial structures of heavy, light chains, variable region, constant region, CDR and frame area were marked by different colours respectively to exhibit the 3D structure on every side. RESULTS: The 3D structure of the heavy-light chain and antigen complex we constructed was consistent well with the theory of antigen binding to antibody molecules. CONCLUSON; The structure of the chimeric antibody we constructed with the bioinformatic method was in accordance with the general structure of antibody, and its antigen binding site was also consistent with the molecular theory. Thus, the model helps to analyze the 3D structure of antibody and antigen-antibody interaction.%目的:对1株嵌合抗体(antiCD28:ch-2F5)进行3D建模,并与其抗原分子进行对接,验证是否与抗原抗体结合理论相符,并提供一种抗原抗体及其识别的空间结构分析方法.方法:在http://www.ncbi.nlm.nih.gov网站提交序列进行分析,综合运用GenBank、Protein data bank、GENO-3D等数据库比对分析,用Swiss-model同源建模服务器模建,运用GRAMM-X Protein Docking Web Server在线进行对接,结果采用Chimera软件对嵌合抗体的重、轻链、

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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...... of natural HibCP antibodies (r = 0.59; P = 0.00002). A possible role of natural exposure for Hib or cross-reactive bacteria on the mucosal surfaces in the shaping of the isotype response to HibCP conjugate vaccines is discussed....

  2. Stabilization of antibody fragments in adverse environments.

    Science.gov (United States)

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  3. 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.

  4. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans;

    1985-01-01

    detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry......Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

  5. 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 ...

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

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

    DEFF Research Database (Denmark)

    Messih, M. A.; Lepore, R.; Marcatili, Paolo;

    2014-01-01

    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 specif......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...... 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....

  13. Screening and identification of neutralizated single-chain antibody of anti-glomerular basement membrane antibody%中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    肖静; 刘章锁; 王沛; 黄留玉; 宋宏彬; 赵明辉

    2010-01-01

    Objective To screen a human single-chain variable fragments(scFv)against antiGBM antibody.Methods Using phage display technique,the phage antibody library was panned by antiglomerular basement membrane(GBM)antibody which was coated in a micro-titer plate,one clone was found to have high affinity to anti-GBM antibody.The DNA sequence of the positive clone was determined.Results Along with the increase of rounds anti-GBM antibody specific phage antibody was highly enriched and screening efficiency was increased 137 folds than the firest round.ELISA and competition inhibition assay showed that the scFv had a specific combination character with anti-GBM antibody.DNA sequencing confirmed that the whole gene of scFv was 750 bp,and in accordance with humanized single-chain variable region antibody sequence structure.Conclusion The results suggested that the scFv fragment to anti-GBM antibody could be successfully selected by recombinant phage antibody technique,which will laid an experimental foundation for further research of the therapy of Goodpasture syndrome.%目的 制备人抗肾小球基底膜(GBM)抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株(C31)阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.

  14. 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...

  15. 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.

  16. Streamlining Demand Fulfilment Chain in Construction Projects : The Case of a Pre Engineered Steel Building Manufacturer

    NARCIS (Netherlands)

    Roosmalen, K.; van Weele, A.; Ashayeri, J.

    2010-01-01

    Practical implications – The results of this paper clearly verify the need for mind change among Pre Engineered and Structural Steel Building firms operating in the Middle-East. Supply chain management methods should be used to improve competitiveness.

  17. 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

  18. 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...

  19. Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-hong; ZHANG Xi-zhen; ZHAO Dong-hai; SHI He-liang; YU Yong-hui; WU Yong-ge; YU Xiang-hui; KONG Wei

    2008-01-01

    Survivin,a novel member of inhibitor ofapoptosis(IAP) protein family,is aberrantly expressed in cancer but undetectable in normal,differentiated adult tissues.The cancer-specific expression of survivin,coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment.Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B.The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21,and the relative molecule mass(Mr) of expressed fusion protein was approximately 21000.The recombinantprotein was purified through Ni2~ affinity chromatography column and characterized by SDS-PAGE and Western blot.The purified recombinant protein was then injected into rabbits,and antisurvivin polyclonal antibody with a high titer was obtained.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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-

  6. 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

  7. Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system.

    Science.gov (United States)

    Ma, Li; Koyota, Souichi; Myoen, Yu; Yamashita, Tetsuro; Yatabe, Naoki; Koizumi, Yukio; Aosasa, Masayoshi; Nishimichi, Norihisa; Matsuda, Haruo; Sugiyama, Toshihiro

    2012-02-24

    Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.

  8. 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.

  9. Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures.

    Science.gov (United States)

    Gao, Feng-shan; Bai, Jing; Zhang, Qiang; Xu, Chong-bo; Li, Yanmin

    2012-07-10

    Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and β(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-β(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-β(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, β-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro.

  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. 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

  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. 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 ...... two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens.......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...

  14. 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 ...

  15. Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry.

    Science.gov (United States)

    Zeglis, Brian M; Mohindra, Priya; Weissmann, Gabriel I; Divilov, Vadim; Hilderbrand, Scott A; Weissleder, Ralph; Lewis, Jason S

    2011-10-19

    A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes (64)Cu and (89)Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with (64)Cu and (89)Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7-5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the (64)Cu-DOTA- and (89)Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the (64)Cu- and (89)Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system-one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators-will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.

  16. 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...

  17. 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.

  18. 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.

  19. Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hairul Bahara, Nur Hidayah; Chin, Siang Tean; Choong, Yee Siew; Lim, Theam Soon

    2016-01-01

    The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

  20. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  1. 通过打造低碳供应链提升农业游品质%Constructing Low Carbon Supply Chain to Improve Quality of Agriculture Tourism

    Institute of Scientific and Technical Information of China (English)

    吴丹

    2011-01-01

    The concept and characteristics of low carbon supply chain were described and the agricultural tourism products supply chain were analyzed. The low carbon agricultural tourism products supply chain was constructed and quality promotion strategy for the low carbon supply chain of agricultural tourism was proposed.%阐述了低碳供应链的概念及特点,对农业游产品供应链进行了分析,构建了低碳农业游产品供应链,提出了通过打造低碳供应链提升农业游品质的策略.

  2. 抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选%Construction and preliminary panning of Fab phage display antibody library against respiratory syncytial virus

    Institute of Scientific and Technical Information of China (English)

    汪治华; 张国成; 李安茂; 周南; 陈一; 李小青; 苏字飞; 邓阳彬; 王治静

    2008-01-01

    Objective To construct a human phage display antibody library,which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic.This can provide a platform for human antibody preparation and diagnosis,prophylaxis and therapy of RSV infection in children.Methods Peripheral blood lymphocytes were isolated from 52 children with RSV infection,cDNA was synthesized from the total RNA of lymphocytes.The light and heavy chain Fd (VH-CH1)fragments of immunoglobulin gene were amplified by RT-PCR.The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue.The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.The plasmids extracted from amplified E.coil were digested with restriction endonucleases Sac 1,Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes.RSV virions were utilized as antigens to screen Fab antibodies.Results By recombination of light and heavy chain genes,an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed,in which 70% clones had light chains and heavy chain Fd genes.The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06 x 1012 pfu/mL The antibody library gained an enrichment in different degrees after the preliminary panning.Condusions Utilizing the technology of phage display,an immune Fab phage display antibody library against RSV was successfully constructed in this study,which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis,therapy and prophylaxis of RSV infection in children

  3. 人源性抗乳腺癌单链抗体库的筛选与初步鉴定%Isolation and identification of single chain Fv antibodies against breast cancer from a human phage display library

    Institute of Scientific and Technical Information of China (English)

    王净; 王慧; 王超; 袁媛; 崔岩; 叶菁; 李青

    2012-01-01

    [目的]本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定.[方法]以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况.[结果]成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白.[结论]本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库.研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础.%AIM: To Isolate and identify single chain Fv (scFv) antibodies against breast cancer from a constructed human phage display library. METHODS: Recombinant phages specific for breast cancer cells were enriched after four-round screening with MCF-7 cells. We selected the antigen-positive ones from the enriched clones by phage ELISA. The positive clones were used to infect E. coli HB2151 to express soluble scFv antibody. The antigen binding activity of the soluble antibodies was detected by West-em blotting. RESULTS; The specific antibodies against MCF-7 cells were enriched after four rounds of affinity selection. SDS-PAGE and Western blotting showed a band at relative molecular mass 30 000 Da, which indicated soluble antibodies were present. ELISA analysis revealed that soluble antibodies had the affinity to a human breast cancer cell line MCF-7 but not to other cancer cell line, which demonstrated scFv could react specifically with breast cancer cells. CONCLUSION; We constructed a scFv phage

  4. Purification and determination of C-reactive protein and inter-α-trypsin inhibitor heavy chain 4 in dogs after major surgery through generation of specific antibodies.

    Science.gov (United States)

    Soler, L; García, N; Unzueta, A; Piñeiro, M; Álava, M A; Lampreave, F

    2016-10-15

    Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) and C-reactive protein (CRP) have been isolated from acute phase dog sera by affinity chromatography with insolubilized polyclonal antibodies anti pig Major Acute phase Protein (Pig-MAP) and with p-Aminophenyl Phosphoryl Choline, respectively. Isolated proteins were used to prepare specific polyclonal rabbit antisera that have allowed quantifying their concentration in serum samples by single radial immunodifussion. Both proteins were quantified in sera from female dogs that had undergone ovariohysterectomy (OVH, n=9) or mastectomy (n=10). The observed increases in CRP concentrations showed that surgical traumas induced an acute phase response of a great magnitude in the dogs. In both surgeries a four-fold increase of ITIH4 concentrations was detected. It can be concluded that ITIH4 is a new positive acute phase protein in dogs, as reported in other species. PMID:27590422

  5. Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli.

    Science.gov (United States)

    Xi, Hualong; Yuan, Ruosen; Chen, Xiaoxu; Gu, Tiejun; Cheng, Yue; Li, Zhuang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-10-01

    An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.

  6. 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.

  7. 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...

  8. 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.

  9. Prokaryotic expression and purification of anti-AEG-1 single-chain variable antibody%抗 AEG-1单链可变区抗体的原核表达及纯化

    Institute of Scientific and Technical Information of China (English)

    路蔓; 刘昕阳; 龙敏; 王琛; 刘冲; 郜赵伟; 欧阳永日; 陈曦; 张惠中

    2015-01-01

    Objective To construct anti-astrocyte elevated gene-1(AEG-1)single-chain variable antibody (V23)prokaryotic ex-pression vector,and to conduct the protein purification and immunological activity detection.Methods The Primer5 software was applied to design the primers aiming at the gene sequence of the antibody anti-AEG-1 single-chain variable region for constructing the prokaryotic expression plasmid of PRsetC/V23.After the enzyme digestion by the restriction enzyme Pst1 and correct DNA se-quencing,the prokaryotic expression plasmid was led to E.coli BL21 ,the prokaryotic expression engineering strain containing the V23 gene was constructed.After the induction with IPTG,the interest protein was purified by the magnetic beads with the HIS tag,and the content of interest protein was determined by the SDS-PAGE electrophoresis.Western blot and ELISA were adopted to detect the immune activity of the nti-AEG-1 single-chain variable region antibody.Results For the constructed prokaryotic expres-sion plasmid PRsetC/V23,the single enzyme digestion and sequencing analysis displayed that the constructed V23 gene was com-pletely consistent to the designing sequences.After IPTG induction,SDS-PAGE electrophoresis showed an apparent protein band at 31×103 ,the Western blot detection showed a specific AEG-1 response band at 80 ×103 ,the ELISA test showed the positive re-sults.Conclusion The PRsetC/V23 prokaryotic expression plasmid and the V23 prokaryotic expression engineering strain are suc-cessfully constructed,this engineering strain can express anti-AEG-1 single-chain variable region antibody protein,and the protein has good immune activity.%目的:构建抗星形细胞上调基因1(AEG-1)单链可变区抗体(V23)的原核表达载体,并对表达蛋白进行纯化及免疫活性检测。方法应用 Primer5软件设计针对抗 AEG-1单链可变区抗体基因序列的引物,构建 PRsetC/V23原核表达质粒,经限制性内切酶 Pst1酶切以及 DNA

  10. 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

  11. 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

  12. Varicella Zoster Virus Myelitis in Two Elderly Patients: Diagnostic Value of Nested Polymerase Chain Reaction Assay and Antibody Index for Cerebrospinal Fluid Specimens

    Directory of Open Access Journals (Sweden)

    Teruyuki Takahashi

    2013-04-01

    Full Text Available Background: Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. Case Presentation: This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. Conclusion: The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.

  13. 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

  14. 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

  15. Design, construction, and validation of a modular library of sequence diversity standards for polymerase chain reaction.

    Science.gov (United States)

    Baum, Paul D; Young, Jennifer J; Zhang, Qianjun; Kasakow, Zeljka; McCune, Joseph M

    2011-04-01

    Methods to measure the sequence diversity of polymerase chain reaction (PCR)-amplified DNA lack standards for use as assay calibrators and controls. Here we present a general and economical method for developing customizable DNA standards of known sequence diversity. Standards ranging from 1 to 25,000 sequences were generated by directional ligation of oligonucleotide "words" of standard length and GC content and then amplified by PCR. The sequence accuracy and diversity of the library were validated using AmpliCot analysis (DNA hybridization kinetics) and Illumina sequencing. The library has the following features: (i) pools containing tens of thousands of sequences can be generated from the ligation of relatively few commercially synthesized short oligonucleotides; (ii) each sequence differs from all others in the library at a minimum of three nucleotide positions, permitting discrimination between different sequences by either sequencing or hybridization; (iii) all sequences have identical length, GC content, and melting temperature; (iv) the identity of each standard can be verified by restriction digestion; and (v) once made, the ends of the library may be cleaved and replaced with sequences to match any PCR primer pair. These standards should greatly improve the accuracy and reproducibility of sequence diversity measurements.

  16. 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.

  17. 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 ...

