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Sample records for multivalent single-chain fv

  1. Tumor Targeting by a Multivalent Single-Chain Fv (scFv) Anti-Lewis Y Antibody Construct

    PubMed Central

    Kelly, Marcus P.; Lee, F.-T.; Tahtis, Kiki; Power, Barbara E.; Smyth, Fiona E.; Brechbiel, Martin W.; Hudson, Peter J.

    2008-01-01

    Abstract The use of single-chain variable fragment (scFv) constructs has been investigated in cancer radioimmunotherapy (RIT) and radioimmunodetection, as these molecules permit rapid tumor penetration and clearance from the serum relative to whole IgG. Multimerization of scFv constructs has demonstrated improvements in functional affinity (i.e., avidity) and maximal tumor uptake. In this paper, we report the first biodistribution and pharmacokinetics studies of a noncovalent, direct-linked scFv (VL-0-VH) trimeric/tetrameric “multimer” of the anti-Lewis Y monoclonal antibody, hu3S193. The in vitro binding and in vivo biodistribution of the hu3S193 multimer was characterized alongside the hu3S193 F(ab′)2 following radiolabeling with the Indium-111 (111In) radioisotope. Immunoreactivities of the radiolabeled multimer and F(ab′)2 were 73% and 53.2%, and binding affinities (Ka) were 1.58 × 107 M−1 and 4.31 × 106 M−1 for the multimer and F(ab′)2, respectively. Maximal tumor uptake in Ley-positive MCF-7 breast cancer xenografted BALB/c nude mice was 12.6 ± 2.5 percent injected dose/per gram (%ID/g) at 6 hours postinjection for the multimer and 15.7 ± 2.1 %ID/g at 24 hours postinjection for the F(ab′)2. However, limited in vitro stability and high renal localization of radiolabeled constructs were observed, which, despite the observed tumor targeting of the hu3S193 multimer, most likely preclude its use in RIT and imaging modalities. PMID:18771345

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

    SciTech Connect

    Surinder Batra, Ph D

    2006-02-27

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

  3. A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy.

    PubMed

    Liang, Hui; Li, Xiaoran; Chen, Bing; Wang, Bin; Zhao, Yannan; Zhuang, Yan; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2015-07-10

    Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.

  4. Paracrine inhibition of prion propagation by anti-PrP single-chain Fv miniantibodies.

    PubMed

    Donofrio, Gaetano; Heppner, Frank L; Polymenidou, Magdalini; Musahl, Christine; Aguzzi, Adriano

    2005-07-01

    Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C). A growing body of evidence suggests that antibodies to PrP(C) can antagonize deposition of PrP(Sc). However, host tolerance hampers the induction of immune responses to PrP(C), and cross-linking of PrP(C) by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP(Sc), even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.

  5. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

    PubMed

    Tavladoraki, P; Benvenuto, E; Trinca, S; De Martinis, D; Cattaneo, A; Galeffi, P

    1993-12-01

    Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development. PMID:8247156

  6. Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

    PubMed

    Sato, Yusui; Tanaka, Yusuke; Inaba, Satomi; Sekiguchi, Hiroshi; Maruno, Takahiro; Sasaki, Yuji C; Fukada, Harumi; Kobayashi, Yuji; Azuma, Takachika; Oda, Masayuki

    2016-10-01

    Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events. PMID:27222286

  7. Multi-channeled single chain variable fragment (scFv) based microfluidic device for explosives detection.

    PubMed

    Charles, Paul T; Davis, Jasmine; Adams, André A; Anderson, George P; Liu, Jinny L; Deschamps, Jeffrey R; Kusterbeck, Anne W

    2015-11-01

    The development of explosives detection technologies has increased significantly over the years as environmental and national security agencies implement tighter pollution control measures and methods for improving homeland security. 2, 4, 6-Trinitrotoluene (TNT), known primarily as a component in munitions, has been targeted for both its toxicity and carcinogenic properties that if present at high concentrations can be a detriment to both humans, marine and plant ecosystems. Enabling end users with environmental detection and monitoring systems capable of providing real-time, qualitative and quantitative chemical analysis of these toxic compounds would be extremely beneficial. Reported herein is the development of a multi-channeled microfluidic device immobilized with single chain fragment variable (scFv) recombinant proteins specific for the explosive, TNT. Fluorescence displacement immunoassays performed under constant flow demonstrated trace level sensitivity and specificity for TNT. The utility of three multi-channeled devices immobilized with either (1) scFv recombinant protein, (2) biotinylated-scFv (bt-scFv) and (3) monoclonal anti-TNT (whole IgG molecule) were investigated and compared. Fluorescence dose response curves, crossreactivity measurements and limits of detection (LOD) for TNT were determined. Fluorescence displacement immunoassays for TNT in natural seawater demonstrated detection limits at sub-parts-per-billion levels (0.5 ppb) utilizing the microfluidic device with immobilized bt-scFv. PMID:26452845

  8. Multi-channeled single chain variable fragment (scFv) based microfluidic device for explosives detection.

    PubMed

    Charles, Paul T; Davis, Jasmine; Adams, André A; Anderson, George P; Liu, Jinny L; Deschamps, Jeffrey R; Kusterbeck, Anne W

    2015-11-01

    The development of explosives detection technologies has increased significantly over the years as environmental and national security agencies implement tighter pollution control measures and methods for improving homeland security. 2, 4, 6-Trinitrotoluene (TNT), known primarily as a component in munitions, has been targeted for both its toxicity and carcinogenic properties that if present at high concentrations can be a detriment to both humans, marine and plant ecosystems. Enabling end users with environmental detection and monitoring systems capable of providing real-time, qualitative and quantitative chemical analysis of these toxic compounds would be extremely beneficial. Reported herein is the development of a multi-channeled microfluidic device immobilized with single chain fragment variable (scFv) recombinant proteins specific for the explosive, TNT. Fluorescence displacement immunoassays performed under constant flow demonstrated trace level sensitivity and specificity for TNT. The utility of three multi-channeled devices immobilized with either (1) scFv recombinant protein, (2) biotinylated-scFv (bt-scFv) and (3) monoclonal anti-TNT (whole IgG molecule) were investigated and compared. Fluorescence dose response curves, crossreactivity measurements and limits of detection (LOD) for TNT were determined. Fluorescence displacement immunoassays for TNT in natural seawater demonstrated detection limits at sub-parts-per-billion levels (0.5 ppb) utilizing the microfluidic device with immobilized bt-scFv.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    PubMed Central

    Shou, Yulin; Graves, Steven W.; Bradbury, Andrew R. M.

    2011-01-01

    Background Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection. Methods Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA. Results Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. Conclusions Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv

  11. Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris.

    PubMed

    Chang, Hyun-Joo; Choi, Sung-Wook; Chun, Hyang Sook

    2008-10-01

    A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff. PMID:18575809

  12. Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vec...

  13. Redesigning of anti-c-Met single chain Fv antibody for the cytoplasmic folding and its structural analysis.

    PubMed

    Edwardraja, Selvakumar; Neelamegam, Rameshkumar; Ramadoss, Vijayaraj; Venkatesan, Subramanian; Lee, Sun-Gu

    2010-06-15

    Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition.

  14. A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv).

    PubMed

    Tu, Chao; Terraube, Virginie; Tam, Amy Sze Pui; Stochaj, Wayne; Fennell, Brian J; Lin, Laura; Stahl, Mark; LaVallie, Edward R; Somers, Will; Finlay, William J J; Mosyak, Lydia; Bard, Joel; Cunningham, Orla

    2016-01-15

    Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.

  15. Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

    PubMed

    Muller, B H; Chevrier, D; Boulain, J C; Guesdon, J L

    1999-07-30

    Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

  16. Radioimmunoguided surgery in colorectal cancer using a genetically engineered anti-CEA single-chain Fv antibody.

    PubMed

    Mayer, A; Tsiompanou, E; O'Malley, D; Boxer, G M; Bhatia, J; Flynn, A A; Chester, K A; Davidson, B R; Lewis, A A; Winslet, M C; Dhillon, A P; Hilson, A J; Begent, R H

    2000-05-01

    In radioimmunoguided surgery (RIGS), a radiolabeled antibody is given i.v. before surgery and a hand-held gamma-detecting probe is used to locate tumor in the operative field. The rapid blood clearance and good tumor penetration of single-chain Fv antibodies (scFv) offer potential advantages over larger antibody molecules used previously for RIGS. A Phase I clinical trial is reported on RIGS with scFv (MFE-23-his) to carcinoembryonic antigen (CEA). Thirty-four patients undergoing surgery for colorectal carcinoma (17 primary tumors, 16 liver metastases, and 1 anastomotic recurrence) and 1 patient with liver metastases of pancreatic carcinoma received 125I-labeled MFE-23-his scFv (125I-MFE-23-his) 24, 48, 72, or 96 h before operation. 125I-MFE-23-his showed biexponential blood clearance with alpha and beta half-lives of 0.32 and 10.95 h, respectively. The abdomen was scanned during surgery with a hand-held gamma detecting probe (Neoprobe Corp.). 125I-MFE-23-his showed good tumor localization; comparison with histology showed overall accuracy of 84%. Highest median ratios for tumor:normal tissue and tumor:blood were recorded 72 or 96 h after scFv injection for patients undergoing resection of liver metastases. High levels of radioactivity were found in the kidneys. Five patients had grade 1 fever, and three had a grade 1 rise in blood pressure according to the Common Toxicity Criteria. There was a significant correlation between these ratios and those measured in excised tissues using a laboratory gamma counter (P < 0.001). MFE-23-his scFv antibody localizes in CEA-producing carcinomas. The short interval between injection and operation, the lack of significant toxicity, and the relatively simple production in bacteria make MFE-23-his scFv suitable for RIGS.

  17. Bacterial cytoplasm production of an EGFP-labeled single-chain Fv antibody specific for the HER2 human receptor.

    PubMed

    Lombardi, Alessio; Gianese, Giulio; Arcangeli, Caterina; Galeffi, Patrizia; Sperandei, Maria

    2011-12-01

    The human epidermal growth factor receptor 2 (HER2) is the main diagnostic marker of breast and ovary cancers. Here, to obtain a rapid and sensitive immunodiagnostic tool a single-chain antibody (scFv800E6) specific for the HER2 was fused to the N-terminus of the enhanced green fluorescent protein (EGFP) by a flexible linker. The soluble production of the novel scFv800E6-EGFP protein in the cytoplasm of Escherichia coli was investigated at different induction temperatures (25, 30 and 37°C); the intrinsic fluorescent properties and the binding activity to HER2 positive tumour cells of the fusion protein were analysed. Western blotting and fluorescence analysis of SDS-PAGE revealed the presence of two scFv800E6-EGFP forms, with different mobility and optical properties, their ratio depending on the induction temperature. The fluorescent form maintained the optical fluorescence properties of EGFP and exhibited a binding activity to the HER2-expressing cells comparable to that of the non-fused scFv800E6. In addition, to provide an insight into the effect of the induction temperature on the molecular structure, the folding of the fusion protein was assessed at atomic level by performing molecular dynamics simulations of the homology-derived model of scFv800E6-EGFP at 300 K and 310 K. The comparison of the data collected at these two temperatures revealed that the higher temperature affects specific structural elements. To improve the production of the soluble and functional scFv800E6-EGFP protein, "in silico" results could be utilised for ad hoc design of the molecular structure. PMID:22066531

  18. Development of high-affinity single chain Fv against foot-and-mouth disease virus.

    PubMed

    Jung, Joon-Goo; Jeong, Gu Min; Yim, Sung Sun; Jeong, Ki Jun

    2016-03-01

    Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (KD=42.7nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV. PMID:26827774

  19. Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus.

    PubMed

    Gould, L Hannah; Sui, Jianhua; Foellmer, Harald; Oliphant, Theodore; Wang, Tian; Ledizet, Michel; Murakami, Akikazu; Noonan, Kristin; Lambeth, Cassandra; Kar, Kalipada; Anderson, John F; de Silva, Aravinda M; Diamond, Michael S; Koski, Raymond A; Marasco, Wayne A; Fikrig, Erol

    2005-12-01

    West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.

  20. Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2).