  18. Development of a monoclonal antibody to urinary degradation products from the C-terminal telopeptide alpha 1 chain of type I collagen. Application in an enzyme Immunoassay and comparison to CrossLaps(TM) ELISA

    DEFF Research Database (Denmark)

    C, Fledelius; I, Kolding; P, Quist;

    1997-01-01

    A monoclonal antibody MAbA7 was raised against a synthetic peptide having a sequence (EKAHDGGR) specific for a part of the C-telopeptide alpha 1 chain of type I collagen. MAbA7 was labelled with horseradish peroxide and used in a competitive one-step enzyme-linked immunosorbent assay (ELISA...

  19. 抗IV型胶原酶单链抗体在毕赤酵母中分泌表达%EXPRESSION OF SINGLE CHAIN ANTIBODY TO COLLAGENASE IV BY PICHIA PASTORIS

    Institute of Scientific and Technical Information of China (English)

    阎锡绿; 汤健; 刁爱侣

    2001-01-01

    To express human single chain antibody to collagenase IV as soluble proteins secreted by pichia pastoris, a recombinant vector scFv-pPIC9 was constructed, and then transformed into pichia pastoris GS115 by electuoporation, The transformants were selected by DNA hybridization and cultured in the media with methanlo. Soluble scFv were secreted into culturesupernatant and characterized by SDS-PAGE and Western Blot. Our results showed that the secreting of soluble scFv in pichia pastoris was as high as 20mg/L. The soluble scFv has similar immuno-activity to its counterpart produced in bacteria, and it is more easily and efficiently purified.%利用毕赤酵母系统表达抗1V型胶原酶人单链抗体。首先把目的基因克隆到毕赤酵母表达载体上,电击转化受体菌。在甲醇诱导下表达单链抗体。SDS-PAGE和免疫印迹显示毕赤酵母分泌表达人单链抗体,表达量约20mg/L酵母培金物。该表达系统与大肠杆菌相比.简化了表达产物的分离纯化程序。

  20. 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

  1. 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.

  2. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  3. Construction of human naive phage antibody library and primary screening of the gab antibodies against gp96%人天然噬菌体抗体库的构建及抗gp96抗体的初步筛选

    Institute of Scientific and Technical Information of China (English)

    马小兵; 畅继武; 李成文; 李慧忠; 王馨

    2009-01-01

    Objective To construct a naive human Fab fragment phage display library,from which the anti-gp96 antibodies may be panned by the gp96 purified from the tissue of urothelial carcinoma in the urinary bladder and provide a basis to new therapy for the malignant tumors.Methods Peripheral blood lymphocytes were isolated from 800 ml of blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lympbocytes were amplified by PCR with variable regions 5' and 3' primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E.coli XL1-Blue by electroperation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac Ⅰ,Xba Ⅰ,Xho Ⅰ and Spe Ⅰ, and both the phage antibody Fabs and phage-raids abstracted from amplified E.coil were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment.The gp96 purified from the urothelial carcinoma tissue of the bladder by affinity chromatog-raphy on eoncanavalin-A sephnrose and DEAE-sephnrose ion exchange chromatography were utilized as antigens to process three rounds of panning to the original Fab antibody library.Results The quantity of total RNA and cDNA were qualified.By combination of light chain and heavy chain genes, an antibody library containing 6.6×106 clones was obtained, and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.The gp96 protein was obtained from urothelial carcinoma tissue in the urinary bladder.After having been panned by gp96, the original antibody library gained enrichment by 68 times.Conclusion Utilizing the technology of phage surface display, specific antibody can be gained from the human

  4. 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

  5. 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.

  6. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    Science.gov (United States)

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

  7. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    Science.gov (United States)

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease. PMID:27494228

  8. Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

    Science.gov (United States)

    Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

    2010-01-01

    Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb. PMID:19597742

  9. 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

  10. Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

    Directory of Open Access Journals (Sweden)

    Camila Appolinário

    2015-10-01

    Full Text Available ABSTRACTRabies virus (RABV isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2,Desmodus rotundus (variant 3, crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT and real time polymerase chain reaction (qRT-PCR for quantification of rabies virus nucleoprotein gene (N gene. Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

  11. Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil.

    Science.gov (United States)

    Appolinário, Camila; Allendorf, Susan Dora; Vicente, Acácia Ferreira; Ribeiro, Bruna Devidé; Fonseca, Clóvis Reinaldo da; Antunes, João Marcelo; Peres, Marina Gea; Kotait, Ivanete; Carrieri, Maria Luiza; Megid, Jane

    2015-01-01

    Rabies virus (RABV) isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2), Desmodus rotundus (variant 3), crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT) and real time polymerase chain reaction (qRT-PCR) for quantification of rabies virus nucleoprotein gene (N gene). Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

  12. B2B collaboration method through trust values for e-supply chain integrator: a case study of Malaysian construction industry

    Science.gov (United States)

    Ab. Aziz, Norshakirah; Ahmad, Rohiza; Dhanapal Durai, Dominic

    2011-12-01

    Limited trust, cooperation and communication have been identified as some of the issues that hinder collaboration among business partners. These one also true in the acceptance of e-supply chain integrator among organizations that involve in the same industry. On top of that, the huge number of components in supply chain industry also makes it impossible to include entire supply chain components in the integrator. Hence, this study intends to propose a method for identifying "trusted" collaborators for inclusion into an e-supply chain integrator. For the purpose of constructing and validating the method, the Malaysian construction industry is chosen as the case study due to its size and importance to the economy. This paper puts forward the background of the research, some relevant literatures which lead to trust values elements formulation, data collection from Malaysian Construction Supply Chain and a glimpse of the proposed method for trusted partner selection. Future work is also presented to highlight the next step of this research.

  13. 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

  14. 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

  15. 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.

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

    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.

  17. 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.

  18. Analysis on the Value Chain of Brand Construction of Huishan Dairy%辉山乳业品牌建设价值链分析

    Institute of Scientific and Technical Information of China (English)

    王政军

    2012-01-01

    为推动辽宁省农产品品牌建设的进程,以沈阳市辉山乳业为例,运用价值链理论深入剖析其产品品牌建设的过程、途径及经验,并从价值链优化角度,探讨其给农产品品牌建设带来的有益启示。%This article, taking Huishan Diary as an example, discusses the brand construction process, approaches, and experience using value chain theory, and probes into the insight it brings to the brand construction for farm products from the perspective of value chain optimization in an effort to stimulate the brands construction in Liaoning province.

  19. 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

  20. 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

  1. 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。

  2. 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).

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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。

  10. 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...

  11. A Method for Constructing Reshaping Single-Domain Antibody%一种构建改形单域抗体的方法

    Institute of Scientific and Technical Information of China (English)

    程巨龙; 王祥斌; 张众; 刘晶; 姚新生; 黄华梁

    2002-01-01

    为验证一种构建改形单域抗体的实用新方法,与以往方法不同的是,该方法不需要对抗体进行空间结构模拟,以确定人源抗体的FRs接受序列及在人源FRs接受序列中哪些氨基酸残基需要突变,并且该方法将抗体的改形与亲和力成熟于同一过程完成.利用该方法构建了改形抗CD28重链单域抗体.根据一种鼠源抗CD28重链单域抗体的氨基酸序列,于GenBank中查得两条与之最同源的人源抗体序列,利用其中一条的FRs作为改形抗体的主框架进行改形构建.将鼠源抗体的CDR区插入到人源FR区后,对人源FR区的一些氨基酸残基进行替换突变,替换的氨基酸残基数及替换原则主要是根据对查到的人源抗体序列、鼠源抗体序列,以及这些序列与Kabat分类中的种属序列进行的比较.为了增加改形抗体基因的多样性,对要被替换的氨基酸残基在基因合成中采用简并的方式,使要被替换的氨基酸残基和替换的氨基酸残基都有机会出现,二者出现的几率各为50%.同时,在将大小不同的合成核苷酸片段采用重叠PCR扩增以获得完整改形抗体基因时,采用高Mg2+浓度下和使用Taq DNA聚合酶,以进一步随机引入突变.利用重叠PCR产物构建了一个噬菌体抗体库,经过3轮淘选后,获得了几个具有较高免疫学活性的改形抗体.对其中的两个抗体进行了进一步研究,将两个抗体的基因在大肠杆菌BL21(DE3)中表达,复性后的表达蛋白仍具有较高的免疫学活性.结果表明该方法是有效可行的.%The aim of this research was to demonstrate a novel and practical method for constructing reshaping Single-domain antibodies. Different from other methods, our method does not need to model the configuration of antibodies with specific sequences to determine the sequences of human acceptor FRs and then determine which amino acid residues in human acceptor FRs should be substituted. Most importantly

  12. 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.

  13. Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening

    Directory of Open Access Journals (Sweden)

    Bernard Couvreur

    2003-06-01

    Full Text Available We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen. We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.

  14. cDNA sequence and Fab crystal structure of HL4E10, a hamster IgG lambda light chain antibody stimulatory for γδ T cells.

    Directory of Open Access Journals (Sweden)

    Petra Verdino

    Full Text Available Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.

  15. Reliability model of organization management chain of South-to-North Water Diversion Project during construction period

    Institute of Scientific and Technical Information of China (English)

    Feng Jingchun; Zhou Yang; Hong Yuzhen; Zhao Shixin; Ren Xiaoqiang

    2008-01-01

    In order to analyze the indispensability of the organization management chain of the South-to-North Water Diversion Project (SNWDP), two basic forms (series connection state and mixed state of both series connection and parallel connection) of the organization management chain can be abstracted. The indispensability of each form has been studied and is described in this paper. Through analysis of the reliability of the two basic forms, reliability models of the organization management chain in the series connection state and the mixed state of both series connection and parallel connection have been set up.

  16. 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.

  17. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    Science.gov (United States)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  18. Competency development in antibody production in cancer cell biology

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.S.

    1998-12-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The main objective of this project was to develop a rapid recombinant antibody production technology. To achieve the objective, the authors employed (1) production of recombinant antigens that are important for cell cycle regulation and DNA repair, (2) immunization and specific selection of antibody-producing lymphocytes using the flow cytometry and magnetic bead capturing procedure, (3) construction of single chain antibody library, (4) development of recombinant vectors that target, express, and regulate the expression of intracellular antibodies, and (5) specific inhibition of tumor cell growth in tissue culture. The authors have accomplished (1) optimization of a selection procedure to isolate antigen-specific lymphocytes, (2) optimization of the construction of a single-chain antibody library, and (3) development of a new antibody expression vector for intracellular immunization. The future direction of this research is to continue to test the potential use of the intracellular immunization procedure as a tool to study functions of biological molecules and as an immuno-cancer therapy procedure to inhibit the growth of cancer cells.

  19. 基于交易成本的建筑供应链构建及运作研究%Creation and Operation of Construction Supply Chain Based on Transaction Cost

    Institute of Scientific and Technical Information of China (English)

    刘跃武; 席洪林; 翟晓飞; 尚美婷

    2011-01-01

    A transaction cost model of the construction supply chain was built up according to the framework of construction supply chain and inner network of contractual relations. Basic conditions of construction supply chain were established and operated from aspect of transaction cost. The network of construction supply chain was further investigated under project management contracting mode. The fluctuation of transaction cost of construction supply chain was quantitatively discussed when project management contractor was in construction supply chain. The precondition of that project management contractor undertook constructing and operating of supply chain was obtained.%针对建筑供应链框架结构及其内部企业契约关系网络,构建了建筑供应链交易成本模型,从交易成本角度研究了构建及运作建筑供应链的基本条件,通过进一步研究项目管理承包模式下的建筑供应链网络结构,定量分析了项目管理承包商进入建筑供应链后交易成本的变化情况,得出了项目管理承包商承担建筑供应链构建及运作任务的前提条件.

  20. 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.

  1. 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; Westergaard, Marie;

    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...... index (SLEDAI)) score of the SLE patients (r = 0.399, p = 0.007). Furthermore, concentrations of FLCs correlated with titers of dsDNA antibodies (r = 0.383, p = 0.009), and FLC levels and SLEDAI scores correlated in the anti-dsDNA-positive SLE patients, but not in anti-dsDNA-negative SLE patients. Total...

  2. 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...

  3. 抗p185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector for Anti-p185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2013-01-01

    本研究构建抗p185erbB2人鼠嵌合抗体慢病毒表达载体,并将其转染293T细胞,明确转染后嵌合抗体基因的表达情况.采用PCR法扩增抗p185erbB2鼠单抗轻、重链可变区基因(vL和vH)和人IgG1的轻、重链恒定区基因(κ和γ1),再利用三引物PCR法将vL和κ,vH和γ1进行拼接,得到嵌合轻链基因(L)和嵌合重链基因(H),分别插入质粒pVAX1/IRES的IRES元件的下、上游.用内切酶将H-IRES-L从pVAX1/H-IRES-L上切下,插入慢病毒载体pWPI中,经相应酶切和测序鉴定,正确构建了慢病毒表达载体pWPI/H-IRES-L.将其转染293T细胞,48 h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,RT-PCR和直接ELISA法检测嵌合抗体的表达.结果显示,转染pWPI/H-IRES-L的293T细胞中有嵌合轻链和嵌合重链基因的共同表达,而且表达的嵌合抗体能够与p185erbB2分子特异性结合,为今后抗p185erbB2工程抗体的研究奠定了基础.%This research was to construct the lentiviral expression vector for anti- pl85erbB2 mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-pl85erbB2 mAb and the constant regions of human IgG1 (Κ and γ1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody

  4. 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; ...

  5. 集群式供应链战略联盟构建策略研究%Construction Strategy for Cluster Supply Chain Strategic Alliance

    Institute of Scientific and Technical Information of China (English)

    袁科峰; 张晓霞

    2015-01-01

    在产业集群化发展、相互渗透发展以及产业持续发展背景下,基于对集群式供应链与战略联盟共性和差异性分析,提出一种新型的网络化战略合作组织即集群式供应链战略联盟,分析影响其发展的主要障碍,并提出构建策略。%The industrial clustering development, mutual penetration and industry under the background of industry cluster development, interinfiltration development and sustainable development, it discusses the similarities and differences of cluster supply chain and strategic alliance, andputs forward a kind of new network strategic cooperation organization, cluster supply chain strategic alliance. Some difficulties are analyzed and some construction strategies are offered.