    PubMed

    Li, Tai-Wei; Cheng, Shu-Fang; Tseng, Yen-Tzu; Yang, Yu-Chih; Liu, Wen-Chun; Wang, Sheng-Cyuan; Chou, Mei-Ju; Lin, Yu-Jen; Wang, Yueh; Hsiao, Pei-Wen; Wu, Suh-Chin; Chang, Ding-Kwo

    2016-01-01

    Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

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

    PubMed Central

    Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

    2008-01-01

    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. PMID:18783590

  2. Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

    NASA Astrophysics Data System (ADS)

    Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

    2014-10-01

    In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

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

    PubMed

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

    2016-03-01

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

  4. Functional stability of 3D8 scFv, a nucleic acid-hydrolyzing single chain antibody, under different biochemical and physical conditions.

    PubMed

    Lee, Joungmin; Park, Hyunjoon; Kim, Minjae; Seo, Youngsil; Lee, Yeonjin; Byun, Sung June; Lee, Sukchan; Kwon, Myung-Hee

    2015-12-30

    3D8 single-chain Fv (scFv) is a catalytic nucleic acid antibody with anti-viral activity against a broad spectrum of viruses. Here we investigated the functional stability of 3D8 scFv to provide a basis for engineering a 3D8 scFv derivative and for developing stable formulations with improved stability and potential use as an anti-viral agent. The stability of 3D8 scFv was assessed by measuring its DNA-hydrolyzing activity under different biochemical and physical conditions using a fluorescence resonance energy transfer (FRET)-based method. In addition, the anti-influenza (H9N2) effect of 3D8 scFv was evaluated in A549 cells. 3D8 scFv was stable at 50°C for 6h at pH 7.2, for 3 days at pH 4-10 at 37°C and 30 days at pH 4-8 at 37°C. The stability was not affected by a reducing condition, freeze-thawing for up to 30 cycles, or lyophilization. Evaluation of the anti-virus effect showed that cells treated with 32-128 units of 3D8 scFv showed a 50% decrease in influenza replication compared to untreated cells. Based on its enzymatic stability in various biochemical and physical environments, 3D8 scFv holds good potential for development as an anti-viral therapeutic. PMID:26536531

  5. Structure of a Single-Chain Fv Bound to the 17 N-Terminal Residues of Huntingtin Provides Insights into Pathogenic Amyloid Formation and Suppression

    PubMed Central

    De Genst, Erwin; Chirgadze, Dimitri Y.; Klein, Fabrice A.C.; Butler, David C.; Matak-Vinković, Dijana; Trottier, Yvon; Huston, James S.; Messer, Anne; Dobson, Christopher M.

    2015-01-01

    Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3–11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12–17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv–peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT. PMID:25861763

  6. Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors.

    PubMed

    Cheng, Ming; Santich, Brian H; Xu, Hong; Ahmed, Mahiuddin; Huse, Morgan; Cheung, Nai-Kong V

    2016-06-01

    Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.

  7. High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

    PubMed

    Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

    2014-12-01

    Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies. PMID:25239038

  8. Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors

    PubMed Central

    Cheng, Ming; Santich, Brian H.; Xu, Hong; Ahmed, Mahiuddin; Huse, Morgan; Cheung, Nai-Kong V.

    2016-01-01

    ABSTRACT Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca2+ flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity. PMID:27471647

  9. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom.

    PubMed

    Castro, J M A; Oliveira, T S; Silveira, C R F; Caporrino, M C; Rodriguez, D; Moura-da-Silva, A M; Ramos, O H P; Rucavado, A; Gutiérrez, J M; Magalhães, G S; Faquim-Mauro, E L; Fernandes, I

    2014-09-01

    BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of

  10. Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv).

    PubMed

    Zhou, N; Paemen, L; Opdenakker, G; Froyen, G

    1997-09-15

    The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

  11. Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.

    PubMed

    Wu, Qian; Wang, Xiaodong; Gu, Yong; Zhang, Xiao; Qin, Yao; Chen, Heng; Xu, Xinyu; Yang, Tao; Zhang, Mei

    2016-07-01

    Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D. PMID:27270580

  12. GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

    PubMed Central

    2010-01-01

    Background Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify. Results In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs. Conclusions Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy. PMID:20923574

  13. Novel anti-Tn single-chain Fv-Fc fusion proteins derived from immunized phage library and antibody Fc domain.

    PubMed

    Kubota, Tsuguo; Matsushita, Takefumi; Niwa, Rinpei; Kumagai, Izumi; Nakamura, Kazuyasu

    2010-09-01

    Tn[GalNAc(α1-3)-Ser/Thr] antigen, a tumor-associated carbohydrate antigen, is highly expressed in various tumors and an attractive candidate for cancer immunotherapy. The generation of an anti-Tn antibody is a first step toward the construction of new anticancer molecules. However, because of the simple and small conformation of the Tn molecule, it is difficult to generate an anti-Tn antibody for therapeutic use by conventional hybridoma technology. The purpose of this study was to isolate anti-Tn single-chain antibody fragments (scFv) by phage display technology from a novel immunised library, to attach an antibody constant region (Fc) and to convert them to scFv-Fc fusion proteins. The scFv-Fcs obtained here showed strict specificity against the Tn antigen and also showed antibody-dependent cellular cytotoxicity. These results suggest a potential use of this antibody generating method by phage display and indicate the potential of Fc-fusion proteins as therapeutic candidates.

  14. The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6.

    PubMed

    Phillips, Damilola D; Garboczi, David N; Singh, Kavita; Hu, Zonglin; Leppla, Stephen H; Leysath, Clinton E

    2013-08-01

    The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

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

    PubMed

    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.

  16. Generation of a Single Chain Antibody Variable Fragment (scFv) to Sense Selectively RhoB Activation

    PubMed Central

    Chinestra, Patrick; Olichon, Aurélien; Medale-Giamarchi, Claire; Lajoie-Mazenc, Isabelle; Gence, Rémi; Inard, Cyril; Ligat, Laetitia; Faye, Jean-Charles; Favre, Gilles

    2014-01-01

    Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation. PMID:25365345

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

    PubMed

    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

  18. Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody.

    PubMed

    Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

    2015-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.

  19. Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

    PubMed Central

    Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

    2015-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer. PMID:25663946

  20. Human Single-Chain Fv Immunoconjugates Targeted to a Melanoma-Associated Chondroitin Sulfate Proteoglycan Mediate Specific Lysis of Human Melanoma Cells by Natural Killer Cells and Complement

    NASA Astrophysics Data System (ADS)

    Wang, Baiyang; Chen, Yi-Bin; Ayalon, Oran; Bender, Jeffrey; Garen, Alan

    1999-02-01

    Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.

  1. Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding.

    PubMed

    Maruta, Yasuhiro; Kuwata, Takeo; Tanaka, Kazuki; Alam, Muntasir; Valdez, Kristel Paola Ramirez; Egami, Yoshika; Suwa, Yoshiaki; Morioka, Hiroshi; Matsushita, Shuzo

    2016-09-21

    The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

  2. In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes.

    PubMed

    Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid

    2014-07-28

    Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression.

  3. Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus.

    PubMed

    Pyo, Hyun-Mi; Kim, In-Joong; Kim, Seong-Hee; Kim, Hyun-Soo; Cho, Soo-Dong; Cho, In-Soo; Hyun, Bang-Hun

    2009-03-23

    Porcine epidemic diarrhea virus (PEDV) is a causative agent of severe diarrhea which leads to death in piglets. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease in Korea. In this study, we developed a prophylactic candidate using single-chain Fvs to prevent the PEDV infection. ScFvs of mouse monoclonal antibody which was verified to neutralize PEDV was expressed in Escherichia coli expression system. After the confirmation of PEDV neutralizing activity of purified recombinant scFvs by VN test, scFvs were expressed on the surface of E. coli cells. The signal sequence and autotransporter beta domain of protease IgA (IgAP) of Neisseria gonorrhoeae were introduced to endow scFvs with the direction to the cell surface and the support as a transmembrane domain. 5x10(6)CFU of E. coli expressing scFvs against PEDV showed promising result of 94% foci reduction compared to wild type E. coli. This result demonstrated that E. coli expressing scFvs on the cell surface retained functional potency of parent antibody and therefore blocked PEDV infection into target cells in vitro. This in vitro assay result proposes the perspective of recombinant E. coli cells expressing scFvs as a novel prophylactic against PEDV infection. PMID:19428826

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

    PubMed

    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.

  5. Expression of V(H)-linker-V(L) orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli.

    PubMed

    Liu, Aiping; Ye, Yang; Chen, Weifeng; Wang, Xiaohong; Chen, Fusheng

    2015-02-01

    Aflatoxin B1 (AFB1) is a toxic secondary metabolic product, which threatens human and animal health. Antibody is a key factor for immunoassay against toxic stuff like AFB1, and single-chain Fv antibody fragment (scFv) has become a popular format of genetically engineered antibody. In this study, four hybridoma cell lines against AFB1 were obtained, and then scFvs 2E6 derived from hybridoma cell line 2E6 were constructed in different V(H)/V(L) orientations. Subsequently, scFvs 2E6 were expressed in E. coli BL21(DE3) mainly in the form of inclusion body. SDS-PAGE, Western blot and ELISA were employed to characterize scFvs 2E6. The results revealed that the yield of inclusion body of scFvs 2E6 in either V(H)/V(L) orientation was similar; however, only the scFv in V(H)-linker-V(L) orientation showed anti-AFB1 bioactivity after refolding. The present study underscores the importance of choosing optimal V(H)/V(L) orientation for scFv construction, and scFv may be favorable for immunoassays in food industry. PMID:25540048

  6. The FGF-1-specific single-chain antibody scFv1C9 effectively inhibits breast cancer tumour growth and metastasis

    PubMed Central

    Shi, Hengliang; Fu, Chunling; Wang, Wei; Li, Yu; Du, Shuang; Cao, Rangjuan; Chen, Jingying; Sun, Dong; Zhang, Zhongyu; Wang, Xingzhi; Zhu, Xiaojuan

    2014-01-01

    Immunotherapy mediated by recombinant antibodies is an effective therapeutic strategy for a variety of cancers. In a previous study, we demonstrated that the fibroblast growth factor 1 (FGF-1)-specific recombinant antibody scFv1C9 arrests the cell cycle at the G0/G1 transition by blocking the intracrine FGF-1 pathway in breast cancer cells. Here, we further show that the overexpression of scFv1C9 in MCF-7 and MDA-MB-231 breast cancer cells by lentiviral infection resulted in decreased tumourigenicity, tumour growth and lung metastasis through FGF-1 neutralization. We found that scFv1C9 resulted in the up-regulation of p21, which in turn inhibited the expression of CDK2 and blocked cell cycle progression. To explore the potential role of scFv1C9 in vivo, we delivered the gene into solid tumours by electroporation, which resulted in significant inhibition of tumour growth. In tumour tissue sections, immunohistochemical staining of the cellular proliferation marker Ki-67 and the microvessel marker CD31 showed a reduction in the proliferative index and microvessel density, respectively, upon expression of scFv1C9 compared with the appropriate controls. Thus, our data indicate a central role for scFv1C9 in blocking the intracrine pathway of FGF-1, therefore, scFv1C9 could be developed in an effective therapeutic for breast cancer. PMID:25124967

  7. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    PubMed Central

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  8. Targeted delivery of a SNARE protease to sensory neurons using a single chain antibody (scFv) against the extracellular domain of P2X(3) inhibits the release of a pain mediator.

    PubMed

    Ma, Hui; Meng, Jianghui; Wang, Jiafu; Hearty, Stephen; Dolly, J Oliver; O'Kennedy, Richard

    2014-09-01

    P2X3 (P2X purinoceptor 3) is predominantly expressed on nociceptive sensory neurons and plays a crucial role in signalling leading to chronic inflammatory pain and some features of neuropathic pain. Thus it represents a potential target for pain therapeutics. BoNT/A (botulinum neurooxin type A) effectively relieves certain types of pain through inhibiting the neuronal release of pain peptides. A recombinant single-chain variable fragment (scFv) antibody designated MH7C was generated against the extracellular domain of P2X3 using phage display. The genes encoding the scFv and activated di-chain form of BoNT/A without the C-terminal-binding subdomain (LC-HN-HCN/A) were ligated and expressed in Escherichia coli cells as a composite fusion protein. The purified protein bound and entered P2X3-containing sensory neurons, cleaved synaptosomal-associated protein of 25 kDa and inhibited the release of a pain peptide. This novel fusion protein designated 'LC-HN-HCN/A-MH7C' has potential clinical applications in the treatment of chronic inflammatory and sympathetically maintained neuropathic pain.