  6. 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。

  7. 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。

  8. Production of High Affinity Human Single-chain Antibody Against PreS1 of Hepatitis B Virus:Comparison of Large Na(i)ve and In vitro Immune Phage Displayed Antibody Library%高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究

    Institute of Scientific and Technical Information of China (English)

    张志超; 胡学军; 包永明; 杨青; 张红梅; 安利佳

    2002-01-01

    A large nave phage displayed human single-chain variable fragments antibody (scFv) library and an in vitro immune library were constructed in parallel conditions, based on the PBLs from healthy and sero-negative blood donors, part of which were in vitro immunized by peptide PreS1 conjugated to BSA. After 3 rounds of panning against PreS1, measurement of antibody-antigen reaction revealed: a scFv specific to PreS1 from the immune library was obtained, which affinity (k=10-7~10-8 M) was higher than that from the nave one (k=10-6~10-7 M). Sequencing of the two scFv showed they were human antibodies, which may be of interest in therapy of Hepatitis B. This investigation also illustrated that the method of in vitro immunization results in antibody library more satisfied even than the large nave one.%以健康人的外周血淋巴细胞为来源,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫.分别从免疫和未经免疫的淋巴细胞提取RNA,扩增抗体基因,构建大容量天然单链抗体(scFv)噬菌体展示文库和体外免疫scFv抗体库.以PreS1肽进行3轮淘选后,抗原抗体反应结果显示,从免疫库中获得了亲和力10-7~10-8 M的抗乙型肝炎病毒PreS1的单链抗体,高于天然库的结果(10-6~10-7 M).测序结果表明两株抗体均为人抗体.为基因工程抗体用于临床治疗乙型肝炎奠定基础.同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库.

  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. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

    Science.gov (United States)

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2008-08-01

    To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

  17. Amplifying variable region gene of light chain(VL)of monoclonal antibody against human papillomavirus 16 L1(HPV16 L1)protein by 5'-RACE and sequence analysis%5'-RACE法扩增抗人乳头瘤病毒16型L1蛋白单克隆抗体轻链可变区基因及序列分析

    Institute of Scientific and Technical Information of China (English)

    王燕; 商庆龙; 陈思佳; 邵迪; 韩聪; 李茉; 谷鸿喜

    2008-01-01

    Objective To acquire the variable region gene of light chain(VL)of monoclonal antibody a-gainst human papillomavirus 16 LI(HPV16 LI)protein.Methods Total RNA waft extract from hybridoma cells secreting specific monoclonal antibody against HPV16L1,transcripted reversely into cDNA with random primers.The variable region of the light chain gene fragments was ampliflied using 5'-RACE.Sequencing was confirmed by agarose gel electrophoresis and sequencing analysis.Results Full letIsth of VL gene Was 336bp that encoding 112 amino acids.The VL gene was homologous with the published gene sequences of mouse antibody variable region.Conclusion The light chain sequence of monoclonal antibody against HPV16L1 protein Was obtained by 5'-RACE,which provides a good basis for construction of a recombinant antibody against HPV16 L1.%目的 获得抗人乳头瘤病毒16型(HPV16)L1蛋白单克隆抗体的轻链可变区(VL)基因并分析序列.方法 从分泌抗HPV16L1蛋白单克隆抗体的杂交瘤细胞中提取总RNA,逆转录形成cDNA,用5'-RACE策略扩增抗体轻链可变区基因,经琼脂糖凝胶电泳鉴定,并测序及进行序列分析.结果 VL基因全长336bp,编码112个氨基酸,基因测序结果符合小鼠抗体轻链可变区特征.结论 5'-RACE法成功获得了抗HPV16L1蛋白的单克隆抗体轻链可变区基因的真实序列,为基因工程抗体研究奠定了良好基础.

  18. 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.

  19. 建筑供应链风险评价研究%Research on the Risks Evaluation of Construction Supply Chain

    Institute of Scientific and Technical Information of China (English)

    刘志强; 张方明

    2012-01-01

    As a new administration mind and method of construction industry, supply chain management can not only improve the core competitiveness of construction enterprises, inerease the profits, but also bring some risks. Thus how to an- alyze and evaluate these risks have been very important problems. This paper firstly summarizes the basie theory of eon- struetion supply chain under engineering-procurement-construction (EPC) mode, and then designs the index system of risks evaluation on the basis of risks analysis, puts forward a risk evaluation model based on support veetor machines theolw and gives an example.%供应链管理作为一种新的建筑管理理念与方法.在提高建筑业企业核心竞争力和增加利润的同时。也会带来一定的风险.如何分析和评价这些风险已成为供应链上核心企业所面临的重要问题。简述了EPC模式下的建筑供应链基本理论.在对EPC建筑供应链进行风险分析的基础上.设计了EPC模式下的建筑供应链风险评价指标体系.构建了基于支持向量机的建筑供应链风险评价模型并进行应用研究。

  20. 单链抗体-包膜蛋白嵌合型单嗜性逆转录病毒构建及靶向感染肿瘤细胞的研究%Tumor cell-specific gene transfer with retroviral vectors displaying single-chain antibody

    Institute of Scientific and Technical Information of China (English)

    汤宇澄; 李煜; 钱关祥

    2002-01-01

    Objective To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope.Methods Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22 Ⅰ/Sal Ⅰ was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypetide. Then the single-chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Ψ2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed.Results The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen.Conclusion These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.%目的将单链抗体基因插入单嗜性逆转录病毒胞膜蛋白,以构建能靶向感染肿瘤细胞的逆转录病毒载体。方法 构建单链抗体融合表达质粒PET20b-scFv,

  1. Integrated Operation Framework Model of Construction Supply Chain%工程项目供应链整合管理运作框架模型

    Institute of Scientific and Technical Information of China (English)

    白玉; 陈建华

    2011-01-01

    通过分析工程项目管理的不同模式,同时结合国内外建筑行业的市场环境、管理体制的差异性,分别从实体层次、基本要素架构和框架模型以及运作基本思想三个方面探究工程项目供应链整合及其管理运作问题.首先提出了核心层、中间层和外围层是工程项目供应链整合的三个基本实体层次;其次从项目"三全"角度出发,基于三个基本实体层次分析了工程项目供应链整合的基本要素作为框架模型的基础;再次具体分析工程项目供应链整合管理运作层次的螺旋式推进过程特征、供应链整合管理运作的阶段性特征以及三个基本实体之间的不同整合水平,剖析了工程项目供应链整合框架的基本环节和过程,最终构建了工程项目供应链整合管理运作的框架模型,并说明该模型运转的基本思想.%The supply chain integration and its management operational issues were explored by analyzing the different models of project management, combined with domestic and international market environment for the construction industry, the management system differences.The work was studied from three aspects of the physical level, the basic elements of architecture and framework of the model and the basic idea of the operation of the project, respectively.Firstly, basic organizational relationship levels consisting of the core level, the mid-level and the outer level of the supply chain integration of construction were presented from project life cycle perspective.From perspective of project total life cycle and total system and total process, an integrated element framework of the management on the supply chain integration was proposed.Considering different organizational relationship levels and integration levels, based on the strategic stages characteristics of the supply chain integration, step-up relations and the spire advancing process of integrated elements in operational level, a basic

  2. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Science.gov (United States)

    Harrington, Kimberly H; Gudgeon, Chelsea J; Laszlo, George S; Newhall, Kathryn J; Sinclair, Angus M; Frankel, Stanley R; Kischel, Roman; Chen, Guang; Walter, Roland B

    2015-01-01

    The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (Pspectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  3. 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...

  4. 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 ...

  5. 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...

  6. 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

  7. 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

  8. 绿色供应链绩效评价体系构建研究%Construction of Praise System for Green Chain of Supply

    Institute of Scientific and Technical Information of China (English)

    王媛媛; 王浩

    2013-01-01

    The paper illustrated the construction of green chain of supply in manufacturing sector in order to solve the man -agement for the chain of supply in the operation of an enterprise .It is inevitable for a manufacturer to extend the product life circle and reduce the production costs .%  近年来我国环境逐渐恶化,资缺乏,制造企业所承担的环保压力逐渐增大,企业已将目光逐渐放在如何降低产品生命周期,降低经营成本。有效的解决途径是在经营过程中实施有效的绿色供应链策略,拟对制造业绿色供应链绩效评价体系的构建进行简要阐述。

  9. 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

    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...

  10. 全人源抗前列腺癌ScFv抗体的筛选及生物学特性研究%Screening and biological characteristics study of human single chain Fv antibody against prostatic cancer

    Institute of Scientific and Technical Information of China (English)

    郑桂喜; 王传新; 张欣; 李伟; 杜鲁涛; 李娟; 刘慧

    2012-01-01

    目的 从已成功建立的前列腺癌噬菌体抗体库中筛选抗人前列腺癌单链抗体(ScFv),并分析其生物学特性.方法 以良性前列腺增生(BPH)细胞与噬菌体抗体库共孵育,去除抗体库中能够与BPH细胞结合的噬菌体抗体,再以前列腺癌细胞系DU145与去除非特异性结合后的噬菌体抗体库共孵育,通过“吸附-洗脱-扩增”对前列腺癌的噬菌体抗体库进行3轮的富集.采用流式细胞术筛选出对人前列腺癌细胞系DU145高亲合力的阳性克隆.阳性噬菌体克隆经亚克隆并转染大肠杆菌TG1,表达出可溶性ScFv抗体.采用流式细胞术、免疫荧光法分析其生物学特性.结果 以前列腺癌细胞系DU145为抗原,筛选出19个噬菌体抗体,经测定C11与DU145细胞亲合力最高,亚克隆至原核表达载体表达纯化为C11 ScFv抗体.流式细胞术和免疫荧光法检测结果均显示,C11ScFv抗体与前列腺癌细胞DU145和PC3结合,与BPH细胞不结合.结论 通过噬菌体展示技术筛选到前列腺癌细胞系高亲合力的可溶性ScFv抗体,为进一步研究抗体的靶向性治疗奠定了基础.%To screen the soluble ScFv antibodies which were specific for human prostatic cancer from the successfully constructed phage antibody library and analyse their biological characteristics. Methods Normal human noncancerous epithelial line BPH was used to deplete the phage library of nonspecific binders. Supernatant containing depleted phage library was then incubated with prostate cancer cells DU145. The phage library was enriched by three cycles of binding, elution and amplification. The prostate cancer cells DU145 was used to screen the positive clones with high affinity using FACS. The screening postive clone was sub-cloned and infected by TGI to get the soluble ScFv antibody. The biological characteristics of ScFv were identified by the method of FACS and immunofluorescence. Results 19 phage antibodies were gotten with the antigens

  11. Potential therapeutic use of antibodies directed towards HuIFN-gamma.

    Science.gov (United States)

    Froyen, G; Billiau, A

    1997-01-01

    IFN-gamma is an important regulator of immune responses and inflammation. Studies in animal models of inflammation, autoimmunity, cancer, transplant rejection and delayed-type hypersensitivity have indicated that administration of antibodies against IFN-gamma can prevent the occurrence of diseases or alleviate disease manifestations. Therefore, it is speculated that such antibodies may have therapeutical efficacy in human diseases. Since animal-derived antibodies are immunogenic in patients several strategies are being developed in order to reduce or abolish this human anti-mouse antibody (HAMA) response. In our laboratory, we have constructed a single-chain variable fragment (scFv) derived from a mouse antibody with neutralizing potential for human IFN-gamma. A scFv consists of only variable domains tethered together by a flexible linker. The scFv was demonstrated to neutralize the antiviral activity of HuIFN-gamma in vitro and therefore might be considered as a candidate for human therapy.

  12. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  13. 抗四环素单链抗体基因的构建与序列测定%Construction and Sequence Analysis of the Single-Chain Variable Fragment Gene Specific for Anti-Tetracycline

    Institute of Scientific and Technical Information of China (English)

    左伟勇; 王永娟; 陆辉; 洪伟鸣; 刘莉

    2013-01-01

    目的:构建抗盐酸四环素的单链抗体(scFv)基因。方法:以抗盐酸四环素单克隆抗体杂交瘤细胞株的总RNA为模板,用RT-PCR法扩增全套抗体轻、重链基因;经重叠延伸反应,以编码柔性多肽(Gly4Ser)3的基因为接头,将轻、重链基因组装为完整的scFv基因,并克隆到pGEMT-Easy载体中进行测序分析。结果:所克隆的四环素scFV基因全长为735 bp,为VH-Linker-VL结构,VH基因为354 bp,Linker为(Gly4Ser)3多肽的核酸序列,VL基因为336 bp。结论:构建了抗四环素的单链抗体基因,为进一步用于四环素的残留检测奠定基础。%Objective: To construct the single chain variable fragment(scFv) gene specific for anti-tetracycline. Methods: The total RNA was isolated from anti-tetracycline monoclonal hybridoma cell mRNA. Variable light and heavy domains of the immunoglobulin genes were amplified by PCR and assembled to produce full length of scFv by overlap extension PCR using a linker primer containing flexible polypeptide (Gly4Ser)3. The scFv fragment was cloned into the vector pGEMT-Easy and sequenced with auto-DNA sequencer. Results: Cloned scFv gene was the structure of VH-Linker-VL, consisting 735 bp. The VH gene was 354 bp, while the VL gene was 336 bp. The sequence of the linker was (Gly4Ser)3. Conclusion: We built the single-chain antibody genes for tetracycline, and lay the foundation for further used for the detection of tetracycline residues.

  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. 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.