  9. 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.

    PubMed

    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

  10. Single-chain Fv antibody fragments retain binding properties of the monoclonal antibody raised against peptide P1 of the human prion protein.

    PubMed

    Skrlj, Nives; Serbec, Vladka Curin; Dolinar, Marko

    2010-03-01

    Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics. PMID:19597999

  11. Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.

    PubMed

    Makvandi-Nejad, Shokouh; Sheedy, Claudia; Veldhuis, Linda; Richard, Gabrielle; Hall, J Christopher

    2010-08-31

    Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.

  12. Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

    PubMed Central

    Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2015-01-01

    Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

  13. 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.

    PubMed

    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.

  14. Specific Single Chain Variable Fragment (ScFv) Antibodies to Angiotensin II AT2 Receptor: Evaluation of the Angiotensin II Receptor Expression in Normal and Tumor-bearing Mouse Lung

    PubMed Central

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

    2010-01-01

    Summary To gain insight into the mechanism by which angiotensin II type 2 receptor (AT2) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT2 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 AT2 receptor protein. The specificity of the antibodies was verified using AT2 over-expressing COS-7 cells and AT2 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 AT2 and AT1 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. PMID:18438736

  15. Method for preparation of single chain antibodies

    DOEpatents

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

    2012-04-03

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

  16. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  17. Using Engineered Single-Chain Antibodies to Correlate Molecular Binding Properties and Nanoparticle Adhesion Dynamics

    PubMed Central

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

    2011-01-01

    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 effect 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. Based on 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 bear

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

    PubMed

    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

  19. Creation and Evaluation of a Single-chain Antibody Tetramer that Targets Brain Endothelial Cells

    PubMed Central

    Zhang, Xiaobin; Wang, Xin Xiang; Shusta, Eric V.

    2014-01-01

    Antibodies that target and internalize into blood-brain barrier (BBB) endothelial cells offer promise as drug delivery agents. Previously, we identified a single-chain antibody (scFvA) capable of binding to the BBB. In an attempt to improve the binding and internalization properties of the single chain antibody (scFvA), a biotinylation tag (Avitag) was fused to scFvA and the protein secreted by yeast. The scFvA-Avitag could be biotinylated by yeast-displayed BirA enzyme and biotinylated scFvA-Avitag could be used to create scFv tetramers. Tetramerization of scFvA improved the internalization of scFvA into BBB endothelial cells, and biotinylated scFvA-Avitag could also be used to target streptavidin-coated quantum dots for BBB endothelial cell internalization. Perfusing the rat brain with scFvA-tetramer confirmed that the antigen targeted by scFvA is distributed on blood side of the BBB, suggesting the potential for downstream application of scFvA in brain-targeted drug delivery. PMID:24659822

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Immobilization strategies for single-chain antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Miller, Keith D.; Pefaur, Noah B.; Gonzalez, Rachel M.; Apiyo, David O.; Engelmann, Heather E.; Srinivastava, Sudhir; Kagan, Jacob; Rodland, Karin D.; Zangar, Richard C.

    2008-06-01

    Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays have great potential for validating biomarkers of disease. ELISA relies on robust affinity reagents that retain activity when immobilized or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional immunoglobin G (IgG). Unfortunately, scFv are typically less stabile than IgG and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv structural modifications to see if we could develop a more robust scFv reagent. Two promising strategies that emerged from these studies: 1) the precapture of epitope-tagged scFv using an anti-epitope antibody and 2) the direct printing of a thioredoxin/scFv fusion protein on glass slides. The use of either strategy improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the anti-epitope precapture method had a risk of reagent transfer. Using the direct printing method, we show that anti-PSA scFv are highly specific when tested against 21 different IgG-based assays. Finally, the scFv microarray PSA assay gave comparable results (R2 = 0.95) to a commercial 96-well ELISA when tested using serum samples. Overall, these results suggest that minor modifications of the scFv protein structure are sufficiently to produce reagents that are suitable for use in multiplex assay systems.

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

    DOEpatents

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

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

  3. Targeting nanodisks via a single chain variable antibody - Apolipoprotein chimera

    SciTech Connect

    Iovannisci, David M.; Beckstead, Jennifer A.; Ryan, Robert O.

    2009-02-06

    Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that {alpha}-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

  4. Advances in single chain technology.

    PubMed

    Gonzalez-Burgos, Marina; Latorre-Sanchez, Alejandro; Pomposo, José A

    2015-10-01

    The recent ability to manipulate and visualize single atoms at atomic level has given rise to modern bottom-up nanotechnology. Similar exquisite degree of control at the individual polymeric chain level for producing functional soft nanoentities is expected to become a reality in the next few years through the full development of so-called "single chain technology". Ultra-small unimolecular soft nano-objects endowed with useful, autonomous and smart functions are the expected, long-term valuable output of single chain technology. This review covers the recent advances in single chain technology for the construction of soft nano-objects via chain compaction, with an emphasis in dynamic, letter-shaped and compositionally unsymmetrical single rings, complex multi-ring systems, single chain nanoparticles, tadpoles, dumbbells and hairpins, as well as the potential end-use applications of individual soft nano-objects endowed with useful functions in catalysis, sensing, drug delivery and other uses. PMID:26505056

  5. Advances in single chain technology.

    PubMed

    Gonzalez-Burgos, Marina; Latorre-Sanchez, Alejandro; Pomposo, José A

    2015-10-01

    The recent ability to manipulate and visualize single atoms at atomic level has given rise to modern bottom-up nanotechnology. Similar exquisite degree of control at the individual polymeric chain level for producing functional soft nanoentities is expected to become a reality in the next few years through the full development of so-called "single chain technology". Ultra-small unimolecular soft nano-objects endowed with useful, autonomous and smart functions are the expected, long-term valuable output of single chain technology. This review covers the recent advances in single chain technology for the construction of soft nano-objects via chain compaction, with an emphasis in dynamic, letter-shaped and compositionally unsymmetrical single rings, complex multi-ring systems, single chain nanoparticles, tadpoles, dumbbells and hairpins, as well as the potential end-use applications of individual soft nano-objects endowed with useful functions in catalysis, sensing, drug delivery and other uses.

  6. Use of Single-Chain Antibody Derivatives for Targeted Drug Delivery

    PubMed Central

    Safdari, Yaghoub; Ahmadzadeh, Vahideh; Khalili, Masoumeh; Jaliani, Hossein Zarei; Zarei, Vahid; Erfani-Moghadam, Vahid

    2016-01-01

    Single-chain antibodies (scFvs), which contain only the variable domains of full-length antibodies, are relatively small molecules that can be used for selective drug delivery. In this review, we discuss how scFvs help improve the specificity and efficiency of drugs. Small interfering RNA (siRNA) delivery using scFv-drug fusion peptides, siRNA delivery using scFv-conjugated nanoparticles, targeted delivery using scFv-viral peptide-fusion proteins, use of scFv in fusion with cell-penetrating peptides for effective targeted drug delivery, scFv-mediated targeted delivery of inorganic nanoparticles, scFv-mediated increase of tumor killing activity of granulocytes, use of scFv for tumor imaging, site-directed conjugation of scFv molecules to drug carrier systems, use of scFv to relieve pain and use of scFv for increasing drug loading efficiency are among the topics that are discussed here. PMID:27249008

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

    PubMed

    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.

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

    PubMed

    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

  9. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.

  10. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases. PMID:22397408

  11. Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections

    PubMed Central

    Moazen, Bahareh; Ebrahimi, Elahe; Nejatollahi, Foroogh

    2016-01-01

    Background: Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents. Objectives: The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis. Materials and Methods: Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay. Results: Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively. Conclusions: Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients

  12. Targeting nanodisks via a single chain variable antibody--apolipoprotein chimera.

    PubMed

    Iovannisci, David M; Beckstead, Jennifer A; Ryan, Robert O

    2009-02-01

    Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that alpha-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

  13. Construction of multiform scFv antibodies using linker peptide.

    PubMed

    Wang, Shihua; Zheng, Cengjie; Liu, Ying; Zheng, Huirong; Wang, Zonghua

    2008-05-01

    Multiform single chain variable fragments (scFvs) including different length linker scFvs and bispecific scFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between V(H) and V(L) of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON scFv and antizearalenone (anti-ZEN) scFv was also constructed through connection by a short peptide linker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecific scFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgenic plants.

  14. Characterization of a single-chain variable fragment specific to Cronobacter spp. from hybridoma based on outer membrane protein A.

    PubMed

    Chen, Qiming; Tao, Tingting; Bie, Xiaomei; Lu, Fengxia; Li, Yuanhong; Lu, Zhaoxin

    2016-10-01

    Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as antigen. The protein sequences of OmpA of Cronobacter spp. were analyzed first. The results showed protein OmpA with length of 347 amino acids was conserved in Cronobacter genus (94.83%-100% of protein identity) and was greater than that observed for the other genera tested (8.28-91.64% of protein identity). Then, purified protein OmpA expressed in E. coli was used to prepare hybridoma and to construct scFv further. The scFv was named scFvH81 and analyzed by bioinformatics. The model of scFvH81 built by homologous modeling had a good quality (residues in disallowed regions: 3%) and showed that scFvH81 had a standard pocket-like site. Purified scFvH81 was prepared by denaturation and renaturation of inclusion body and it showed a good specificity and its affinity of Ka=2.39×10(6)M(-1). Therefore, it could be used in the detection and the pathogenesis study of Cronobacter spp. PMID:27498230

  15. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

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

    PubMed

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

    2015-01-01

    The purposes was to establish optimal modeling of single-chain antibody molecules based on similarity algorithm and seek the connecting peptides that had the minimal effect on the structure and bioactivity of the variable region of heavy chain (VH) and that of light chain (VL) in a single-chain antibody against liver cancer. After the Linker with different lengths (n=0~7) had been added into single chain fragment variable (ScFv), modeling of the overall sequences of VH, VL and ScFv were conducted respectively. Meanwhile, the peptide chain structure of (Gly4Ser)n was adopted for the connecting peptide. Then the spatial spherical shell layer alignment algorithm based on spherical polar coordinates was utilized for comparing the structural similarity of VH and VL before and after adding connecting peptide. Equally, in order to determine the stability of VH and VL, MATLAB was applied for analysis of the fore and aft distances and the diffusion radius. Indirect ELISA method was used to detect single-chain antibody immunological activity of Linker with different lengths. The MTT assay was utilized for the examination of the inhibition rate of single-chain antibody with different lengths of Linker to liver cancer cell. When n=4, the structural similarity between VH together with VL and their original ones was the highest. When n=3, the influence of connecting peptide on the stability of VH and VL was minimum. When n>3, the fore and aft distances changed little due to the increase and fold of the length of peptide chain. The results of ELISA detection showed that when n=4, affinity of single chain antibody to liver cancer cells was much higher. The MTT test also indicated that when n=4, the inhibition rate of the connecting peptide on hepatoma carcinoma cell reached the highest, and that came second when n=3. When n=4, the structural stability and biological functions of anti-hepatoma single-chain antibody were both favorable. This study has provided a basis for the design

  17. Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

    PubMed

    Jang, Jun-Hyuck; Kim, Dong-Hyung; Paek, Se-Hwan; Woo, Eui-Jeon; Kim, Young-Wan

    2016-01-01

    Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

  18. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv. PMID:26041237

  19. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    PubMed

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers. PMID:26256421

  20. Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

    PubMed Central

    Mukherjee, K. J.; Rowe, D. C. D.; Watkins, N. A.; Summers, D. K.

    2004-01-01

    Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from λPL under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from λPR. Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD600] of 3.5) was 37 mg liter−1, compared to a maximum of 13 mg liter−1 in the control culture (final OD600 of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD600 of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter−1. A control culture with a similar feed reached an OD600 of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter−1. Quiescent cells at an OD600 of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter−1 was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins. PMID:15128562

  1. Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

    PubMed

    Patel, Rekha; Andrien, Bruce A

    2010-01-01

    Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field. PMID:19720041

  2. Expression of sclerostin scFv and the effect of sclerostin scFv on healing of osteoporotic femur fracture in rats.

    PubMed

    Yao, Qi; Ni, Jie; Hou, Yu; Ding, Lixiang; Zhang, Licheng; Jiang, Hua

    2014-06-01

    Osteoporosis is a systemic metabolic disease characterized by low bone mass with deterioration of the bony microstructure which leads to both bone brittleness and increased risk of fracture. Sclerostin is a protein encoded by the SOST gene which is specifically expressed in osteocyte. Monoclonal antibodies of sclerostin can promote bone formation by antagonizing its inhibitory action. However, the effectiveness of monoclonal antibodies to exert such effects are limited by the large molecular mass and high immunogenicity. Here, we report that we purified a high immune affinity, single-chain antibody of SOST: SOST-single-chain Fv (scFv). Real-time polymerase chain reaction amplification of the variable regions of the heavy- and light-chain gene from a secretory anti-SOST antibody was performed. Animal experiments showed that SOST-scFv promoted bone healing in a rat model of osteoporosis.

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

    PubMed

    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.

  4. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    PubMed

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).

  5. Chemotactic Signaling by Single-Chain Chemoreceptors

    PubMed Central

    Mowery, Patricia; Ames, Peter; Reiser, Rebecca H.; Parkinson, John S.

    2015-01-01

    Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit

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

    SciTech Connect

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    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.

  7. Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System

    PubMed Central

    Cheng, Chiu-Min; Tzou, Shey-Cherng; Zhuang, Ya-Han; Huang, Chien-Chiao; Kao, Chien-Han; Liao, Kuang-Wen; Cheng, Ta-Chun; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Tai, Ming-Hong; Cheng, Tian-Lu

    2014-01-01

    Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein. PMID:24824752

  8. Production of an anti-idiotypic antibody single chain variable fragment vaccine against Edwardsiella tarda.

    PubMed

    Qin, Hong; Jin, Xiaohang; Huang, Weiquan; Liu, Yulin

    2010-02-01

    Edwardsiella tarda is the pathogen responsible for edwardsiellosis, a serious infectious disease of freshwater and marine fish species, and currently recognized to be the species pathogenic for human. An anti-idiotypic monoclonal antibody (mAb), 1E11, has been developed. It mimics the protective epitope of E. tarda and can prevent fish from infection of E. tarda. In this study, the correct variable heavy (VH) and variable light (VL) genes were obtained from 1E11 by using bioinformatics methods, and a 15 amino acid (Gly4Ser)3 linker was used to hold the two V domains together for the construction of VL-linker-VH form of single chain variable fragment (scFv) gene. Then, the scFv was subcloned into the vector pET-28a, expressed in the Escherichia coli BL21 cells, and identified by SDS-PAGE and western blotting. Red drum (Sciaenops ocellatus L.) weighing about 50 g was subjected to challenge with different E. tarda strains after 4 weeks followed by vaccination, the mortality rates and relative percentage survival were recorded and calculated, and the survival rate of fish in the scFv subgroups was obviously higher than that of control subgroups (P<0.01). Enzyme-linked immunosorbent assay results show that after 4 weeks of post-vaccination, the level of specific antibody in fish sera of scFv groups was significantly higher than control groups. This study indicates that the recombinant antibody scFv was successfully developed, and it may serve as an effective vaccine candidate against E. tarda. PMID:20119624

  9. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    PubMed Central

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  10. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    SciTech Connect

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung'u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.

    2010-04-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  11. Flow Cytometry-Based Methods for Assessing Soluble scFv Activities and Detecting Antigens in Solution

    PubMed Central

    Gray, Sean A.; Weigel, Kris M.; Miller, Keith D.; Ndung'u, Joseph; Büscher, Philippe; Tran, Thao; Baird, Cheryl; Cangelosi, Gerard A.

    2010-01-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. Alimiting step in the isolation of scFv from non-immune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast-displayed and -secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast-displayed scFv that bind non-overlapping or non-competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast-displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries. PMID:19953671

  12. Targeted inhibition of tumour cell growth by a bispecific single-chain toxin containing an antibody domain and TGF alpha.

    PubMed Central

    Schmidt, M.; Wels, W.

    1996-01-01

    Overexpression of the epidermal growth factor receptor (EGFR) and ErbB-2 has been observed in a variety of human tumours, making these receptors promising targets for directed tumour therapy. Since many tumour cells express both ErbB-2 and EGFR and these receptors synergise in cellular transformation, therapeutic reagents simultaneously binding to ErbB-2 and EGFR might offer advantages for tumour therapy. We have previously described the potent anti-tumoral activity of a bispecific antibody toxin that contains ErbB-2- and EGFR-specific single-chain Fv (scFv) domains. Here we report the construction and functional characterisation of a novel bispecific recombinant toxin, scFv(FRP5)-TGF alpha-ETA. The fusion protein consists of the antigen-binding domain of the ErbB-2-specific MAb, FRP5, and the natural EGFR ligand, TGF alpha, inserted at different positions in truncated Pseudomonas exotoxin A. ScFv(FRP5)-TGF alpha-ETA protein displayed binding to EGFR and ErbB-2, thereby inducing activation of the receptors, which was dependent on the cellular context and the level of EGFR and ErbB-2 expression. The bispecific molecule was cytotoxic in vitro for tumour cells expressing various levels of the target receptors. In vivo scFv(FRP5)-TGF alpha-ETA potently inhibited the growth of established A431 tumour xenografts in nude mice. Images Figure 1 Figure 2 Figure 5 PMID:8826849

  13. Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate.

    PubMed

    Bourin, P; Servat, A; Lataillade, J J; Goyffon, M; Vaux, D; Billiald, P

    2000-02-01

    G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.

  14. Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems.

    PubMed

    Malpiedi, Luciana P; Nerli, Bibiana B; Taqueda, Maria E S; Abdalla, Dulcineia S P; Pessoa, Adalberto

    2015-07-01

    In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid-liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.

  15. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

    PubMed Central

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A.; Motamedi-Shad, Neda; Irving, James A.; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J.; Miranda, Elena; Lomas, David A.

    2015-01-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity. PMID:25757566

  16. Design, expression and characterization of a single chain anti-CD20 antibody; a germline humanized antibody derived from Rituximab.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Hosseinpour Feizi, Mohammad Ali; Khavarinejad, Ramazan Ali

    2014-10-01

    CD20 is a B cell lineage specific surface antigen involved in various B cell malignancies. So far, several murine and chimeric antibodies have been produced against this antigen among which Rituximab is a commercially approved antibody widely used in treatment of cancers associated with CD20 overexpression. The current study reports the production and characterization of a humanized single chain version of Rituximab through CDR grafting method. For either heavy or light chain variable domains, a human antibody with the highest sequence homology to Rituximab was selected from human germline sequences and used as framework donors. Vernier zone residues in framework regions were replaced with those of Rituximab to retain the antigen binding affinity of parental antibody. The reactivity of humanized single chain antibody with CD20 was examined by ELISA and dot blot assays. The ability of antibody to suppress the growth of CD20 overexpressing Raji cells was tested by MTT assay. Analysis of reactivity with CD20 antigen revealed that the humanized single chain antibody reacted to the target antigen with high affinity. Proliferation inhibition assay showed that humanized scFv could suppress the proliferation of Raji cells efficiently in a dose-dependent manner. This successful production of a humanized scFv with the ability to inhibit growth of CD20-expressing cancer cell may provide a promising alternative strategy for CD20 targeted therapy.

  17. Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli.

    PubMed

    Akbari, Vajihe; Sadeghi, Hamid Mir Mohammad; Jafarian-Dehkordi, Abbas; Abedi, Daryoush; Chou, C Perry

    2015-12-01

    A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications.

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

    PubMed

    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.

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

    PubMed

    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

  20. Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle.

    PubMed

    Poungpair, Ornnuthchar; Bangphoomi, Kunan; Chaowalit, Prapaipit; Sawasdee, Nunghathai; Saokaew, Nichapatr; Choowongkomon, Kiattawee; Chaicumpa, Wanpen; Yenchitsomanus, Pa-thai

    2014-01-01

    Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1-14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules. PMID:24492300

  1. Production of scFv-Conjugated Affinity Silk Powder by Transgenic Silkworm Technology

    PubMed Central

    Sato, Mitsuru; Kojima, Katsura; Sakuma, Chisato; Murakami, Maria; Aratani, Eriko; Takenouchi, Takato; Tamada, Yasushi; Kitani, Hiroshi

    2012-01-01

    Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers. PMID:22496833

  2. Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants.

    PubMed

    Lai, Huafang; He, Junyun; Hurtado, Jonathan; Stahnke, Jake; Fuchs, Anja; Mehlhop, Erin; Gorlatov, Sergey; Loos, Andreas; Diamond, Michael S; Chen, Qiang

    2014-10-01

    Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ∆XF plants, a glycosylation mutant lacking plant-specific N-glycan residues. Glycan analysis revealed that ∆XF plant-derived pE16scFv-CH (∆XFpE16scFv-CH) and pE16 (∆XFpE16) both displayed a mammalian glycosylation profile. ∆XFpE16 and ∆XFpE16scFv-CH demonstrated equivalent antigen-binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell-produced E16 (mE16). A single dose of ∆XFpE16 or ∆XFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost-effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases. PMID:24975464

  3. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells.

    PubMed

    Epel, Malka; Ellenhorn, Joshua D; Diamond, Don J; Reiter, Yoram

    2002-11-01

    Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy. PMID:12384808

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

    PubMed

    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.

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

    PubMed Central

    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

  6. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    SciTech Connect

    Bradbury, Andrew M; Velappan, Nileena; Schmidt, Jurgen G

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  7. Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

    PubMed Central

    Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khoram Khorshid, Hamid Reza; Samadi, Nasser; Ahdi Khosroshahi, Shiva; Zarei Jaliani, Hossein

    2015-01-01

    Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies. PMID:26793607

  8. Single Chain Variable Fragment against Nicastrin Inhibits the γ-Secretase Activity*

    PubMed Central

    Hayashi, Ikuo; Takatori, Sho; Urano, Yasuomi; Iwanari, Hiroko; Isoo, Noriko; Osawa, Satoko; Fukuda, Maiko A.; Kodama, Tatsuhiko; Hamakubo, Takao; Li, Tong; Wong, Philip C.; Tomita, Taisuke; Iwatsubo, Takeshi

    2009-01-01

    γ-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid β precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the γ-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the γ-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the γ-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the γ-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the γ-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins. PMID:19684016

  9. Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants.

    PubMed

    Panjideh, Hossein; Coelho, Vânia; Dernedde, Jens; Fuchs, Hendrik; Keilholz, Ulrich; Thiel, Eckhard; Deckert, P Markus

    2008-10-01

    Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.

  10. Antineoplastic effect of intracellular expression of a single-chain antibody directed against type IV collagenase.

    PubMed

    Wang, W; Zhou, J; Xu, L; Zhen, Y

    2000-01-01

    It has been shown that the type IV collagenase with its two subtypes, 72 kDa/ MMP-2 and 92 kDa/MMP-9, plays an important role in tumor invasion and metastasis formation that occur through a mechanism of proteolytic degradation of collagen IV in the basement membrane. One possible method to specifically inhibit the function of the targeted protein of a cell is to express intracellular antibody combining site that can block the function or prevent the expression of the targeted molecule. Accordingly, intracellular antibodies against type IV collagenase may have a therapeutic use against tumor invasion and metastasis. As described in our previous reports, an anti-type IV collagenase monoclonal antibody (3D6) was obtained using the hybridoma approach, and its functional single-chain Fv fragment (scFv) named M97 was constructed based on recombinant phage display techniques. In this study, the endoplasmic reticulum (ER)-retained scFv antibody fragment was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acid (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector containing the CMV early-intermediate promoter/enhancer. The resulting plasmid was sequenced and then introduced by the lipofectamine method into PG cells, a highly metastatic human lung cancer cell line and G418-resistant cells were obtained by G418 selection. After transfection, the M97 mRNA expression was observed and the type IV collagenase expression was downregulated significantly as measured by ELISA. The biological behavior of PG cells, such as the ability of in vitro invasion of colony formation on soft agar through Matrigel, were also inhibited by scFv M97 transfection. The results indicate that intracellular antibody technology represents a novel and efficient way to selectively abrogate the activity of type IV collagenase, at least in vitro. We

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

    PubMed

    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.