  16. 抗黄曲霉毒素M1单链抗体基因克隆与蛋白结构模拟%Construction and structure prediction of murine single chain Fv gene against aflatoxin M1

    Institute of Scientific and Technical Information of China (English)

    李鑫; 李培武; 张奇; 张文; 雷佳文

    2012-01-01

    To construct the gene of single chain Fv ( scFv ) fragment against aflatoxin M, ,to predict secondary and tertiary structures of its encoding protein by computer assisted modeling and to analyze its physical and chemical properties. The gene of anti-aflatoxin M, scFv was constructed with 1B12 hybridoma cells that is able to secret monoclonal antibodies (mAb) with high activity and specificity against aflatoxin M1 as the raw material,and sequenced by splicing overlap exten-sion(SOE) PCR assay. Furthermore,its amino acid sequence was deduced and the secondary and tertiary structures were predicted using biological information software. Meanwhile, its physical and chemical properties were analyzed. The full-length scFv gene had 737 bp and encoded 245 amino acids. The relative molecular weight of the scFv was 25 897. 4, its predictive isoelectric point(pl) was 6. 90. The predicted secondary structure indicated that the protein contained 38 α-he-lix,24β-sheet,74 extended strand and 109 random coli. The mimic tertiary structure model of the scFv indicted that all the six CDRs of VL and VH were contribute to the antigen-binding site. The successful construction of a scFv gene against aflatoxin M, can laid the foundation for the further research into the expression, purification and bioactivity of genetic engineering antibody.%为构建抗黄曲霉毒素M1单链抗体(scFv)基因,并进行蛋白质结构模拟及理化特性分析.采用重叠延伸PCR方法,以能特异性分泌抗黄曲霉毒素M1单克隆抗体的杂交瘤细胞株1B12为原料,构建其单链抗体基因,进行测序,采用生物信息软件进行氨基酸序列推导、结构预测以及理化性质分析.抗黄曲霉毒素M1 scFv基因全长737 bp,对应245个氨基酸,单链抗体分子量约为25 897.4,等电点预测值6.90;二级结构显示抗黄曲霉毒素M1单链抗体含α螺旋38处,β折叠24处,延伸链74处,随机卷曲109处.三级结构建模显示重链(VH)和轻链(VL)的6个

  17. 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 ...

  18. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Directory of Open Access Journals (Sweden)

    Kimberly H Harrington

    Full Text Available The CD33/CD3-bispecific T-cell engaging (BiTE antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML, we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001 but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003. AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  19. 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

  20. 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. 

  1. 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

  2. scFv Antibody: Principles and Clinical Application

    Directory of Open Access Journals (Sweden)

    Zuhaida Asra Ahmad

    2012-01-01

    Full Text Available To date, generation of single-chain fragment variable (scFv has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

  3. MICA抗体与器官移植%Antibodies against major histocompatibility complex class Ⅰ-related chain A and organ transplantation

    Institute of Scientific and Technical Information of China (English)

    刘晓华; 杜占慧; 吴振军; 焦淑贤

    2014-01-01

    MICA是主要组织相容性复合体Ⅰ类分子链相关基因(major histocompatibility complex class Ⅰ chain related gene,MIC)家族的1个成员.MICA蛋白可与C型凝集素样活化性受体NKG2D结合,从而调节多种免疫效应细胞的功能.近年来MICA在器官移植中的重要作用引起越来越多的关注,一系列的研究结果证实MICA抗体与肾移植的存活率下降有关.本文就MICA基因的多态性,MICA蛋白的表达,MICA抗体的产生以及在器官移植中的作用进行了综述.

  4. [Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein].

    Science.gov (United States)

    Zhou, Rongping; Sun, Lina; Liu, Yang; Wu, Wei; Li, Chuan; Liang, Mifang; Qiu, Peihong

    2016-01-01

    The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

  5. 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.

  6. 纯化和标记抗人轻链β2m单克隆抗体的方法%Efficient purification and label of anti-human beta 2-microglobulin light chain monoclonal antibody

    Institute of Scientific and Technical Information of China (English)

    阮光萍; 姚翔; 庞荣清; 汪兴明; 戴莹; 潘兴华

    2011-01-01

    BACKGROUND: The inoculation of hybridoma cell strain onto mouse abdominal cavity may obtain ascites containing mass antibody. Previous method to purify monoclonal antibody in ascites is complex and difficult to operate.OBJECTIVE: To prepare, purify and label anti-human leukocyte antigen (HLA)-I class molecule light chain monoclonal antibody, and to detect the expression of tumor cell surface HLA-I class molecules. METHODS: Hybridoma cells were inoculated onto the mouse abdominal cavity. Ascites containing anti-human light chain beta2-microglobulin antibody were obtained and purified with the modified caprylic acid-ammonium sulfate method. The purified monoclonal antibody was labeled with fluorescein isothiocyanate to detect peripheral blood mononuclear cells, T2 cells expressing blank HLA-A2 molecule and K562 cells surface HLA-I class molecules. The expression of HLA-I class molecules was determined by using flow cytometry and fluorescent microscopy. RESULTS AND CONCLUSION: The purified anti-human light chain beta2-microglobulin-fluorescein isothiocyanate monoclonal antibodies accounted for 96% purity. Flow cytometry results showed that, the HLA-I class molecules were highly expressed in peripheral blood mononuclear cells surface, lowly expressed in T2 cells, and not expressed in K562 cells surface. It is a simple and convenient method to purify ascites with the modified caprylic acid-ammonium sulfate method, and according prepare anti-human light chain beta2-microglobulin-fluorescein isothiocyanate. This method is effective to distinguish the levels of HLA-I class molecules expressed in various cells.%背景:通过杂交瘤细胞株接种小鼠腹腔可获得含大量抗体的腹水,但以往纯化腹水中单克隆抗的方法较复杂,不易操作.目的:制备、纯化和标记抗人类白细胞抗原Ⅰ类分子轻链的单克隆抗体,以检测肿瘤细胞表面的人类白细胞抗原Ⅰ类分子的表达.方法:将杂交瘤细胞接种至小

  7. 甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析%Nested PCR Amplifying and DNA Sequence Analysis of Light and Heavy Chain Variable Region Gene of Monoclonal Antibody Against H1N1 Influenza Virus Hemagglutinin

    Institute of Scientific and Technical Information of China (English)

    李慧瑾; 胡军; 李研; 孙晶莹; 高锦伟; 赵向绒; 封青; 谭天天; 胡巧侠; 李元

    2013-01-01

    Objective: To clone and analysis light and heavy chain genes of monoclonal antibody against H1N1 influenza virus hemagglutinin by Nested PCR, then construct a common method for cloning light and heavy chain variable region genes of mouse anti-human H1N1 influenza virus hemagglutinin monoclonal antibody. Methods:We designed 22 pairs of primers for amplifying Igκ light and heavy chain variable region gene, and cloned light and heavy chain variable region genes of 6 mouse anti-human H1N1 influenza virus hemagglutinin monoclonal anti⁃bodies. Cloning and subsequent sequences analysis of immunoglobulin gene were performed, sequence alignment with mouse immunoglobulin in NCBI. Results: The nucleotide and corresponding amino acid sequences were in line with characteristics of mouse immunoglobulin variable region. Conclusion: In this study, we cloned 6 mouse antibody variable region genes, and found a common method for cloning immunoglobulin light and heavy chain variable region genes. It provides a basis for late amplifying monoclonal antibody variable region, and experimental data for analysis of H1N1 influenza virus hemagglutinin and antibody binding sites.%  目的:采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法:设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果:巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论:建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了

  8. 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

  9. 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.

  10. Catalyst-Directed Diastereoselective Isomerization of Allylic Alcohols for the Stereoselective Construction of C(20) in Steroid Side Chains: Scope and Topological Diversification.

    Science.gov (United States)

    Li, Houhua; Mazet, Clément

    2015-08-26

    The stereoselective construction of C20 in steroidal derivatives by a highly diastereoselective Ir-catalyzed isomerization of primary allylic alcohols is reported. A key aspect of this strategy is a straightforward access to geometrically pure steroidal enol tosylate and enol triflate intermediates for subsequent high yielding stereoretentive Negishi cross-coupling reactions to allow structural diversity to be introduced. A range of allylic alcohols participates in the diastereoselective isomerization under the optimized reaction conditions. Electron-rich and electron-poor aryl or heteroaryl substituents are particularly well-tolerated, and the stereospecific nature of the reaction provides indifferently access to the natural C20-(R) and unnatural C20-(S) configurations. Alkyl containing substrates are more challenging as they affect regioselectivity of iridium-hydride insertion. A rationale for the high diastereoselectivities observed is proposed for aryl containing precursors. The scope of our method is further highlighted through topological diversification in the side chain and within the polycyclic domain of advanced and complex steroidal architectures. These findings have the potential to greatly simplify access to epimeric structural analogues of important steroid scaffolds for applications in biological, pharmaceutical, and medical sciences.

  11. 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.

  12. Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease.

    Science.gov (United States)

    Bresson, D; Chardès, T; Chapal, N; Bès, C; Cerutti, M; Devauchelle, G; Bouanani, M; Mani, J C; Péraldi-Roux, S

    2001-01-01

    Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.

  13. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  14. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  15. 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。

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 抗人CD25嵌合抗体基因的构建及其瞬时表达研究%Study on construction and transient expression of human-mouse chimeric antibody gene against human CD25

    Institute of Scientific and Technical Information of China (English)

    胡迪超; 张爱华; 潘勇兵; 詹珊珊; 杨晓明

    2011-01-01

    目的:构建抗人CD25嵌合抗体基因并在哺乳动物细胞中进行瞬时表达和初步鉴定.方法:采用RLM-RACE法克隆WuTac抗体可变区和信号肽序列,并利用基因拼接法构建嵌合抗体基因.用脂质体法瞬时转染三种哺乳动物细胞,并使用ELISA、FCM、WB、Dot blot和免疫荧光法进行检测.结果:成功克隆WuTac抗体可变区和信号肽序列,并构建了抗人CD25嵌合抗体表达质粒.瞬时转染结果表明所表达的嵌合抗体保留了亲本抗体WuTac的抗原结合力.结论:成功构建了抗人CD25嵌合抗体基因,为其进一步研究打下基础.%Objective:To construct chimeric antibody gene against human an CD25 angigen,and prelin inarily identify the expressed prod-ucts produced from transiently transfected mammalian cells in order to facilitate the further study of stable expression.Methods:The RLM-RACE was employed to clone variable region genes and leader sequences,and the Overlap PVR method was used to construct the chimeric anti-body gene.After transiently transfected in three mammalian cells with liposome method, the expressed products were determined by ELISA,FCM,W B,Dotblot and immunofluorescence assay.Results:The variable region genes and leader sequences were successfully amplified,and the eukayotic expression plasmids were constructed.The results from transient transfection indicate the expressed products retain the antigen binding capacity of parental antibody WuTac.Conclusion:The successfully constructed chimeric antibody gene against human CD25 lays sound foun-dation for further study.

  1. Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

    Science.gov (United States)

    Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J

    2002-06-28

    MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for

  2. 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...

  3. 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。

  4. Construction and identification of an expressing vector for a large human naive phage antibody library%用于大容量人源天然噬菌体抗体库的表达载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    潘博; 陈斌; 童贻刚; 付文卓; 王晓娜; 张宝中; 米志强; 安小平; 刘大斌; 李存; 姜焕焕

    2012-01-01

    目的 构建一个应用于大容量Fab段天然噬菌体抗体库的表达载体.方法 用定点突变技术将表面呈现噬菌粒载体pDF上的BssHⅡ酶切位点改为BglⅡ酶切位点,然后分别在抗体轻链和重链位置插入自杀基因SacB,构建含自杀基因的噬菌粒载体pDF-D-SacB;利用抗乙肝表面抗原抗体的基因为模板,PCR扩增重链和轻链基因片段.将PCR扩增的轻链基因和重链基因分别插入载体pDF-D-SacB内,利用电转化的方法将其转入Transl-Blue大肠杆菌,构建2个初级质粒,再利用初级质粒超感染BS1365菌,使其轻链与重链发生重组,获得重组质粒,进一步获得抗乙肝表面抗原抗体的噬菌体.之后利用该噬菌体感染大肠杆菌Transl-Blue进行扩增,得到大量抗乙肝表面抗原的噬菌体抗体.最后,通过酶联免疫吸附剂测定检测所获得的抗体.结果 通过向pDF重轻链区域插入SacB基因,改造抗体基因克隆位点,构建了pDF-D-SacB载体;经检测,pDF-D-SacB可以表达具有功能的Fab噬菌体抗体,可以在分泌Cre蛋白酶的细菌胞内发生预期的Cre-Loxp介导的定点重组.结论 所获得的含自杀基因的噬菌粒载体pDF-D-SacB适用于构建大容量噬菌体抗体库.%Objective To construct an expression vector that can be applied to the construction of large Fab fragment phage display library. Methods BssH II restriction site in phagemid vector pDF was mutated to Bgl Ⅱ restriction sites by site-directed mutagenesis techniques, and a suicide gene SacB was inserted into both the heavy and light chain region, resulting in phagemid vector pDF-D-SacB. Heavy chain (VH) and light chain (VL) genes of anti-hepatitis B surface antigen (HBsAg) antibody were inserted into the phagemid vector pDF-D-SacB separately. The recombinant VH and VL phagemids were transformed into Transl-Blue E, coli by electroporation respectively. By co-infecting the VH and VL phagemids into bacteria BS1365 at high multiplicity

  5. 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.

  6. 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

  7. A Discussion on Information Flow Management of Construction Supply Chain Based on BIM Technology%基于 BIM技术的建筑供应链信息流管理的探讨

    Institute of Scientific and Technical Information of China (English)

    张韬; 孙波

    2016-01-01

    BIM 技术是目前建筑行业中的一种信息技术,正处于快速发展阶段,在 BIM 技术的基础上对建筑供应链信息流管理进行分析探讨不仅具有非常重要的理论意义,同时也具有较大的实践价值。以 BIM 技术为基础的建筑供应链管理是目前的发展趋势。本文根据BIM 技术在供应链信息流管理中应用所存在的问题,提出相应的解决措施,从而为建筑行业中的供应链信息流管理工作提供一些具有参考价值的建议。%As a kind of information technology in the construction industry ,BIM technology is in a rapid development stage .Discussions on information flow management of construction supply chain based on the BIM technology not only has a very important theoretical significance ,but also has a great practical value . Management of construction supply chain based on the BIM technology is the growing trend .This paper analyzes the existing problems in the information flow management of supply chain based on the BIM tech -nology and puts forward the corresponding measures to provide some suggestions of reference value for in -formation flow management of supply chain in the construction industry .