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

    PubMed

    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.

  13. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    PubMed

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. PMID:25757566

  14. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    PubMed

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.

  15. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    SciTech Connect

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.

    2011-10-01

    A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

  16. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    PubMed

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

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

  17. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    PubMed

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein.

  18. Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

    PubMed

    Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

    2015-03-10

    Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification.

  19. 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.

    PubMed

    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

  20. Green fluorescent-conjugated anti-CEA single chain antibody for the detection of CEA-positive cancer cells.

    PubMed

    Salavatifar, Maryam; Amin, Shadi; Jahromi, Zahra Moghaddassi; Rasgoo, Nasrin; Rastgoo, Nasrin; Arbabi, Mehdi

    2011-06-01

    According to World Health Organization (WHO), cancer is a leading cause of death worldwide, accounting for 7.4 million deaths (around 13% of all deaths) in 2004. Monoclonal/recombinant antibodies, which specifically target clinical biomarkers of disease, have increasingly been applied as powerful tools in cancer imaging and therapy, a fact that is highlighted by some nine FDA-approved monoclonal antibodies (MAbs) or their immunoconjugates (as of December 2008) for use in cancer treatment. In this study, five monoclonal antibodies (MAbs) were generated and characterized against carcinoembryonic antigen (CEA), which is widely used clinically as both a blood and tissue tumor marker of epithelial malignancy. Variable domains (VH and VL) of one the stable MAbs with highest affinity were PCR-amplified and assembled as single-chain antibody fragment (scFv). Following the cloning and expression of scFv antibody fragments in Escherichia coli, the functional binding and specificity of the recombinant antibody were confirmed by ELISA. To develop a direct in vitro detection of CEA-positive cancer cells, scFv DNA was genetically fused to enhanced green fluorescent protein (EGFP) gene and expressed in bacteria. The chimeric fluorescent protein is able to specifically detect CEA-positive cell lines; no cross-reactivity was observed with a negative control cell line. This strategy will likely allow the establishment of a rapid, single-step detection assay of CEA, which is considered to be one of the best predictors of malignancy among all other tumor markers.

  1. Single-Chain Probes for Illuminating Androgenicity of Chemicals.

    PubMed

    Kim, Sung-Bae; Tao, Hiroaki

    2016-01-01

    The present protocol introduces a single-chain probe carrying a functional peptide in the N-terminal domain of the androgen receptor (AR NTD) for illuminating androgenicity of ligands. In the single-chain probe, a functional peptide in the AR NTD was genetically fused to the ligand-binding domain of AR (AR LBD) via a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. This single-chain probe exerts (1) a high signal-to-background ratio and (2) sensitive discrimination between agonists and antagonists, where the dimerization of AR LBD is not involved. The present protocol guides a fundamental methodology on how to discriminate weak protein-protein (peptide) binding, and provides a new insight into the intramolecular folding inside monomeric AR. PMID:27424901

  2. Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments.

    PubMed

    Opperman, Pamela A; Maree, Francois F; Van Wyngaardt, Wouter; Vosloo, Wilna; Theron, Jacques

    2012-08-01

    Recombinant single-chain variable fragments (scFvs) of antibodies make it possible to localize antigenic and immunogenic determinants, identify protective epitopes and can be exploited for the design of improved diagnostic tests and vaccines. A neutralizing epitope, as well as other potential antigenic sites of a SAT2 foot-and-mouth disease virus (FMDV) were identified using phage-displayed scFvs. Three unique ZIM/7/83-specific scFvs, designated scFv1, scFv2 and scFv3, were isolated. Further characterization of these scFvs revealed that only scFv2 was capable of neutralizing the ZIM/7/83 virus and was used to generate neutralization-resistant virus variants. Sequence analysis of the P1 region of virus escaping neutralization revealed a residue change from His to Arg at position 159 of the VP1 protein. Residue 159 is not only surface exposed but is also located at the C-terminal base of the G-H loop, a known immunogenic region of FMDV. A synthetic peptide, of which the sequence corresponded to the predicted antigenic site of the VP1 G-H loop of ZIM/7/83, inhibited binding of scFv2 to ZIM/7/83 in a concentration-dependent manner. This region can therefore be considered in the design of SAT2 vaccine seed viruses for the regional control of FMD in Africa.

  3. Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice.

    PubMed

    Wang, W; Zhang, S; Li, Y; Xu, L; Zhou, J; Zhen, Y

    2000-08-01

    It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)-retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer showed that scFv M97 transfection significantly prolonged the survival time of nude mice. The results indicate that intracellular antibody technology represents a novel and efficient way to abrogate selectively the activity of type IV collagenase. PMID:18726348

  4. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

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

    SciTech Connect

    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.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    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

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

    PubMed Central

    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

  9. In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe

    PubMed Central

    Mazzocco, Claire; Fracasso, Giulio; Germain-Genevois, Coralie; Dugot-Senant, Nathalie; Figini, Mariangela; Colombatti, Marco; Grenier, Nicolas; Couillaud, Franck

    2016-01-01

    We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragment, was labeled using a near-infrared fluorophore to generate a specific imaging probe (X770-scFvD2B). PSMA-unrelated scFv-X770 was used as a control. Probes were injected intravenously into mice with prostate tumors and fluorescence was monitored in vivo by fluorescence molecular tomography (FMT). In vitro assays showed that X770-scFvD2B specifically bound to PSMA and was internalized in PSMA-expressing LNCaP cells. After intravenous injection, X770-scFvD2B was detected in vivo by FMT in the prostate region. On excised prostates the scFv probe co-localized with the cancer cells and was found in PSMA-expressing cells. The PSMA-unrelated scFv used as a control did not label the prostate cancer cells. Our data demonstrate that scFvD2B is a high affinity contrast agent for in vivo detection of PSMA-expressing cells in the prostate. NIR-labeled scFvD2B could thus be further developed as a clinical probe for imaging-guided targeted biopsies. PMID:26996325

  10. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    PubMed

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

  11. Anti-Staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice.

    PubMed

    Wang, Man; Zhang, Yan; Zhu, Jianguo

    2016-03-01

    Staphylococcus aureus is a leading causative agent of bovine mastitis, which can result in significant economic losses to the dairy industry. However, available vaccines against bovine mastitis do not confer adequate protection, although passive immunization with antibodies may be useful to prevent disease. Hence, we constructed a bovine single-chain variable region fragment (scFv) phage display library using cDNAs from peripheral blood lymphocytes of cows with S. aureus-induced mastitis. After four rounds of selection, eight scFvs that bound S. aureus antigens with high affinity were obtained. The framework regions of the variable domains (VH and VL) of the eight scFvs were highly conserved, and the complementarity-determining regions (CDRs) displayed significant diversity, especially CDR3 of the VH domain. All eight scFvs inhibited S. aureus growth in culture medium. Lactating mice were challenged by injecting S. aureus into the fourth mammary gland. Histopathological analysis showed that treatment with these scFvs prior to bacterial challenge maintained the structure of the mammary acini, decreased infiltration of polymorphonuclear neutrophils, increased levels of interferon-gamma and interleukin-4, and reduced tumor necrosis factor-alpha levels in mammary tissues, as compared with mice treatment with physiological saline (P < 0.05). These novel bovine scFvs may be suitable candidates for therapeutic agents for the prevention of S. aureus-induced bovine mastitis.

  12. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

    PubMed Central

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2014-01-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

  13. Enzyme-mediated site-specific bioconjugation of metal complexes to proteins: sortase-mediated coupling of copper-64 to a single-chain antibody.

    PubMed

    Paterson, Brett M; Alt, Karen; Jeffery, Charmaine M; Price, Roger I; Jagdale, Shweta; Rigby, Sheena; Williams, Charlotte C; Peter, Karlheinz; Hagemeyer, Christoph E; Donnelly, Paul S

    2014-06-10

    The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins. PMID:24777818

  14. De novo design of a single-chain diphenylporphyrin metalloprotein.

    PubMed

    Bender, Gretchen M; Lehmann, Andreas; Zou, Hongling; Cheng, Hong; Fry, H Christopher; Engel, Don; Therien, Michael J; Blasie, J Kent; Roder, Heinrich; Saven, Jeffrey G; DeGrado, William F

    2007-09-01

    We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes.

  15. Development trends for generation of single-chain antibody fragments.

    PubMed

    Farajnia, Safar; Ahmadzadeh, Vahideh; Tanomand, Asghar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2014-10-01

    Recombinant antibodies are increasingly being employed as therapeutic agents especially in combination with anti-cancer drugs. The single-chain antibody fragments are small antigen-binding proteins which provide the most commonly used antibody formats for diagnostic and therapeutic purposes. These antibody fragments have more rapid tumor penetration and clearance from the serum relative to full-length monoclonal antibodies. There are in vitro antibody-display technologies such as phage display, cell surface display, ribosome display and mRNA display that can be used to isolate high specificity and affinity single-chain antibodies against a wide variety of targets. We review these strategies for generation of stable and active antibody fragments in the present article.

  16. Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: a review.

    PubMed

    Lê, H Q A; Sauriat-Dorizon, H; Korri-Youssoufi, H

    2010-07-26

    An electrochemical label-free immunosensor based on a biotinylated single-chain variable fragment (Sc-Fv) antibody immobilized on copolypyrrole film is described. An efficient immunosensor device formed by immobilization of a biotinylated single-chain antibody on an electropolymerized copolymer film of polypyrrole using biotin/streptavidin system has been demonstrated for the first time. The response of the biosensor toward antigen detection was monitored by surface plasmon resonance (SPR) and electrochemical analysis of the polypyrrole response by differential pulse voltammetry (DPV). The composition of the copolymer formed from a mixture of pyrrole (py) as spacer and a pyrrole bearing a N-hydroxyphthalimidyl ester group on its 3-position (pyNHP), acting as agent linker for biomolecule immobilization, was optimized for an efficient immunosensor device. The ratio of py:pyNHP for copolymer formation was studied with respect to the antibody immobilization and antigen detection. SPR was employed to monitor in real time the electropolymerization process as well as the step-by-step construction of the biosensor. FT-IR demonstrates the chemical copolymer composition and the efficiency of the covalent attachment of biomolecules. The film morphology was analyzed by electron scanning microscopy (SEM). Results show that a well organized layer is obtained after Sc-Fv antibody immobilization thanks to the copolymer composition defined with optimized pyrrole and functionalized pyrrole leading to high and intense redox signal of the polypyrrole layer obtained by the DPV method. Detection of specific antigen was demonstrated by both SPR and DPV, and a low concentration of 1 pg mL(-1) was detected by measuring the variation of the redox signal of polypyrrole.

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

    PubMed

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    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. PMID:27338340

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

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    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. PMID:27338340

  19. Multivalent Vaccine for Lymphatic Filariasis

    PubMed Central

    Samykutty, Abhilash; Dakshinamoorthy, Gajalakshmi; Kalyanasundaram, Ramaswamy

    2011-01-01

    Lymphatic filariasis is a mosquito borne parasitic infection that cause severe economic burden in several parts of the world. Currently there is no vaccine available to prevent this infection in human. Multidrug therapy is effective, however, requires annual treatment and there is significant concern of drug resistance. In this manuscript we describe development of a multivalent DNA based vaccine comprising BmALT-2 and BmHSP antigens of lymphatic filariasis. Challenge experiments using third stage infective larvae of Brugia malayi in a mouse model suggested that nearly 90% protection can be achieved using the multivalent formulation in a DNA prime protein boost approach. The vaccination regimen induced significant IgG antibody responses and ELISPOT analysis for secreted cytokines from the spleen cells of vaccinated animals showed that these cells produce significant amount of IL-4. Results from this study thus show that a multivalent vaccine formulation of BmALT-2 and BmHSP is an excellent vaccine for lymphatic filariasis and significant protection can be achieved against a challenge infection with B. malayi in a mouse model. PMID:21709765

  20. Multivalent Vaccine for Lymphatic Filariasis.

    PubMed

    Samykutty, Abhilash; Dakshinamoorthy, Gajalakshmi; Kalyanasundaram, Ramaswamy

    2010-01-01

    Lymphatic filariasis is a mosquito borne parasitic infection that cause severe economic burden in several parts of the world. Currently there is no vaccine available to prevent this infection in human. Multidrug therapy is effective, however, requires annual treatment and there is significant concern of drug resistance. In this manuscript we describe development of a multivalent DNA based vaccine comprising BmALT-2 and BmHSP antigens of lymphatic filariasis. Challenge experiments using third stage infective larvae of Brugia malayi in a mouse model suggested that nearly 90% protection can be achieved using the multivalent formulation in a DNA prime protein boost approach. The vaccination regimen induced significant IgG antibody responses and ELISPOT analysis for secreted cytokines from the spleen cells of vaccinated animals showed that these cells produce significant amount of IL-4. Results from this study thus show that a multivalent vaccine formulation of BmALT-2 and BmHSP is an excellent vaccine for lymphatic filariasis and significant protection can be achieved against a challenge infection with B. malayi in a mouse model.