  8. 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

  9. 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

  10. 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

  11. Discussion on Key Problems and Counter Measures of Logistics Management in Construction Supply Chains%工程供应链中物流管理的关键问题与对策研究

    Institute of Scientific and Technical Information of China (English)

    李真; 杜建国; 孟庆峰

    2014-01-01

    Construction supply chain logistics management is quite important for performance. Firstly,according to the actual operation of project logistics,we build a conceptual model of construction supply chain logistics management. The characteristic and key issues in process of construction supply chain logistics management are proposed. In order to improve performance of construction supply chain logistics management,some strategies are proposed: constructing logistics operation principles like"integration","coordination" and "sharing";unified planning logistics systems and building reliable logistics network;building strategic partnerships with suppliers and integrating logistics transport capacity;building information sharing mechanisms between construction contractor and materials supplier. Those strategies aim to provide a theoretical basis for improving logistics and supply chain management project performance.%工程供应链物流管理对于工程系统绩效具有十分重要的意义。根据工程物流的实际运作情况,构建出工程供应链物流管理的概念模型并对其进行系统分析。在此基础上,提出了工程供应链物流管理的特点及其面临的关键问题;并遵循工程供应链物流管理的协调优化思想给出几点策略建议:构建“集成”、“协调”与“共享”的物流运作原则;统一规划物流运作系统,构建可靠的物流运作网络;与供应商构建战略伙伴关系,整合物流运营商的运输能力;构建施工承包商与物资供应商的信息共享机制等,旨在为提高工程供应链中物流管理的绩效提供理论依据。

  12. 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

  13. 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...

  14. 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...

  15. 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

  16. 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.

  17. 基于敏捷供应链管理的上海国际贸易中心建设%On Construction of Shanghai International Trade Center Based on Agile Supply Chain Management

    Institute of Scientific and Technical Information of China (English)

    孙浩

    2014-01-01

    国际贸易中心是全球贸易供应链网络中重要的枢纽点。从供应链管理的角度看,建设具有全球资源配置能力的上海国际贸易中心必须加强其供应链敏捷力建设。论文在分析企业敏捷供应链的含义与特点的基础上,剖析了上海国际贸易中心敏捷供应链的内涵,并结合上海国际贸易中心建设“十二五”发展规划,从构造制造型企业敏捷供应链,具有敏捷性的物流、贸易服务商、政府管理服务系统四个方面分别进行阐述,最后强调了构建一个统一的敏捷的电子信息平台系统应该具备的基本功能。%The international trade center is a crucial hub in the global trade supply chain network.In order to build Shanghai into an international trade center with global resource al-location ability,it is vital to enhance the supply chain agility in the trade network.This paper analyzes the connotation of agile supply chain of Shanghai International Trade Center,and e-laborates the ideas on how to construct the international trade center with agile supply chain from four aspects,i.e.,the construction of agile supply chain of manufacturing enterprises, the construction of agile logistics service system,trade service system and government man-agement system.Finally,this paper emphasizes the necessity of building a unified electronic information platform for agile supply chain management of Shanghai International Trade Cen-ter,and the basic functions of this unified electronic information platform.

  18. 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...

  19. 含F/2A序列的抗 P185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector Containing F/2AFragment for Anti-P185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2012-01-01

    目的 构建含 F/2A 序列的抗 P185erbB2 人鼠嵌合抗体慢病毒表达载体,观察其在 293T 细胞中的表达.方法 用具有自我剪切能力的弗林蛋白酶(Furin)/口蹄疫病毒2A多肽(F/2A)连接人鼠嵌合抗体的重链和轻链,形成一个开放阅读框 (ORF),插入慢病毒表达载体pWPI,构建重组抗P185erbB2全长人鼠嵌合抗体表达载体pWPI/H-F2A-L.以已构建的慢病毒表达载体pWPI/H-IRES-L为对照质粒.应用磷酸钙沉淀法将慢病毒载体 3 质粒系统共转染入 293T 细胞进行包装,测定病毒滴度.再感染 293T 细胞,荧光显微镜下观察 GFP 的表达和转染效率,RT-PCR、ELISA 方法分别检测嵌合抗体 mRNA和蛋白的表达.结果 经测序鉴定,pWPI/H-F2A-L与预期设计一致;pWPI/H-F2A-L组的病毒滴度为4.3×105 TU/ml,而pW-PI/H-IRES-L 组的病毒滴度为3.5×105 TU/ml;两组重组慢病毒的转染效率分别为 87.68%和 79.08%:两组重组慢病毒感染 293T 细胞后,都有嵌合重链和嵌合轻链的表达,由F/2A介导的嵌合抗体的表达水平要高于由 IRES 介导的嵌合抗体.结论 成功构建了含F/2A序列的抗P185erbB2人鼠嵌合抗体慢病毒表达载体,为今后抗P185erbB2工程抗体的研究奠定了基础.%Objective To construct the lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody , and detect its expression in 293T cells. Methods The heavy and light chains of chimeric antibody were joined by Furin and 2A ( F/ 2A) self-cleavage peptide and cloned into a lentiviral vector of pWPI , to generate the lentiviral ex-pression vector, pWPI/H-F2A-L Another lentiviral expression vector , pWPI/H4RES-L that had been generated already , was used as control plasmid. 293 T cells were co-transfected with the 3 helper plasmid system by using calcium phosphate precipitation , and then the virus titer was exam-ined. The 293 T cells were infected by the obtained lentiviral particles. The expression of

  20. 高职院校创业型连锁经营管理人才培养模式构建%Construction of Management Talents Training Mode of Entrepreneurial Chain Operation in Higher Vocational Colleges

    Institute of Scientific and Technical Information of China (English)

    陈晖

    2014-01-01

    构建人才培养模式是根据实际情况和社会需求,对人才培养目标、课程模式、教学设计、培养途径以及师资队伍等要素进行优化组合。文章基于连锁行业发展现状,对连锁经营管理专业人才的需求现实和连锁经营管理专业人才培养目标定位,提出了构建创业型连锁经营管理专业人才培养模式。%The construction of talents training mode which is based on the actual situation and needs of society is the optimization and combination of the talent training goal, curriculum model, teaching design, training methods and teachers. Based on the present development situation of chain industry, This paper locates the reality of talents demand and talents training goal of chain operation management, and puts forward the construction of management talent training mode of entrepreneurial chain operation.

  1. 生鲜乳制品冷链物流模式评价及构建研究%Researching on the Evaluation and Construction of Cold-chain Logistics Mode of Dairy Products

    Institute of Scientific and Technical Information of China (English)

    张建军

    2015-01-01

    Logistics mode of cold-chain concerning fresh milk and dairy products is an important content of cold-chain logistics about dairy products, which is the key to determine the security of quality about fresh milk and dairy products, stabilize the price of dairy products, improve cold-chain circulation rate of dairy product and ensure the economic benefit and service quality of the cold-chain logistics about dairy product. This paper constructs the evaluation index system of cold-chain logistics mode about fresh dairy product, uses the method of analytic hierarchy process to calculate the weight of each index, introduces the main factors that influence the f cold-chain logistics mode of fresh dairy product. Then , based on the main factors and the visualization of the perspective of supply chain, this paper puts forward the path to build a cold-chain logistics mode of dairy product, namely to construct the visual supply chain mode which should deem the dairy products production enterprise as the core enterprise , the third party or the fourth party cold-chain logistics as the main organization form. This mode can optimize the main factors concerning cold-chain logistics mode of dairy product, which is worthy of study and reference.%生鲜乳制品冷链物流模式是乳制品冷链物流的重要内容,是决定生鲜乳制品质量安全、稳定乳制品价格、提高乳制品冷链流通率和保证乳制品冷链物流经济效益和服务质量的关键。文章应用平衡计分卡原理构建生鲜乳制品冷链物流模式评价指标体系、利用层次分析法计算各评价指标的权重,进而分析得出影响生鲜乳制品冷链物流模式的主要因素。紧接着,围绕影响生鲜乳制品冷链物流模式的主要因素,基于可视化供应链的角度,提出构建乳制品冷链物流模式的路径,即构建以乳制品加工企业为核心企业,以第三方、第四方冷链物流为主要组织形

  2. Combinatorial antibody libraries: new advances, new immunological insights.

    Science.gov (United States)

    Lerner, Richard A

    2016-08-01

    Immunochemists have become quite proficient in engineering existing antibody molecules to control their pharmacological properties. However, in terms of generating new antibodies, the combinatorial antibody library has become a central feature of modern immunochemistry. These libraries are essentially an immune system in a test tube and enable the selection of antibodies without the constraints of whole animal or cell-based systems. This Review provides an overview of how antibody libraries are constructed and discusses what can be learnt from these synthetic systems. In particular, the Review focuses on new biological insights from antibody libraries - such as the concept of 'SOS antibodies' - and the growing use of intracellular antibodies to perturb cellular functions.

  3. 零售企业绿色供应链的构建%The Construction of Green Supply Chain in Retail Enterprises

    Institute of Scientific and Technical Information of China (English)

    马桂兰

    2011-01-01

    随着全球环保意识的增强和供应链管理的广泛应用,零售企业实施绿色供应链管理成为降低成本、挖掘利润、实现可持续发展的有效方法当前我国零售企业实施绿色供应链管理,以零售商为主导的绿色供应链主要包括的四个环节:绿色采购、绿色仓储、绿色营销和绿色逆向物流。%With the extensive application of environmental protection and the extensive application of supply chain management,the implementation of green supply chain managemcnt becomcs an cffective way for retail enterprises to reduce cost,mine profit,and achieve sustainable conditions for retail enterprises to implement green supply chain management,and proposes that green supply chain lcaded by retail cnterprises mainly includes four links:green procurement.green storage,green marketing and green reverse logistics.

  4. 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.

  5. Research on Construction and Application of the Model of Food Supply Chain Risk Evaluation%食品供应链风险评判模型的构建及应用研究

    Institute of Scientific and Technical Information of China (English)

    董千里; 李春花; 关高峰

    2012-01-01

    According to the phenomenon that emergency frequently happen in China's food industry, used related theory of Analytic Hierarchy Process, Fuzzy Evaluation Method, Membership and System Analysis, established a risk evaluation model, which reflected essential characteristics of food supply chain. Based on the formation mechanism and system analysis for food supply chain risk, formed a food supply chain risk evaluation index system,and constructed the food supply chain risk multi-grade fuzzy comprehensive evaluation model, judged risk degree of one class index factors and food supply chain system, with every factor risk degree sequence. And as an example, applied the model to a dairy production enterprise in food supply chain, for the risk of evaluation. The results of the study show that the accurate evaluation of food supply chain risk can determine more reasonable risk level, and help provide enterprise with reliable basis, reduce the happening of food supply chain emergency.%针对中国食品行业突发事件的频发现象,运用层次分析法、模糊评判法、隶属度和系统分析的相关理论,建立一个反映食品供应链本质特征的风险评判模型。基于对食品供应链风险的形成机理和系统分析,形成食品供应链风险评判指标体系,构建食品供应链风险多级模糊综合评判模型,判定一级指标各因素风险度和食品供应链系统风险度,并对各因素风险度排序。以某乳制品生产企业所处的食品供应链为例,对模型进行应用。研究结果表明,食品供应链风险的准确评判,可以确定更加合理的风险等级,有助于为企业提供可靠的风险规避依据,减少食品供应链突发事件的发生。

  6. 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.

  7. 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。

  8. 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。

  9. Targeting gene transfer to ovarian epithelial carcinoma cells with lentivirus displaying single-chain antibody%单链抗体导向卵巢上皮癌进行靶向基因 转移的实验研究

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective:To investigate the targeting gene transfer of Anti-MUC-1(ScFv) directed lentivirus to MUC-1+ ovarian epithelial carcinoma cells.Methods:MUC-1 single-chain antibody gene constructed in the plasmid pM.TC.G were transferred to packaging cell line 293T to produce anti-MUC-1-directed lentivirus using Ca3(PO4)2 precipitation method. Ovarian epithelial cancer cell line SKOV-3(MUC-1+) and normal human gingival fibroblast cell GF(MUC-1-)were co-cultured, then were infected by lentivirus. The infection result was observed through fluorescence microscopy. The competition assay that was the addition of the anti-MUC-1 single-chain antibody prior to lentivirus infection showed whether anti-MUC-1 Ab could inhibite infection. Results: After lentivirus infetion, cell SKOV-3 and GF which were co-cultured had remarkably different infection rate. The addition of the MUC-1 Ab prior to the lentivirus infection showed anti-MUC-1 Ab can inhibite infection.This competition assay showed that the infection is mediated by the antibody moiety and, therefore, is antibody specific. Conclusions:Anti-MUC-1(ScFv) directed lentivirus can targetingly transfer gene into MUC-1+ ovarian cancer cell line.%目的:探讨靶向性基因转移载体—含抗MUC-1单链抗体的慢病毒对MUC-1+卵巢上皮癌细胞系SKOV-3细胞进行基因转移的特异性,为卵巢上皮癌的靶向性基因治疗提供实验基础。方法:将针对SKOV-3细胞上抗原MUC-1的单链抗体基因构建到慢病毒包膜结构质粒(pM.TC.G),以报告基因绿色荧光蛋白(GFP)基因作为目的基因。用磷酸钙沉淀法进行Anti-MUC-1(ScFv)慢病毒包装,感染共培养的卵巢癌细胞SKOV-3(MUC-1+)和正常人成纤维细胞GF(MUC-1-),荧光显微镜下观察感染效果。竞争抑制实验即病毒感染前加入Anti-MUC-1单抗是否抑制病毒感染。结果:荧光显微镜下观察到Anti-MUC-1(ScFv)介导的慢病毒对共培养的SKOV-3(MUC-1+

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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。

  15. 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

  16. Falling chains

    Science.gov (United States)

    Wong, Chun Wa; Yasui, Kosuke

    2006-06-01

    The one-dimensional fall of a folded chain with one end suspended from a rigid support and a chain falling from a resting heap on a table is studied. Because their Lagrangians contain no explicit time dependence, the falling chains are conservative systems. Their equations of motion are shown to contain a term that enforces energy conservation when masses are transferred between subchains. We show that Cayley's 1857 energy nonconserving solution for a chain falling from a resting heap is incorrect because it neglects the energy gained when a link leaves a subchain. The maximum chain tension measured by Calkin and March for the falling folded chain is given a simple if rough interpretation. Other aspects of the falling folded chain are briefly discussed.

  17. 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...

  18. Early and late antibody responses to full-length and truncated constructs of outer surface protein A of Borrelia burgdorferi in Lyme disease.

    Science.gov (United States)

    Kalish, R A; Leong, J M; Steere, A C

    1995-06-01

    The immunoglobulin G (IgG) antibody response to outer surface protein A (OspA) of Borrelia burgdorferi has been reported to occur late in the course of Lyme disease. To learn when reactivity to particular epitopes of OspA develops and whether the strength of particular responses correlates with the duration of arthritis and HLA-DR specificities, we determined the IgM and IgG responses by enzyme-linked immunosorbent assay in 128 patients with various manifestations of Lyme disease to full-length recombinant OspA and three OspA fragments which divided the protein approximately into thirds. Among the 10 patients who were followed serially, an early IgM response was often found to epitopes in all three fragments of OspA, sometimes accompanied by a weak IgG response, primarily to an epitope in the middle third of the protein. Months to years later, the seven patients who had prolonged or moderate episodes of arthritis developed strong IgG responses to OspA, especially to epitopes in the N-terminal and C-terminal fragments, that paralleled the course of the arthritis. In single serum samples from 128 patients, a similar pattern of IgM and IgG reactivity with OspA epitopes was seen in patients with early or late manifestations of the illness. Of the 80 patients with arthritis, 62 (78%) had IgG responses to OspA, usually with the strongest reactivity to the C-terminal fragment. In these patients, the strength of the IgG response to OspA correlated with the duration of arthritis; in HLA-DR4-positive patients, most of whom had chronic arthritis, this association was attributable to reactivity with the C-terminal fragment. Thus, patients with Lyme disease often have early responses to OspA, but those with prolonged arthritis do not develop IgG responses to certain epitopes of the protein until late in the illness. In patients with HLA-DR4, the strength of IgG reactivity with one or more epitopes in the C-terminal fragment of OspA correlates with the duration of arthritis.

  19. 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

  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. 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.

  2. 基于跨境供应链整合的第三方物流海外仓建设%Third-party Logistics Construction of Oversea Location based on Cross-border Supply Chain Integration

    Institute of Scientific and Technical Information of China (English)

    鲁旭

    2016-01-01

    Thought the construction subject of oversea locations can be diversified, the strategic value of the third-party logistics’oversea location is increasingly prominent. From the perspective of cross-border supply chain, the service function extension commencing on middle-end warehousing and developing on tail-end distribution will have the qualitative change effect of integration on the whole cross-border supply chain if it continually expands to head-end transportation. However, the cross-border supply chain integration of the third party logistics is easily influenced by forwarder’s thought, expansion competition, oversea cooperation mode and international line’s development so that the inverse trend of integration is also inevitable. Only with the help of normalizing innovations, boosting construction, reducing intervention and adapting to environment, the oversea location ’s construction can develop along the direction of cross-border supply chain integration and thus play its important strategic role in the cross-border e-commerce era.%尽管海外仓的建设主体可以多样化,但是,以第三方物流公司为主体的海外仓建设的战略价值日益凸显。从跨境供应链的角度来看,第三方物流公司开始于中端仓储、发展于尾程配送的服务功能拓展,如果继续向头程运输延伸的话,就能够对整个跨境供应链产生整合质变的效果。然而,第三方物流公司的跨境供应链整合容易受到货代思维、扩张竞争、海外合作方式以及国际专线发展的影响,从而逆整合的发展趋势难以避免。只有规范海外仓创新、助力海外仓建设、减少海外仓干预、适应海外仓环境,第三方物流公司的海外仓建设才能沿着跨境供应链整合的方向发展,从而在跨境电子商务时代发挥其重要的战略作用。

  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. Construction and implementation the Data-Warehouse of Chain-SuperMarket Management Analysis System%超市经营分析系统数据仓库的创建与实现

    Institute of Scientific and Technical Information of China (English)

    侯超; 丁岳伟; 侯勇

    2011-01-01

    连锁超市在信息化建设的过程中,随着超市规模的扩大和数据的积累,超市数据仓库的建设已经尤为重要。本文根据连锁超市的现状和特点,设计并实现了针对超市管理的数据仓库,利用OLAP技术实现对数据的分析。在构建数据仓库过程中,重点研究了数据接口实现,数据模型构建,ETL分析等方面的内容。本系统在某超市的实际应用中,取得了良好的效果。%In the process of Infromation-Building in Chain-SuperMarket , data warehouse construction has been particularly important。Accord to chain-supermarkets under the present situation and characteristics, this paper design and implement data warehouse for the supermarket management . Also this system Use OLAP technology for data analysis . Duiring building the data-warehouse process , this paper pay attation on data model construction ,ETL analysis and interface . In the practical of a supermarket ,the system achieved satisfied result.

  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. A Critical Success Factors Model for Construction Supply Chain of EPC: Development and Empirical Study%总承包模式下工程项目供应链关键影响因素研究

    Institute of Scientific and Technical Information of China (English)

    段志成; 陈通; 张巧云

    2012-01-01

    基于文献检索和专业访谈,提炼出20项总承包模式下工程项目供应链关键影响因素,并提出假设.通过问卷调查,使用SPSS进行描述性分析和R型聚类分析,验证并修正了假设.结果表明:20项关键因素体现在战略一致性、协作的有效性、过程有效性、态度有效性和资源保障等5个维度.最后给出了提高工程项目供应链绩效的管理策略.%Based on literature reviews and interviews with project managers, 20 factors were identified as critical success factors of construction supply chain under EPC, and the hypothesis was given. With development of questionnaire, the data was analyzed by the SPSS to verify the hypothesis, finding that 20 factors were critical and could be divided into five dimensions: strategy, cooperation, process, attitude and recourse. Finally, management strategies were suggested to improve the performance of construction supply chain.

  7. 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.

  8. Process and system innovation in the building and construction industry: Developing a model for integrated value chain and life cycle management of built objects

    NARCIS (Netherlands)

    Ridder, H.A.J. de; Vrijhoef, R.

    2004-01-01

    The building and construction industry has a large contribution and impact on society, e.g. economical and environmental, involving a vast spectrum of stakeholders. However, the value delivering performance of the industry has often been criticized. The predictability of the value, price and costs o

  9. 农村连锁超市客户配送体系建设研究%Study on Construction of Rural Chain Supermarket Customer Distribution System

    Institute of Scientific and Technical Information of China (English)

    郭玉杰

    2012-01-01

    在商务部“万村千乡市场工程”的推动下,我国农村连锁超市有了巨大的发展.农村地区由于受特殊的经济和社会条件限制,对连锁超市的客户配送有着与城市不同的要求.只有建设完善的客户配送体系,农村连锁超市才能不断向纵深发展,创造更多的经济和社会效益.%In this paper we argued that due to the unique economic and social conditions of the rural area, customer distribution of chain supermarkets there are under different requirements as that in the cities and only through building a complete customer distribution system could chain supermarkets in rural areas create more economic and social benefits.

  10. 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.

  11. 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.

  12. 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

  13. 基于网络治理视角的供应链企业社会责任研究%Research on Enterprise Social Responsibility Construction in Supply Chain Based on the Perspectives of Network Governance

    Institute of Scientific and Technical Information of China (English)

    周翼翔

    2015-01-01

    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。%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.

  14. 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。

  15. 风险环境中基于Supply-Hub的建筑工程供应链协同模型优化%Optimization of Construction Project Supply Chain Collaboration Model in Risky Environment Based on Supply-hub

    Institute of Scientific and Technical Information of China (English)

    卫飚; 李毅鹏

    2014-01-01

    In this paper, we proposed the collaboration model of a construction project supply chain based on supply-hub, then adopted the Monte Carlo simulation process to simulate the whole process of the supply chain, and found that, as compared to the traditional non-collaborative models, the supply-hub based collaborative model could remarkably reduce the supply chain cost and improve the total profit and performance of the supply chain.%考虑由多个建筑材料供应商、单个总承包商和业主所组成的建筑工程供应链。在供应商的材料供应和业主的需求都存在不确定性的环境中,总承包商采取了三种不同的应对风险的态度偏好:风险中性、延误规避和浪费规避。提出了基于Supply-Hub的建筑工程供应链协同模型,并采用Monte Carlo仿真模拟供应链的运作全过程。数据结果显示:采用了Supply-Hub的协同模型相比于传统的非协同模型,有效地减低了供应链的成本、提高了供应链的总利润和绩效、整个建筑工程供应链达到了帕累托改善。

  16. 现代服务业供应链整合与上海国际贸易中心建设%Integration of supply chain of modern service industry and the construction of Shanghai's international trade centre

    Institute of Scientific and Technical Information of China (English)

    黄丙志

    2012-01-01

    In the context of economic globalization, supply chain is increasingly becoming a mainstream mode of production. The capabilities of supply chain management are particularly important. This article investigated Shanghai's business services, financial services, logistics services and information services, analyzed the present situation and problems of the development of the integration of the supply chain during the construction of Shanghai's international trade centre and put forward the suggestions to enhance the ability to integrate the supply chain of modern service industry in Shanghai.%在当今经济全球化背景下,供应链日益成为产业组织的一种主流生产模式,高端与专业性服务业的供应链整合尤为重要。服务企业能否形成以其为核心的供应链,关键取决于各自的供应链管理能力。通过考察上海商贸、金融、物流、计算机与信息服务业等涉及国际贸易中心建设的关键服务行业,在分析上海国际贸易中心建设中现代服务业供应链整合发展的现状与存在问题的基础上,提出上海增强服务业供应链整合能力的对策建议。

  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. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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...

  3. 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.

  4. Unwrapping Chains

    CERN Document Server

    Cambou, A D; Hamm, E; Hanna, J A; Menon, N; Santangelo, C D; Walsh, L

    2012-01-01

    A loop of chain can move along its own tangents, maintaining a steady shape. An open-ended chain undergoing a nontrivial motion must change its shape. One consequence is that chains pulled around objects will fail to follow the contours of the objects, unwrapping themselves instead. This short note accompanies a fluid dynamics video submission (83068) to the APS DFD Gallery of Fluid Motion 2012.

  5. Falling chains

    CERN Document Server

    Wong, C W; Wong, Chun Wa; Yasui, Kosuke

    2006-01-01

    The one-dimensional falling motion of a bungee chain suspended from a rigid support and of a chain falling from a resting heap on a table is studied. Their Lagrangians are found to contain no explicit time dependence. As a result, these falling chains are conservative systems. Each of their Lagrange's equations of motion is shown to contain a term that enforces energy conservation when masses are transferred between subchains. We show in particular that Cayley's 1857 energy nonconserving solution for a chain falling from a resting heap is incorrect because it neglects the energy gained when the transferred link is emitted by the emitting subchain. The maximum chain tension measured by Calkin and March for the falling bungee chain is given a simple if rough interpretation. In the simplified one-dimensional treatment, the kinetic energy of the center of mass of the falling bungee chain is found to be converted by the chain tension at the rigid support into the internal kinetic energy of the chain. However, as t...

  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. 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

  8. 建筑供应链中基于空间约束的多供应商横向协同研究%Research on Multi-suppliers Horizontally Synchronization Based on Space-constrained Construction Supply Chain

    Institute of Scientific and Technical Information of China (English)

    李毅鹏; 马士华

    2013-01-01

    A construction supply chain consisting of multi-suppliers and one main contractor is considered, in the context of constrained building material space. How multi uncertain suppliers horizontally synchronized supply to reduce the main contractor's expected total cost is identified. Through information sharing among supply chain, multi-suppliers horizontally synchronized basic model is presented. Then through capacity outsourcing or expediting, multi-suppliers horizontally synchronized advanced model is presented. Finally, the whole proceed of building material supply is simulated by Monte Carlo methodology. The result shows: compared with traditional supply model, these two synchronized model can decrease the main contractor's expected total cost effectively. The basic model's cost is minimum, and the advanced model can reduce the cost of construction duration delay. Pareto improvement of construction supply chain is achieved.%针对由多个建筑材料供应商和一个总承包商组成的建筑供应链,研究了在建筑材料存放空间约束的条件下,不确定的多个供应商如何进行横向地协同供应,从而使得总承包商的期望总成本最低的问题.通过供应链中的信息共享,建立了多供应商横向协同的基本模型;再通过产能外包或加班赶工的方式,建立了多供应商横向协同的高级模型,并给出了模型的求解步骤.最后使用了Monte Carlo方法来模拟材料供应的整个过程,仿真结果显示:相对于传统的供应模型,两种协同模型均能有效地减低总承包商的期望总成本.基本模型所花费的代价最小,高级模型能够显著降低建筑工期延误成本,从而实现建筑供应链的帕累托改善.

  9. ELUCID - Exploring the Local Universe with reConstructed Initial Density field I: Hamiltonian Markov Chain Monte Carlo Method with Particle Mesh Dynamics

    CERN Document Server

    Wang, Huiyuan; Yang, Xiaohu; Jing, Y P; Lin, W P

    2014-01-01

    Simulating the evolution of the local universe is important for studying galaxies and the intergalactic medium in a way free of cosmic variance. Here we present a method to reconstruct the initial linear density field from an input non-linear density field, employing the Hamiltonian Markov Chain Monte Carlo (HMC) algorithm combined with Particle Mesh (PM) dynamics. The HMC+PM method is applied to cosmological simulations, and the reconstructed linear density fields are then evolved to the present day with N-body simulations. The constrained simulations so obtained accurately reproduce both the amplitudes and phases of the input simulations at various $z$. Using a PM model with a grid cell size of 0.75 Mpc/h and 40 time-steps in the HMC can recover more than half of the phase information down to a scale k~0.85 h/Mpc at high z and to k~3.4 h/Mpc at z=0, which represents an improvement of a factor of two to three in spatial scale over similar reconstruction models in the literature, and indicates that our model ...