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

    PubMed

    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

  2. A Single-Chain Antibody Using LoxP511 as the Linker Enables Large-Content Phage Library Construction via Cre/LoxP Recombination.

    PubMed

    Zhang, Yan; Wang, Wei; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Yali; Shen, Beifen; Ma, Yuanfang; Li, Yan; Qiao, Chunxia; Feng, Jiannan

    2014-07-01

    To obtain natural or "me-better" antibodies (e.g., affinity-maturated antibodies), phage display libraries are widely used. However, the likelihood of obtaining satisfactory antibodies depends on the library content. Here, we used computer-aided design to model the use of the LoxP511 site as a linker between the heavy and light variable domains of an antibody for construction of a large single-chain fragment (scFv) antibody phage library by using the Cre/LoxP recombinant system. Then, we constructed two novel scFvs based on 2C4, namely, AH_scFv15 (15 amino acid [aa] linker; common [SG4]3 sequence) and AH_scFv21 (21-aa linker; LoxP511 sequence), to verify the use of the LoxP511 site as a linker. Our results indicate that LoxP511 could be used effectively for the construction of a large (e.g., 5 × 10(12)) phage display library of scFv antibodies from which it was possible to isolate an antibody with the same epitope as 2C4 but with higher affinity.

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

    PubMed

    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

  4. Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency.

    PubMed Central

    Ren, X; Gao, S; You, D; Huang, H; Liu, Z; Mu, Y; Liu, J; Zhang, Y; Yan, G; Luo, G; Yang, T; Shen, J

    2001-01-01

    Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240-1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251-255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5-10%, 15-20% and 25-30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8 M urea by Co(2+)-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46 x 10(5) M(-1) and 1.03 x 10(5) M(-1) respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394 units/micromol, which approaches the activity of rabbit liver GPX. PMID:11583583

  5. Reducing heterophilic antibody interference in immunoassays using single chain antibodies

    SciTech Connect

    Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.

    2011-12-15

    Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

  6. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.

    SciTech Connect

    Miller, Keith D.; Feldhaus, Jane M.; Gray, Sean A.; Siegel, Robert W.; Feldhaus, Michael J.

    2005-08-01

    Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0-99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mg L-1 culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was also used to compare scFv production levels from the periplasm, inclusion bodies, and culture media. The E. coli production system was then used to produce variants of several scFv to determine structure function relationships.

  7. Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments

    PubMed Central

    Stech, Marlitt; Hust, Michael; Schulze, Corina; Dübel, Stefan; Kubick, Stefan

    2014-01-01

    Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the cell-free expression of functional single-chain antibody variable fragments (scFvs) in a eukaryotic and endotoxin-free in vitro translation system based on Spodoptera frugiperda (Sf21) insect cell extracts. Three scFv candidates with different specificities were chosen as models. The first scFv candidate SH527-IIA4 specifically discriminates between its phosphorylated (SMAD2-P) and nonphosphorylated antigens (SMAD2) (where SMAD is mothers against decapentaplegic homolog 2), whereas the second scFv candidate SH527-IIC10 recognizes both, SMAD2-P and SMAD2. The third scFv candidate SH855-C11 binds specifically to a linear epitope of the CXC chemokine receptor type 5. The translocation of antibody fragments into the lumen of endogenous microsomal vesicles, which are contained in the lysate, was facilitated by fusion of scFv genes to the insect cell specific signal sequence of honeybee melittin. We compared the binding capabilities of scFv fragments with and without melittin signal peptide and detected that translocated scFv fragments were highly functional, whereas scFvs synthesized in the cytosol of the cell extract showed strongly decreased binding capabilities. Additionally, we describe a cell-free protein synthesis method for the incorporation of noncanonical amino acids into scFv molecules in eukaryotic cell lysates. We demonstrate the successful cotranslational labeling of de novo synthesized scFv molecules with fluorescent amino acids, using residue-specific as well as site-specific labeling. PMID:25821419

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

    PubMed Central

    Saeed, Mesha; van Brakel, Mandy; Zalba, Sara; Schooten, Erik; Rens, Joost AP; Koning, Gerben A; Debets, Reno; ten Hagen, Timo LM

    2016-01-01

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

  9. Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean.

    PubMed

    Wang, Bing; Swaminathan, Sivakumar; Bhattacharyya, Madan K

    2015-01-01

    Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed. PMID:26709700

  10. Anti-ABCG2 scFv antibody of lung adenocarcinoma increases chemosensitivity and induces apoptosis through the activation of mitochondrial pathway.

    PubMed

    Zhao, Wen-Si; Luo, Yi; Li, Bo-Yi; Zhou, Han-Jing; Zhang, Tao

    2016-01-01

    ABCG2 is a multidrug resistance efflux pump expressed in many diverse tumors. The overexpression of ABCG2 is associated with resistance to a wide variety of anticancer agents, providing a noticeable setback to successful cancer therapy. Therapies targeting ABCG2 may therefore be a promising candidate for reversal of chemoresistance. The anti-ABCG2 single-chain variable fragment (scFv) antibody was constructed by phage display peptide library technology. Immunoblotting, ELISA and immunocytochemistry were used to evaluate the soluble expression and immunoreactivity of the scFv. The effects of scFv on cell function and chemosensitization were confirmed by colony formation, cell migration and CCK-8 assays. Flow cytometry was used to analyse the cell cycle and apoptosis. Radioimmunoimaging and nude mouse tumorigenicity assays were taken to determine the biodistribution and antitumor capacity of the scFv antibody. We have successfully screened out the candidate scFv antibody with an apparent molecular weight of 34 kDa. The scFv demonstrated favourable binding ability to lung adenocarcinoma cells and ABCG2 antigen, and the radioactivity was specifically aggregated at the tumor location. Furthermore, the internalized scFv resulted in antibody-mediated downregulation of ABCG2, proliferation inhibition, apoptosis and cisplatin (DDP) sensitivity. The anti-ABCG2 scFv antibody possesses good tumoraffin and antitumor activity and may therefore be an effective therapeutic agent for lung adenocarcinoma that is dependent on ABCG2 for drug resistance and survival. PMID:27293996

  11. Anti-ABCG2 scFv antibody of lung adenocarcinoma increases chemosensitivity and induces apoptosis through the activation of mitochondrial pathway

    PubMed Central

    Zhao, Wen-Si; Luo, Yi; Li, Bo-Yi; Zhou, Han-Jing; Zhang, Tao

    2016-01-01

    ABCG2 is a multidrug resistance efflux pump expressed in many diverse tumors. The overexpression of ABCG2 is associated with resistance to a wide variety of anticancer agents, providing a noticeable setback to successful cancer therapy. Therapies targeting ABCG2 may therefore be a promising candidate for reversal of chemoresistance. The anti-ABCG2 single-chain variable fragment (scFv) antibody was constructed by phage display peptide library technology. Immunoblotting, ELISA and immunocytochemistry were used to evaluate the soluble expression and immunoreactivity of the scFv. The effects of scFv on cell function and chemosensitization were confirmed by colony formation, cell migration and CCK-8 assays. Flow cytometry was used to analyse the cell cycle and apoptosis. Radioimmunoimaging and nude mouse tumorigenicity assays were taken to determine the biodistribution and antitumor capacity of the scFv antibody. We have successfully screened out the candidate scFv antibody with an apparent molecular weight of 34 kDa. The scFv demonstrated favourable binding ability to lung adenocarcinoma cells and ABCG2 antigen, and the radioactivity was specifically aggregated at the tumor location. Furthermore, the internalized scFv resulted in antibody-mediated downregulation of ABCG2, proliferation inhibition, apoptosis and cisplatin (DDP) sensitivity. The anti-ABCG2 scFv antibody possesses good tumoraffin and antitumor activity and may therefore be an effective therapeutic agent for lung adenocarcinoma that is dependent on ABCG2 for drug resistance and survival. PMID:27293996

  12. High-level expression of a phage display-derived scFv in Pichia pastoris.

    PubMed

    Damasceno, Leonardo M; Lee, Frank; Ritter, Gerd; Old, Lloyd; Batt, Carl

    2009-01-01

    Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification of A33scFv was optimized for the methylotrophic yeast P. pastoris. P. pastoris with a Mut(S) phenotype was selected to express A33scFv under regulation of the methanol-inducible AOX1 promoter. Here we describe a large-scale fed-batch fermentation process with an efficient online closed-loop methanol control for the production of the recombinant protein. Purification of A33scFv from clarified culture medium was done using a two-step chromatographic procedure using anion exchange and hydrophobic interaction chromatography, resulting in a final product with more than 90% purity. This chapter provides protocols that can be used as a base for process development of recombinant protein expression in P. pastoris and purification of these proteins for use in further functionality studies and in diagnostic and therapeutic applications.

  13. Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes.

    PubMed

    Yodsheewan, Rungrueang; Maneewatch, Santi; Srimanote, Potjanee; Thueng-In, Kanyarat; Songserm, Thaweesak; Dong-Din-On, Fonthip; Bangphoomi, Kunan; Sookrung, Nitat; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

    2013-10-01

    Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed Escherichia coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2-64-fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN-β genes by 14.75 and 4.95-fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza.

  14. Insilico analysis of three different tag polypeptides with dual roles in scFv antibodies.

    PubMed

    Mohammadi, Mozafar; Nejatollahi, Foroogh; Sakhteman, Amirhossein; Zarei, Neda

    2016-08-01

    Single chain fragment variable (scFv) antibodies are composed of variable heavy (VH) and variable light (VL) domains that are joined by a polypeptide linker. Typically, [(Gly4Ser) n] sequence is used as a linker to retain the integrity of the antigen-binding domain. Due to its low immunogenicity, this sequence cannot be used as a tag for scFv detection and purification. Several evidences have shown that the addition of an N or C-terminal tag for scFv detection and purification will result in the decreased expression and binding capacity of this antibody fragment. In this study, we substituted the traditional linker (GGGGS) with His-tag, C-myc or E-tag sequences through molecular modeling. Stability and integrity of all models were assessed by molecular dynamic (MD) simulation. Based on MD simulation analysis, the model containing E-tag sequence as a linker indicated more stability compared to other molecules. The results suggest that E-tag not only can be substituted for the traditional linker, also eliminates the necessity of using additional tag for scFv detection and purification. PMID:27113782

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

    PubMed

    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.

  16. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

    PubMed

    Bublin, Merima; Kostadinova, Maria; Fuchs, Julian E; Ackerbauer, Daniela; Moraes, Adolfo H; Almeida, Fabio C L; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. PMID:26579717

  17. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

    PubMed Central

    Fuchs, Julian E.; Ackerbauer, Daniela; Moraes, Adolfo H.; Almeida, Fabio C. L.; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R.; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. PMID:26579717

  18. Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

    PubMed

    Indrawattana, Nitaya; Sookrung, Nitat; Kulkeaw, Kasem; Seesuay, Watee; Kongngoen, Thida; Chongsa-nguan, Manas; Tungtrongchitr, Anchalee; Chaicumpa, Wanpen

    2010-03-01

    Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called tetanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant

  19. Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles, an antibody conjugate with potent and selective anti-hepatocellular carcinoma activity.