  10. o-, m-, and p-Pyridyl isomer effects on construction of 1D loop-and-chains: Silver(I) coordination polymers with Y-type tridentate ligands

    Science.gov (United States)

    Kim, Jeong Gyun; Cho, Yoonjung; Lee, Haeri; Lee, Young-A.; Jung, Ok-Sang

    2016-10-01

    Self-assembly of silver(I) hexafluorophosphate with unique Y-type tridentate ligands (2,6-bis[(2-picolinoyloxy-5-methylphenyl)methyl]-p-tolylpicolinate (o-L), 2-nicotinoyloxy- (m-L), and 2-isonicotinoyloxy- (p-L)) produces single crystals consisting of 1D loop-and-chain coordination polymers of [Ag(o-L)](PF6)·Me2CO·CHCl3, [Ag(m-L)](PF6)·Me2CO, and [Ag3(p-L)2](PF6)3·2H2O·2C2H5OH·4CH2Cl2 with quite different trigonal prismatic, trigonal, and linear silver(I) coordination geometry, respectively. Coordinating ability of the three ligands for AgPF6 is in the order of p-L > o-L > m-L. The solvate molecules of [Ag(o-L)](PF6)·Me2CO·CHCl3 can be removed, and be replaced reversibly in the order of acetone ≫ chloroform ≈ dichloromethane ≫ benzene, without destruction of its skeleton.

  11. Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments.

    Science.gov (United States)

    Nishi, Michiru; Jian, Nan; Yamamoto, Keiko; Seto, Haruyo; Nishida, Yuichi; Tonoyama, Yasuhiro; Shimizu, Nobuyoshi; Nishi, Yoshisuke

    2014-10-01

    In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×10(8)cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or

  12. 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

  13. 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

  14. 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.

  15. 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。

  16. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies.

    Science.gov (United States)

    Stöger, E; Vaquero, C; Torres, E; Sack, M; Nicholson, L; Drossard, J; Williams, S; Keen, D; Perrin, Y; Christou, P; Fischer, R

    2000-03-01

    This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

  17. 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.

  18. 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

  19. Resource Service Chain Construction for Networked Manufacturing Based on FAHP and Improved Ant Colony Algorithm%基于FAHP和改进蚁群算法的网络化制造资源链构建研究

    Institute of Scientific and Technical Information of China (English)

    王正成; 谢先文

    2012-01-01

    网络化制造资源集成共享与优化配置主要包括协同制造总任务的分解、单任务驱动的制造资源的评价选择、时序约束关联单任务链驱动制造资源链的构建三个阶段。首先根据协同制造总任务分解的特点,提出了跨组织协同制造任务分解过程模型和相应的分解算法。然后提出了单任务约束驱动网络化制造资源评价指标体系与评价算法,在此基础上对单任务驱动检索的候选资源集,提出了基于改进蚁群算法的时序约束关联单任务链驱动网络化制造资源服务链构建模型与算法。最后通过仿真算例说明了研究成果的有效性。%The nature of networked manufacturing resource integration and optimized configuration is a complex process.In this process,total collaborative manufacturing tasks can be decomposed to construct a single-task-driven chain according to time and sequence constraints in cross-organizational.The process mainly included three stages:the decomposition of the collaborative manufacturing tasks;the evaluation and selection of the manufacturing resources and the construction of the single-task-driven manufacturing resource chain.Firstly,according to the features of the collaborative task decomposition,a process model for cross-organizational collaborative manufacturing and corresponding decomposition algorithm were proposed.Secondly it also put forward an index system and evaluation algorithm for the networked manufacturing resources evaluation.Then the networked manufacturing resource service chain model and the algorithm which was based on improved ant colony algorithm with single task-driven related timing constrains were proposed.Finally,a simulation was taken to verify the feasibility of this research.

  20. 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

  1. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  2. 链条式实践类公共选修课程建设与思考%Construction and Thinking of Chain-style Practical Common Elective Courses

    Institute of Scientific and Technical Information of China (English)

    王伞; 赵旦峰; 刘文智; 刘日起

    2012-01-01

    The construction of practical common elective courses and the promotion of students' science and technology innovation is a novel idea in teaching reform. Based on the example of the chain-type common elective courses in the Electrical and Electronic Experiment Teaching Center of Harbin Engineering University, this paper solved the problems of the construction scheme, the content, and operation mode of these courses. It put forward the idea of offering open courses and changing some courses into contests. At the same time, it elaborated on creative education.%建设实践类公共选修课程,推进学生科技创新是教学改革的一个新思路.现以哈尔滨工程大学电工电子实验教学中心链条式公共选修课为例,解答了该类课程的建设方案、内容设置、运行模式等问题;提出了课程开放、课程变竞赛等创新点,同时针对创新教育等话题进行探讨.

  3. 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。

  4. Anti-DNA antibodies--quintessential biomarkers of SLE.

    Science.gov (United States)

    Pisetsky, David S

    2016-02-01

    Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

  5. Research into Construction and Optimization about Knowledge Network in Industry Technology Chain%新能源产业技术链知识网络构建及优化研究

    Institute of Scientific and Technical Information of China (English)

    王晰巍; 李向军; 王维; 李连子; 王科理

    2012-01-01

    在对产业技术链及知识网络的国内外研究现状进行整理分析的基础上,分析了新能源产业技术链进行知识网络研究的必要性;构建了新能源产业技术链的概念模型和知识网络三层框架。并分析了知识网络传递中存在的瓶颈问题;运用仿生学中粒子群算法构建了新能源产业技术链的知识网络优化模型,并对模型优化结果进行了分析。在理论层面延展了产业技术链在特定产业领域的纵深化研究,在实践层面可指导新能源不同组织之间更好地构建知识网络,以实现新能源产业领域的知识创新。%Based on analyzing current research status about the industry technology chain and knowledge network, the paper analyzes the necessity of knowledge networks in the New Energy Industrial Technology chain; and construct conceptual model and three levels framework about knowledge network in it, and analysis existing bottlenecks in the transmission of knowledge network. It constructs optimization model of knowledge network using particle swarm optimization in bionics field, and analyze the optimization results of model. It extends the depth of research in the field of specific-industry from theoretical level, and guide to construct knowledge network among different organizations of the new energy from practical level, in order to achieve knowledge innovation about in the field of new energy industry.

  6. Specificity of human anti-variable heavy (VH ) chain autoantibodies and impact on the design and clinical testing of a VH domain antibody antagonist of tumour necrosis factor-α receptor 1.

    Science.gov (United States)

    Cordy, J C; Morley, P J; Wright, T J; Birchler, M A; Lewis, A P; Emmins, R; Chen, Y Z; Powley, W M; Bareille, P J; Wilson, R; Tonkyn, J; Bayliffe, A I; Lazaar, A L

    2015-11-01

    During clinical trials of a tumour necrosis factor (TNF)-R1 domain antibody (dAb™) antagonist (GSK1995057), infusion reactions consistent with cytokine release were observed in healthy subjects with high levels of a novel, pre-existing human anti-VH (HAVH) autoantibody. In the presence of HAVH autoantibodies, GSK1995057 induced cytokine release in vitro due to binding of HAVH autoantibodies to a framework region of the dAb. The epitope on GSK1995057 was characterized and dAbs with reduced binding to HAVH autoantibodies were generated; pharmacological comparability was determined in human in-vitro systems and in-vivo animal experiments. A Phase I clinical trial was conducted to investigate the safety and tolerability of the modified dAb (GSK2862277). A significant reduction in HAVH binding was achieved by adding a single alanine residue at the C-terminus to create GSK2862277. Screening a pool of healthy donors demonstrated a reduced frequency of pre-existing autoantibodies from 51% to 7%; in all other respects, GSK2862277 and the parent dAb were comparable. In the Phase I trial, GSK2862277 was well tolerated by both the inhaled and intravenous routes. One subject experienced a mild infusion reaction with cytokine release following intravenous dosing. Subsequently, this subject was found to have high levels of a novel pre-existing antibody specific to the extended C-terminus of GSK2862277. Despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects associated with the presence of a novel pre-existing antibody response specific to the modified dAb framework were identified and highlight the challenge of developing biological antagonists to this class of receptor.

  7. Detection of oligoclonal IgG kappa and IgG lambda bands in cerebrospinal fluid and serum with Hevylite™ antibodies. comparison with the free light chain oligoclonal pattern

    OpenAIRE

    Zeman David; Hradílek Pavel; Švagera Zdeněk; Mojžíšková Eva; Woznicová Ivana; Zapletalová Olga

    2012-01-01

    Abstract Background Oligoclonal IgG bands in cerebrospinal fluid that are absent in serum indicate intrathecal IgG synthesis and are a sensitive marker of CNS inflammatory diseases, in particular multiple sclerosis. It may be of interest to determine whether these bands are predominantly IgGκ or IgGλ. Methods We have used Hevylite™ antibodies and developed a technique for detection of oligoclonal IgGκ and IgGλ bands by means of isoelectric focusing followed by immunoblotting. The same techniq...

  8. 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.

  9. Engineering Construction of Military Supply Chain on the Basis of Informatization Warfare%基于信息化战争的军事供应链工程建设

    Institute of Scientific and Technical Information of China (English)

    祝尔坚

    2012-01-01

      The modern warfare is more dependent on the war energy. It is not only focus on the use of high-tech, including information technology and other tools, to increase the efficiency in the use of war energy in the battlefield, but also work on the conversion efficiency and quality from the economy energy to the war energy. In this regard, we shall pay particular attention to military acquisition which is acting as a bridge in the process of transition from economy energy to war energy. The acquisition department will be in close contact with other functionalities, e.g.: logistics, technology support and other services departments to display the supporting function of the military supply chain in future battlefield. The modern information warfare is becoming to be more and more stereoscopic with a high speed pattern which is in depth and excluding the line operation. It is driving the war movement into a multi-dimensional area. The original line-operation model, i.e. working on the front line, did not exist any more. The joint service on war is much more preferred. The recent practice showed that the logistics support is expected to play a very special role in the future war. No matter how advanced the weaponry will come to be, as long as there is no immediate, sufficient and precise support from logistics side, the tactical arrangements/operations as well as the combat effectiveness cannot be commenced smoothly. The information technology as a philosophy, which is influencing and infiltrating the various fields of this era, is maturing itself in a dynamic development. In the field of logistics support, the application of information technology is an enhanced technology to develop the resource space, improve the efficiency of allocation and to ensure an effective materials transportation and resource usage. The main focus and further development of information technology is aiming at achieving the optimal efficiency in military economy by constructively settling

  10. 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。

  11. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  12. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  13. Antibody structural modeling with prediction of immunoglobulin structure (PIGS)

    DEFF Research Database (Denmark)

    Marcatili, Paolo; Olimpieri, Pier Paolo; Chailyan, 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 appl...... 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. 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.

  15. Chain Gang

    Science.gov (United States)

    2006-01-01

    6 August 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a chain of clustered and battered craters. These were formed by secondary impact. That is, somewhere to the south (beyond the bottom of this image), a large impact crater formed. When this occurred, material ejected from the crater was thrown tens to hundreds of kilometers away. This material then impacted the martian surface, forming clusters and chains of smaller craters. Location near: 15.8oN, 35.6oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Northern Spring

  16. 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%.

  17. Design and practice of regional health informatization by constructing a"collaborative service chain"%区域医疗信息化“协同服务链”建设模式的设计与实践

    Institute of Scientific and Technical Information of China (English)

    蒋璐; 赵强; 王竞; 董瑞国

    2014-01-01

    The paper first analyzes the development of regional health informatization and the existing construction mode .Modern Education Technology Center of Xuzhou Medical College , using its advantages , unique and innovative ideas , planned , implemented and completed the construction of "Viaduct-Association-New Specialty".It built a service chain collaborative construction mode that can meet the requirements of local regional health informatization as well as a network viaduct and information integration platform for regional health informatization , established a management organization and carried out personnel training .Practice proves that these can help improve the quality of health care and promote cooperation and collaboration between medical institutions and medical personnel and the construction of regional health informatization .%在分析区域医疗信息化发展与现有建设模式基础上,徐州医学院现代教育技术中心经历了约6年时间的探索与努力,利用自身优势,以独特的构想、创新的理念,策划、实施并完成了“高架桥-协会-新专业”的建设,构建了符合当地区域医疗信息化要求的“基础设施-信息资源-人才发展”服务链式的协同建设模式及为区域医疗信息化搭建网络高架桥和信息整合平台,建立组织管理机构,进行人才培养。实践证明,该模式有利于提高医疗质量、促进各医疗机构、医护人员的协同合作及区域医疗信息化的建设。

  18. Detection of oligoclonal IgG kappa and IgG lambda bands in cerebrospinal fluid and serum with Hevylite™ antibodies. comparison with the free light chain oligoclonal pattern

    Directory of Open Access Journals (Sweden)

    Zeman David

    2012-02-01

    Full Text Available Abstract Background Oligoclonal IgG bands in cerebrospinal fluid that are absent in serum indicate intrathecal IgG synthesis and are a sensitive marker of CNS inflammatory diseases, in particular multiple sclerosis. It may be of interest to determine whether these bands are predominantly IgGκ or IgGλ. Methods We have used Hevylite™ antibodies and developed a technique for detection of oligoclonal IgGκ and IgGλ bands by means of isoelectric focusing followed by immunoblotting. The same technique was used for oligoclonal free κ and free λ detection. Among several techniques tested, affinity immunoblotting appears to be the most sensitive; it can detect less than 1 ng of IgGκ or IgGλ paraprotein. We compared oligoclonal IgG profiles with those of oligoclonal IgGκ and IgGλ. There was good agreement concerning the presence or absence of intrathecal synthesis. We observed the ratios between oligoclonal IgGκ and IgGλ bands, and they did not always match the ratios between free κ and free λ bands. We were also able to detect antigen-specific CSF-restricted oligoclonal IgGκ and IgGλ bands in neuroborreliosis. It remains to be determined subsequently by a clinically-oriented prospective study, whether predominant IgGκ/IgGλ or free κ/free λ can be observed more frequently in particular diseases with oligoclonal IgG synthesis. Discussion Very sensitive detection of oligoclonal IgGκ and IgGλ bands in cerebrospinal fluid with Hevylite antibodies is feasible; detection of antigen-specific IgGκ or IgGλ is possible as well. In particular situations, e.g. when difficulties arise in distinguishing between oligoclonal and monoclonal pattern, the test may be of considerable clinical value.

  19. 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.

  20. 多中心特异性MICA抗体的检测结果及临床应用价值分析%The analysis of anti-major histocompatibility complex class Ⅰ-related chain A(MICA) specific antibodies testing results in multicenter and its clinical applications

    Institute of Scientific and Technical Information of China (English)

    袁晓妮; 何军; 侯建全; 鲍晓晶; 徐超; 李杨; 缪竞诚

    2014-01-01

    Objective To research the consistency of testing results with three different antimajor histocompatibility complex class Ⅰ-related chain A(MICA) specific antibody reagents in order to evaluate their clinical application's value.Method An collaborative study of 18 laboratories was undertaken at the 16th International HLA and Irnmunogenetics Workshop.Total of 16 sera(4 batchs)were tested for anti-MICA antibodies by Luminex method with three different reagents (Kit-A,-B and -C).Result Anti-MICA antibodies were found in 15 sera,except one sera(no.S04) ; No.S10 sera showed positive results in all the laboratories.The anti-MICA antibodies were divided into MICA-G1 group (MICA01,02,07,12,17 and 18) and MICA-G2 group (MICA 04,06,08/27,09 and 19).MICA-G1 group specific antibodies were detected in 5 sera with Kit-A and-B reagent; but there were false-positive results of anti-MICA08/27 and MICA19 antibodies in this 5 sera with Kit-C.MICA-G2group specific antibodies can be detected in other 5 sera with Kit-A and-B,But the MICA specific antibodies testing gave different results with Kit-A,-B and-C in all the last 5 sera samples.Testing of MICA08/27 showed highest consistency results (86.67%,13/15) with Kit-A,-B and-C; and testing of MICA19 showed lowest consistency results (40%,6/15) with this 3 reagents.There were 80% consistency results of anti-MICA specific antibodies in 13 sera with Kit-B.Conclusion There are the same effect to judgment positive or negative result for anti-MICA antibodies with 3 different reagents,but the results of anti-MICA specific antibodies are not the same.Therefore,it's better to use two or more reagents to test anti-MICA specific antibodies,or choose reagent with wide detection range.%目的 对比3种人主要组织相容性复合物Ⅰ类相关链A位点(MICA)特异性抗体检测试剂结果的一致性,预测其临床推广应用的价值.方法 18家实验室参加16届国际组织相容性和免疫协作组,均采用免疫磁珠流式

  1. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  2. The Construction and Innovation of Business Models of Mobile Payment on The Basis of Industry Chain Integration%基于产业链整合的移动支付商业模式的构建与创新

    Institute of Scientific and Technical Information of China (English)

    芦阳

    2012-01-01

    随着移动支付技术的快速发展和应用范围的不断扩大,我国移动支付产业也进入到培育商业模式的关键时期。由于移动支付产业链的复杂性和多样性,运营模式尚不统一。本文提出,应当构建起银行、运营商、第三方支付机构三方结盟的模式,从而多方共赢。%The development of mobile payment in our country has been the critical period to form the business models along with the rapid development of technology and expansion of business scope.Because of the complexity and diversity of mobile payment industry chain and factors such as policy and market circumstances,the efficient business models have not formed.It is to suggest that it should construct the integrated business models of strategic alliances which is based on the cooperation of mobile operators and banks and is assisted by the organizations of third-party payment to share resource and complement each other's advantages.

  3. Research on Construction Process of Manufacturing Collaborative Chain for Task with Time Sequence Constraint%面向时序约束任务的协同制造链构建过程研究

    Institute of Scientific and Technical Information of China (English)

    程方启

    2013-01-01

    In order to realize multi-enterprise collaboration for manufacturing task , it builds the model of direct-ed graph for task with time sequence constraint based on the concept of collaborative manufacturing chain (CMC).Based on the study of the mathematical model of construction process for CMC , it analyzes the building process and the characteristics of canonical and non -canonical CMCs .The analyzing results for the different forms of CMC are benefit to the multi -enterprise collaboration for manufacturing task .%为实现制造任务的多企业协作,基于协同制造链的概念建立了时序约束任务有向图模型,系统研究了协同制造链构建过程的数学建模问题,分析了规范型与非规范型协同制造链的构建过程及其特点。通过对协同制造链的不同构建模式的分析,有助于实现多企业间制造任务的协作。

  4. 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.

  5. 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

  6. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene

    2012-02-01

    Full Text Available Biologics such as monoclonal antibodies (mAb and soluble receptors represent new classes of therapeutic agents for treatment of several diseases. High affinity and high specificity biologics can be utilized for variety of clinical purposes. Monoclonal antibodies have been used as diagnostic agents when coupled with radionuclide, immune modulatory agents or in the treatment of cancers. Among other limitations of using large molecules for therapy the actual cost of biologics has become an issue. There is an effort among chemists and biologists to reduce the size of biologics which includes monoclonal antibodies and receptors without a reduction of biological efficacy. Single chain antibody, camel antibodies, Fv fragments are examples of this type of deconstructive process. Small high-affinity peptides have been identified using phage screening. Our laboratory used a structure-based approach to develop small-size peptidomimetics from the three-dimensional structure of proteins with immunoglobulin folds as exemplified by CD4 and antibodies. Peptides derived either from the receptor or their cognate ligand mimics the functions of the parental macromolecule. These constrained peptides not only provide a platform for developing small molecule drugs, but also provide insight into the atomic features of protein-protein interactions. A general overview of the reduction of monoclonal antibodies to small exocyclic peptide and its prospects as a useful diagnostic and as a drug in the treatment of cancer are discussed.

  7. 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...

  8. Towards Intelligent Supply Chains

    DEFF Research Database (Denmark)

    Siurdyban, Artur; Møller, Charles

    2012-01-01

    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...... 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......, 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....

  9. 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.

  10. 抗人喉癌单克隆抗体轻链可变区基因的克隆%Cloning of the light chain variable region gene of monoclonal antibody against human laryngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    管国芳; 黄华梁; 孙丽丽; 王安兆; 王祥斌; 石毅; 孙立群; 金春顺; 李野; 文连姬

    2006-01-01

    目的 分离抗人喉癌单克隆抗体LC9-G5轻链可变区(liht chain variable region,VL)基因,并测定分析其核苷酸序列.方法 使用PCR方法体外扩增抗人喉癌单克隆抗体LC9-G5 VL基因,将其克隆入PMD18-T载体,重组子测序,将其序列使用数据库NCBI和Kabat进行分析.结果 这段序列与小鼠卵巢癌抗体VL基因的同源性为99%,基因全长336 bp,属于小鼠免疫球蛋白轻链第Ⅱ亚类,该VL基因序列已被Gene Bank收录,收录号为U60463.结论 该基因为抗人喉癌单克隆抗体LC9-G5轻链可变区基因.

  11. Extrasynaptic location of laminin beta 2 chain in developing and adult human skeletal muscle

    DEFF Research Database (Denmark)

    Wewer, U M; Thornell, L E; Loechel, F;

    1997-01-01

    and Becker muscular dystrophy. Immunoaffinity chromatography of muscle extracts with a monoclonal antibody to the laminin alpha 2 chain followed by immunoblotting with various antibodies to the beta 2 chain demonstrated the presence of the laminin-4 (alpha 2-beta 2-gamma 1) isoform. Together the present...

  12. 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

  13. 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.

  14. 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...

  15. 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.

  16. 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

  17. 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...

  18. An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

    Directory of Open Access Journals (Sweden)

    Furuichi Yasuhiro

    2009-12-01

    Full Text Available Abstract Background Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv antibody by using a target-related molecule that favored selection ofrecombinant antibodies. Results To produce more selective and specific anti-idiotypic scFv-antibodies from a cDNA library, constructed from HM-1 killer toxin (HM-1-neutralizing monoclonal antibodies (nmAb-KT, the method was modified by using an elution buffer supplemented with HM-1 that shares structural and functional similarities with the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT allowed easy and quick dissociation of scFv-displayed phages from immobilized nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After modified panning, 80% clones (40/50 showed several times higher binding affinity to nmAb-KT than regular panning. The major populations (48% of these clones (scFv K1 were genotypically same and had strong cytocidal activity against Saccharomyces and Candida species. The scFv K1 (Kd value = 4.62 × 10-8 M had strong reactivity toward nmAb-KT, like HM-1 (Kd value = 6.74 × 10-9 M as judged by SPR analysis. Conclusion The scFv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. A simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. Our method offers an attractive platform to discover potential therapeutic candidates.

  19. Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

    Science.gov (United States)

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

    2015-01-01

    A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

  20. Rational Design of CXCR4 Specific Antibodies with Elongated CDRs

    Science.gov (United States)

    2015-01-01

    The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a Kd of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future. PMID:25041362

  1. 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

  2. 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...

  3. 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.

  4. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  5. 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...

  6. 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.

  7. Study on the Construction of Emergency Logistics Control System based on the Perspective of Supply Chain%基于供应链视角下的应急物流控制体系建设研究

    Institute of Scientific and Technical Information of China (English)

    张臻竹

    2014-01-01

    monitoring systems, early warning and risk prevention systems, information systems and incentive evaluation systems. The emergency logistics control system, which is based on supply-chain perspective, even should be considered from the long-term, overall and strategic perspective, in order to make them more planning and coordinative. Based on the summarization, which is the various issues for current emergency logistics control system in our country , the construction of emergency logistics system from the perspective of supply chain is proposed, and more systematic exposition would like to use in strategic coordination, supervision and evaluation of early warning systems.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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)

  14. Study of the system construction of the whole tourism value chain based on industry integration%基于产业融合的旅游全价值链体系构建研究

    Institute of Scientific and Technical Information of China (English)

    马勇; 陈慧英

    2012-01-01

    Industry integration has become a kind of tourism development trend, and at the same time, industrial integration is also an important way of the sustainable development of tourism innovation. The integration of tourism and other industries based on the whole tourism value chain will inject new vitality to tourism industry for the sustainable development. This article from the perspective of industry integration, studies of the nine elements of the whole tourism value chain, that is the resource integration value chain, the market integration value chain, the function integration value chain, the brand integration value chain, the cul- ture integration value chain, the motion integration value chain, the capital integration value chain, the technology integration value chain and human integration value chain. At last the article puts forward the system framework of the whole tourism value chain, which provides guidance for tourism development, positioning and value upgrade.%产业融合已成为旅游发展的一种必然趋势,也是旅游创新可持续发展的重要途径。而基于旅游全价值链的旅游业与其他产业进行融合,会给旅游可持续发展注入新的活力。文章从产业融合的视角切入,研究了旅游全价值链的九大构成要素,即资源融合价值链、市场融合价值链、功能融合价值链、品牌融合价值链、文化融合价值链、情感融合价值链、资金融合价值链、技术融合价值链、人才融合价值链。最后提出了基于产业融合的旅游全价值链体系,这将为我国旅游产业发展、定位与价值提升提供全新的理念指导。

  15. Food Safety System Construction Based on Supply Chain Management%基于供应链管理的食品安全体系构建问题研究

    Institute of Scientific and Technical Information of China (English)

    冯晓曼; 刘永悦; 高艳

    2015-01-01

    The food supply chain management in China has problems with material, information and cash flow. Post-event supervision and segmented supervision are more popular rather than beforehand prevention and overall process control. To build a food safety system, a procurement system and traceable supply system must be established and the market access system must be improved. In this connection, the assignment of responsibility of governmental departments must be clear to improve the organization system for supervision. Enterprises should enhance the awareness of corporate responsibility with a sound accountability system. Food inspection resources could be integrat-ed. Risk evaluation for the safety should be optimized. The emergency disposal system should be improved. And the staff expenditure should be increased to accelerate the organizational construction at the primary level for intensifying the supervision at key links and in-creasing the efficiency of special projects.%目前,我国基于供应链管理的食品安全体系中存在着物流、信息流和资金流等方面的问题,普遍重事后监管和分段监管,轻事前预防与全过程控制。食品安全体系的构建包括建立食品安全采购系统和食品供应链可追溯系统,完善市场准入制度。这就应该明确政府部门职责分工,完善监管组织体系;健全责任体系,强化企业责任意识;进一步整合食品检验检测资源;优化安全风险评估,健全应急处置体系;加大人员经费投入,健全基层组织机构建设和加强重点环节监管,提高专项整治质量,努力开创食品安全监管的新局面,确保消除食品安全隐患。

  16. 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

  17. 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

  18. 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.

  19. 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.

  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. 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.

  2. 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.

  3. 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.

  4. 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

    2015-05-01

    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.

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

    Science.gov (United States)

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

    2013-01-01

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

  6. 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

  7. 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

  8. 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...

  9. 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...

  10. Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin.

    Science.gov (United States)

    Sarnovsky, Robert; Tendler, Tara; Makowski, Matheusz; Kiley, Maureen; Antignani, Antonella; Traini, Roberta; Zhang, Jingli; Hassan, Raffit; FitzGerald, David J

    2010-05-01

    Immunotoxins are antibody-toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.

  11. Design and optimization of a linker for fusion protein construction

    Institute of Scientific and Technical Information of China (English)

    Jianhua Zhang; Jun Yun; Zhigang Shang; Xiaohui Zhang; Borong Pan

    2009-01-01

    Bivalent, bispecific single-chain antibody fusion protein construction appears to be a promising tool for tumor therapy. One of its drawbacks is that the function and activity of the fusion proteins have varied affinity and/or anti-tumor activity compared with the ori-ginal molecules from which they are derived. A more optimized linker for fusion proteins would confer more favorable biological activ-ities upon bispecific single-chain antibodies. Thus, it is critical to design and optimize an inter-peptide linker. With different functional domains and optimized linkers, fusion proteins provide a solid base for targeted immune therapy for malignancies. In this paper, we review the inter-peptide linker studies and the design of an optimized linker using genetic algorithms. The spatial structure of the fusion protein can be predicted by using genetic and bioinformatics research. Based on current research, the future focus will be on different correlation models to perform simulations of spatial structures and drug molecule design.

  12. 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.

  13. 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

    not always bind efficiently to passive adsorption surfaces, and we developed a simple method to quantify the binding capacity of surfaces with the peptides. Background binding (the binding of scFvs to the background matrix) is an obstacle for successful selection, and we eval