    PubMed

    Xiangbao, Yin; Linquan, Wu; Mingwen, Huang; Fan, Zhou; Kai, Wang; Xin, Yu; Kaiyang, Wang; Huaqun, Fu

    2014-06-01

    Low sensitivity of tumor tissue, targeting and sustained release of the drug are bottlenecks of the effect of chemotherapy on hepatocellular carcinoma. In this study, we used the ribosome display technology to screen human anti-VEGFR 2-single-chain antibody (ScFv) that could target directly to VEGFR2, and nanotechnology to prepare As2O3-nanoparticles. Then we built anti-VEGFR-2ScFv-As2O3-stealth nanoparticles using molecular coupling technology, which significantly increased anti-tumor effect while reducing toxicity. The in vivo tissue targeting distribution and anti-tumor effects of the anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles were investigated. Our results showed that anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles could inhibit the development of liver cancer xenograft as a targeting agent and also significantly inhibit angiogenesis.

  20. Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles, an antibody conjugate with potent and selective anti-hepatocellular carcinoma activity.

    PubMed

    Xiangbao, Yin; Linquan, Wu; Mingwen, Huang; Fan, Zhou; Kai, Wang; Xin, Yu; Kaiyang, Wang; Huaqun, Fu

    2014-06-01

    Low sensitivity of tumor tissue, targeting and sustained release of the drug are bottlenecks of the effect of chemotherapy on hepatocellular carcinoma. In this study, we used the ribosome display technology to screen human anti-VEGFR 2-single-chain antibody (ScFv) that could target directly to VEGFR2, and nanotechnology to prepare As2O3-nanoparticles. Then we built anti-VEGFR-2ScFv-As2O3-stealth nanoparticles using molecular coupling technology, which significantly increased anti-tumor effect while reducing toxicity. The in vivo tissue targeting distribution and anti-tumor effects of the anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles were investigated. Our results showed that anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles could inhibit the development of liver cancer xenograft as a targeting agent and also significantly inhibit angiogenesis. PMID:24855034

  1. An anti-Aβ (amyloid β) single-chain variable fragment prevents amyloid fibril formation and cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway.

    PubMed

    Marín-Argany, Marta; Rivera-Hernández, Geovanny; Martí, Joaquim; Villegas, Sandra

    2011-07-01

    Aβ (amyloid β) immunotherapy has been revealed as a possible tool in Alzheimer's disease treatment. In contrast with complete antibodies, the administration of scFvs (single-chain variable fragments) produces neither meningoencephalitis nor cerebral haemorrhage. In the present study, the recombinant expression of scFv-h3D6, a derivative of an antibody specific for Aβ oligomers, is presented, as well as the subsequent proof of its capability to recover the toxicity induced by the Aβ1-42 peptide in the SH-SY5Y neuroblastoma cell line. To gain insight into the conformational changes underlying the prevention of Aβ toxicity by this antibody fragment, the conformational landscape of scFv-h3D6 upon temperature perturbation is also described. Heating the native state does not lead to any extent of unfolding, but rather directly to a β-rich intermediate state which initiates an aggregation pathway. This aggregation pathway is not an amyloid fibril pathway, as is that followed by the Aβ peptide, but rather a worm-like fibril pathway which, noticeably, turns out to be non-toxic. On the other hand, this pathway is thermodynamically and kinetically favoured when the scFv-h3D6 and Aβ1-42 oligomers form a complex in native conditions, explaining how the scFv-h3D6 withdraws Aβ1-42 oligomers from the amyloid pathway. To our knowledge, this is the first description of a conformational mechanism by which a scFv prevents Aβ-oligomer cytotoxicity.

  2. Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.

    PubMed

    Sun, Dongbo; Shi, Hongyan; Chen, Jianfei; Shi, Da; Zhu, Qinghe; Zhang, Hong; Liu, Shengwang; Wang, Yunfeng; Qiu, Huaji; Feng, Li

    2012-06-01

    Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×10(7) recombinant phage clones, and the titer of the amplified library was 3.6×10(9)pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described.

  3. Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water.

    PubMed

    Zhang, Xiuyuan; He, Kuo; Zhao, Ruiping; Wang, Lixia; Jin, Yandan

    2016-10-01

    Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv-AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 μgL(-1) and 0.13 ± 0.03 μgL(-1), respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way.

  4. Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water.

    PubMed

    Zhang, Xiuyuan; He, Kuo; Zhao, Ruiping; Wang, Lixia; Jin, Yandan

    2016-10-01

    Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv-AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 μgL(-1) and 0.13 ± 0.03 μgL(-1), respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way. PMID:27380224

  5. An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification.

    PubMed

    Sotelo, Pablo; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

    2012-01-01

    Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

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

    SciTech Connect

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

    2011-08-09

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

  7. Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation.

    PubMed

    Paopang, Porntip; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Seesuriyachan, Phisit; Butr-Indr, Bordin

    2016-01-01

    The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett-Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.

  8. Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

    PubMed Central

    Miethe, Sebastian; Rasetti-Escargueil, Christine; Avril, Arnaud; Liu, Yvonne; Chahboun, Siham; Korkeala, Hannu; Mazuet, Christelle; Popoff, Michel-Robert; Pelat, Thibaut; Thullier, Philippe; Sesardic, Dorothea; Hust, Michael

    2015-01-01

    Background Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure. Results In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay. Conclusion These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development. PMID:26440796

  9. Multivalent glycoconjugates as anti-pathogenic agents†

    PubMed Central

    Bernardi, Anna; Jiménez-Barbero, Jesus; Casnati, Alessandro; De Castro, Cristina; Darbre, Tamis; Fieschi, Franck; Finne, Jukka; Funken, Horst; Jaeger, Karl-Erich; Lahmann, Martina; Lindhorst, Thisbe K.; Marradi, Marco; Messner, Paul; Molinaro, Antonio; Murphy, Paul V.; Nativi, Cristina; Oscarson, Stefan; Penadés, Soledad; Peri, Francesco; Pieters, Roland J.; Renaudet, Olivier; Reymond, Jean-Louis; Richichi, Barbara; Rojo, Javier; Sansone, Francesco; Schäffer, Christina; Turnbull, W. Bruce; Velasco-Torrijos, Trinidad; Vidal, Sébastien; Vincent, Stéphane; Wennekes, Tom; Zuilhof, Han; Imberty, Anne

    2015-01-01

    Multivalency plays a major role in biological processes and particularly in the relationship between pathogenic microorganisms and their host that involves protein–glycan recognition. These interactions occur during the first steps of infection, for specific recognition between host and bacteria, but also at different stages of the immune response. The search for high-affinity ligands for studying such interactions involves the combination of carbohydrate head groups with different scaffolds and linkers generating multivalent glycocompounds with controlled spatial and topology parameters. By interfering with pathogen adhesion, such glycocompounds including glycopolymers, glycoclusters, glycodendrimers and glyconanoparticles have the potential to improve or replace antibiotic treatments that are now subverted by resistance. Multivalent glycoconjugates have also been used for stimulating the innate and adaptive immune systems, for example with carbohydrate-based vaccines. Bacteria present on their surfaces natural multivalent glycoconjugates such as lipopolysaccharides and S-layers that can also be exploited or targeted in anti-infectious strategies. PMID:23254759

  10. Single Chain Fragment Variable Recombinant Antibody Functionalized Gold Nanoparticles for a Highly Sensitive Colorimetric Immunoassay

    PubMed Central

    Liu, Yang; Liu, Yi; Raymond, Raymond L.; Zeng, Xiangqun

    2009-01-01

    In this report, the peptide linker connecting scFv VH and VL domains were genetically modified to contain different amino acids (i.e. cysteine (scFv-cys) or histidines ( scFv-his)) to enable the scFv to adsorb or self-assemble onto the gold nanoparticles (NPs). The scFv-cys stabilized gold NPs were used to develop a highly sensitive colorimetric immunosensor. The scFv-cys stabilized gold NPs were characterized by UV-vis spectra, transmission electron microscope (TEM) and FT-IR. After adding the antigen rabbit IgG, the solution of scFv-cys stabilized gold NPs shows obvious visible color change from deep red to light purple due to the aggregation of the gold nanoparticles. Based on the colorimetric aggregation of scFv-cys stabilized gold NPs, the immunosensor exhibits high sensitivity with detection limit of 1.7 nM and good specificity. The good properties of the colorimetric aggregation immunosensor would be attributed to the small size of scFv and the covalent link between the scFv and gold NPs that improve the better orientation and enhance the probe density. With the advantages of speed, simplicity and specificity, the colorimetric immunoassay based on the functionalized scFv stabilized gold NPs represents a promising approach for protein analysis and clinical diagnostics. PMID:19327975

  11. Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

    NASA Astrophysics Data System (ADS)

    Liu, Shuang

    Multivalent interactions are characterized by the simultaneous binding between multiple ligands and multiple binding sites, either in solutions or at interfaces. In biological systems, most multivalent interactions occur between protein receptors and carbohydrate ligands through hydrogen-bonding and hydrophobic interactions. Compared with weak affinity binding between one ligand and one binding site, i.e. monovalent interaction, multivalent interactioins provide greater avidity and specificity, and therefore play unique roles in a broad range of biological activities. Moreover, the studies of multivalent interactions are also essential for producing effective inhibitors and effectors of biological processes that could have important therapeutic applications. Synthetic multivalent ligands have been designed to mimic the biological functions of natural multivalent interactions, and various types of scaffolds have been used to display multiple ligands, including small molecules, linear polymers, dendrimers, nanoparticle surfaces, monolayer surfaces and liposomes. Studies have shown that multivalent interactions can be highly affected by various architectural parameters of these multivalent ligands, including ligand identities, valencies, spacing, ligand densities, nature of linker arms, scaffold length and scaffold conformation. Most of these multivalent ligands are chemically synthesized and have limitations of controlling over sequence and conformation, which is a barrier for mimicking ordered and controlled natural biological systems. Therefore, multivalent ligands with precisely controlled architecture are required for improved structure-function relationship studies. Protein engineering methods with subsequent chemical coupling of ligands provide significant advantages of controlling over backbone conformation and functional group placement, and therefore have been used to synthesize recombinant protein-based materials with desired properties similar to natural

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

    PubMed

    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.

  13. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

    PubMed

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  14. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

    PubMed Central

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A.; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R.

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  15. Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

    NASA Astrophysics Data System (ADS)

    Liu, Shuang

    Multivalent interactions are characterized by the simultaneous binding between multiple ligands and multiple binding sites, either in solutions or at interfaces. In biological systems, most multivalent interactions occur between protein receptors and carbohydrate ligands through hydrogen-bonding and hydrophobic interactions. Compared with weak affinity binding between one ligand and one binding site, i.e. monovalent interaction, multivalent interactioins provide greater avidity and specificity, and therefore play unique roles in a broad range of biological activities. Moreover, the studies of multivalent interactions are also essential for producing effective inhibitors and effectors of biological processes that could have important therapeutic applications. Synthetic multivalent ligands have been designed to mimic the biological functions of natural multivalent interactions, and various types of scaffolds have been used to display multiple ligands, including small molecules, linear polymers, dendrimers, nanoparticle surfaces, monolayer surfaces and liposomes. Studies have shown that multivalent interactions can be highly affected by various architectural parameters of these multivalent ligands, including ligand identities, valencies, spacing, ligand densities, nature of linker arms, scaffold length and scaffold conformation. Most of these multivalent ligands are chemically synthesized and have limitations of controlling over sequence and conformation, which is a barrier for mimicking ordered and controlled natural biological systems. Therefore, multivalent ligands with precisely controlled architecture are required for improved structure-function relationship studies. Protein engineering methods with subsequent chemical coupling of ligands provide significant advantages of controlling over backbone conformation and functional group placement, and therefore have been used to synthesize recombinant protein-based materials with desired properties similar to natural

  16. Evaluation of rMETase-Loaded Stealth PLGA/Liposomes Modified with Anti-CAGE scFV for Treatment of Gastric Carcinoma.

    PubMed

    Xin, Lin; Caot, Jia-Qing; Liu, Chuan; Zeng, Fei; Cheng, Hua; Hu, Xiao-Yun; Shao, Jiang-Hua

    2015-07-01

    Stealth PLGA/Liposome nanoparticles (NPs) modified with tumor-targeting single-chain antibody fragment (scFV-P/L) for systemic delivery of recombinant methioninase (rMETase) for gastric cancer were prepared. The morphologies and therapeutic effects of rMETase-loaded scFV-P/L (scFV-rMETase-P/L) in vitro were analyzed. Functional scFV-P/L NPs composed of PLGA, DOPC and DSPE-PEG display low cell cytoxicity in SGC-7901 cells, and has more cell uptake ability than P/L NPs. scFV-rMETase-P/L was more effective in inhibiting tumor growth in the subcutaneous gastric carcinoma tumor model than free rMETase in solution (p < 0.05) and rMETase-loaded P/L (rMETase-P/L) (p < 0.05). Our findings collectively support the utility of scFV-targeted P/L NPs as a potentially effective drug delivery system.

  17. Dendritic Cells Transfected with scFv from Mab 7.B12 Mimicking Original Antigen gp43 Induces Protection against Experimental Paracoccidioidomycosis

    PubMed Central

    Ferreira, Karen S.; Maranhão, Andrea Q.; Garcia, Maria C. C.; Brígido, Marcelo M.; Santos, Suelen S.; Lopes, José D.; Almeida, Sandro R.

    2011-01-01

    Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model. PMID:21249212

  18. Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells.

    PubMed

    Wang, Zhujun; Chen, Yuanyuan; Li, Sisi; Cheng, Yuping; Zhao, Haizhao; Jia, Ming; Luo, Zebin; Tang, Yongmin

    2014-02-01

    CD45RA has been found highly expressed on leukemia cells and may be a potential target of the disease. In this study, an anti-CD45RA single-chain antibody fragment (scFv3A4) was genetically linked to the N terminus of the enhanced green fluorescent protein (EGFP) to generate a scFv3A4-EGFP fusion protein. The scFv3A4-EGFP with a molecular weight of 57kDa was stably expressed and secreted from the transfected CHO cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was 1350μg/L. Flow cytometry analysis showed that the scFv3A4-EGFP had the same binding site and a very similar reactivity pattern with its parental murine monoclonal antibody (mAb) 3A4. Furthermore, comparing to conventional labeled 3A4-FITC antibody, the scFv3A4-EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection. Therefore, the scFv3A4-EGFP fusion protein can be a powerful tool to investigate the targeting of CD45RA on leukemia cells, biological activity of the target and possibly for the genetic manipulation of the antibody.

  19. Designing multivalent probes for tunable superselective targeting

    PubMed Central

    Curk, Tine; Auzély-Velty, Rachel; Frenkel, Daan; Richter, Ralf P.

    2015-01-01

    Specific targeting is common in biology and is a key challenge in nanomedicine. It was recently demonstrated that multivalent probes can selectively target surfaces with a defined density of surface binding sites. Here we show, using a combination of experiments and simulations on multivalent polymers, that such “superselective” binding can be tuned through the design of the multivalent probe, to target a desired density of binding sites. We develop an analytical model that provides simple yet quantitative predictions to tune the polymer’s superselective binding properties by its molecular characteristics such as size, valency, and affinity. This work opens up a route toward the rational design of multivalent probes with defined superselective targeting properties for practical applications, and provides mechanistic insight into the regulation of multivalent interactions in biology. To illustrate this, we show how the superselective targeting of the extracellular matrix polysaccharide hyaluronan to its main cell surface receptor CD44 is controlled by the affinity of individual CD44–hyaluronan interactions. PMID:25901321

  20. EM Algorithm for Mapping Quantitative Trait Loci in Multivalent Tetraploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multivalent tetraploids that include many plant species, such as potato, sugarcane and rose, are of paramount importance to agricultural production and biological research. Quantitative trait locus (QTL) mapping in multivalent tetraploids is challenged by their unique cytogenetic properties, such ...

  1. Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.

    PubMed

    Fujita, Koji; Yamaguchi, Yoshitaka; Mori, Tsuyoshi; Muramatsu, Naomi; Miyamoto, Takahito; Yano, Masashi; Miyata, Hironori; Ootsuyama, Akira; Sawada, Makoto; Matsuda, Haruo; Kaji, Ryuji; Sakaguchi, Suehiro

    2011-10-01

    We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

  2. [Construction of combinatorial immune library of single chain human antibodies to orthopoxviruses and selection from this library antibodies to recombinant protein prA30L of variola virus].

    PubMed

    Dubrovskaia, V V; Ulitin, A B; Laman, A G; Gileva, I P; Bormotov, N I; Il'ichev, A A; Brovko, F A; Shchelkunov, S N; Belanov, E F; Tikunova, N V

    2007-01-01

    A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.

  3. Conversion of scFv peptide-binding specificity for crystal chaperone development

    SciTech Connect

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  4. Small anion with higher valency retards the compaction of DNA in the presence of multivalent cation.

    PubMed

    Saito, Takuya; Iwaki, Takafumi; Yoshikawa, Kenichi

    2009-02-01

    It has been established that, upon the addition of multivalent cations, long DNA chains in an aqueous solution exhibit a remarkable discrete transition from a coil state to a compact state at the level of a single chain. In this study, we investigated the polyelectrolyte nature of DNA with the experimental methodology of single-DNA observation, and provide a theoretical interpretation. We examined the effects of co-ions with different valencies (Cl(-), SO4(2-), PO4(3-)) on DNA compaction. As a result, we found that co-ions with a greater valency induce the coil state rather than the compact state. Based on a simple model with mean-field approximation that considered ion pairing, we show how the increase in entropy of small ions contributes to the stability of the compact state, by overcoming entropic penalties such as elastic confinement of the chain and a decrease in the translational freedom of counterions accompanied by charge neutralization.

  5. Emerging trends in enzyme inhibition by multivalent nanoconstructs.

    PubMed

    Kanfar, Nasreddine; Bartolami, Eline; Zelli, Renaud; Marra, Alberto; Winum, Jean-Yves; Ulrich, Sébastien; Dumy, Pascal

    2015-10-21

    Multivalent nanoconstructs, extensively used for enhancing the recognition of biomolecular targets, have been recently exploited for enzyme inhibition showing interesting properties such as improvement of inhibitory potency and selectivity. We review herein the recent results highlighting the potential of multivalent nanoconstructs for the inhibition of different enzymes, and the emerging trends in the generation and identification of multivalent clusters as enzyme inhibitors.

  6. Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

    PubMed

    Liu, Jing; Zhang, Hui C; Duan, Chang F; Dong, Jun; Zhao, Guo X; Wang, Jian P; Li, Nan; Liu, Jin Z; Li, Yu W

    2016-11-01

    The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

  7. Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

    PubMed Central

    Levenhagen, Marcelo Arantes; de Almeida Araújo Santos, Fabiana; Fujimura, Patrícia Tiemi; Caneiro, Ana Paula; Costa-Cruz, Julia Maria; Goulart, Luiz Ricardo

    2015-01-01

    Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis. PMID:25994608

  8. Selection and characterisation of recombinant single-chain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries.

    PubMed

    Moghaddam, A; Løbersli, I; Gebhardt, K; Braunagel, M; Marvik, O J

    2001-08-01

    Selection of antibodies from large repertoire phage display libraries has become a common technique for isolation of specific antibodies to antigens. Many of these libraries are shown to contain antibodies specific to haptens, but only when these haptens are derivatised or conjugated to an immobilising molecule, such as bovine serum albumin (BSA). There has been little demonstration of the suitability of naive recombinant antibody libraries for isolating antibodies that bind low molecular weight haptens in the absence of a carrier molecule and few have addressed the problems associated with selecting antibodies that only recognize the combination of hapten and the carrier molecule. We have panned two-phage antibody libraries against AflatoxinB1-BSA and screened single-chain antibody fragments for binding to AflatoxinB1-BSA and Aflatoxin-B1. Many of the antibodies isolated specifically bound AflatoxinB1-BSA, but not soluble Aflatoxin-B1 or BSA. Modification of the protocol led to isolation of single-chain fragment variable antibody domain (scFv) antibodies that specifically bound soluble Aflatoxin-B1 with an affinity of 6x10(-9) M. PMID:11406162

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

    PubMed

    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.

  10. Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk.

    PubMed

    Welbeck, Katherine; Leonard, Paul; Gilmartin, Niamh; Byrne, Barry; Viguier, Caroline; Arora, Sushrut; O'Kennedy, Richard

    2011-02-01

    Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from naïve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.

  11. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

    PubMed Central

    Andrade, Fernanda B.; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D.; Yamamoto, Bruno B.; Luz, Daniela; Abreu, Patrícia A. E.; Horton, Denise S. P. Q.; Elias, Waldir P.; Ramos, Oscar H. P.; Piazza, Roxane M. F.

    2015-01-01

    Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Methods and Findings Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis. PMID:26154103

  12. The single-chain immunotoxin MCSP-ETA’, targeting melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells

    PubMed Central

    Schwenkert, Michael; Birkholz, Katrin; Schwemmlein, Michael; Kellner, Christian; Peipp, Matthias; Nettelbeck, Dirk M.; Schuler-Thurner, Beatrice; Schaft, Niels; Dörrie, Jan; Ferrone, Soldano; Kämpgen, Eckhart; Fey, Georg H.

    2009-01-01

    A recombinant immunotoxin was constructed by fusing a single-chain Fv (scFv) antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas Exotoxin A (ETA’), carrying a C-terminal KDEL peptide for improved intracellular transport. The resulting immunotoxin, MCSP-ETA’, induced antigen-specific, potent apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80 % of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nM for both cell lines. MCSP-ETA’ also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease, with net cell death reaching up to 70 % within 96 h after treatment with a single dose of 14 nM. MCSP-ETA’ induced cell death synergistically with Cyclosporin A (CsA), both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with CsA justify further evaluation of this novel agent with regard to its potential applications for the treatment of melanoma and other MCSP-positive malignancies. PMID:18337643

  13. Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans.

    PubMed

    Greineder, Colin F; Hood, Elizabeth D; Yao, Anning; Khoshnejad, Makan; Brenner, Jake S; Johnston, Ian H; Poncz, Mortimer; Gottstein, Claudia; Muzykantov, Vladimir R

    2016-03-28

    Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans. PMID:26855052

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

    PubMed

    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

  15. The use of scFv-displaying yeast in mammalian cell surface selections.

    PubMed

    Wang, Xin Xiang; Shusta, Eric V

    2005-09-01

    Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.

  16. Dynamics of Single Chains of Suspended Ferrofluid Particles

    NASA Technical Reports Server (NTRS)

    Cutillas, S.; Liu, J.

    1999-01-01

    We present an experimental study of the dynamics of isolated chains made of super-paramagnetic particles under the influence of a magnetic field. The motivation of this work is to understand if the chain fluctuations exist and, if it does, how does the fluctuation affect chain aggregation. We find that single chains strongly fluctuate and that the characteristic frequency of their fluctuations is inversely proportional to the magnetic field strength. The higher the field the lower the characteristic frequency of the chain fluctuations. In the high magnetic field limit, chains behave like rigid rods without any internal motions. In this work, we used ferrofluid particles suspended in water. These particles do not have any intrinsic magnetization. Once a magnetic field is applied, a dipole moment is induced in each particle, proportional to the magnetic field. A dipolar magnetic interaction then occurs between particles. If dipole-dipole magnetic energy is higher than the thermal energy, the result is a structure change inside the dipolar fluid. The ratio of these two energies is expressed by a coupling constant lambda as: lambda = (pi(a(exp 3))(chi(exp 2))(mu(sub 0))(H(sub 0))(exp 2))/18kT Where a is the particle radius, mu(sub 0) is the vacuum magnetic permeability, H(sub 0) the applied magnetic field, k the Boltzmann constant and T the absolute temperature. If lambda > 1, magnetic particles form chains along the field direction. The lateral coalescence of several chains may form bigger aggregates especially if the particle volume fraction is high. While many studies and applications deal with the rheological properties and the structural changes of these dipolar fluids, this work focuses on the understanding of the chain dynamics. In order to probe the chain dynamics, we used dynamic light scattering (DLS) in self-beating mode as our experimental technique. The experimental geometry is such that the scattering plane is perpendicular to the magnetic field

  17. Anti-EGFR scFv tetramer (tetrabody) with a stable monodisperse structure, strong anticancer effect, and a long in vivo half-life.

    PubMed

    Asano, Ryutaro; Koyama, Noriaki; Hagiwara, Yasuyo; Masakari, Yosuke; Orimo, Ryota; Arai, Kyoko; Ogata, Hiromi; Furumoto, Shozo; Umetsu, Mitsuo; Kumagai, Izumi

    2016-06-01

    The development of single-chain variable fragments (scFvs) as therapeutic agents has the potential to reduce the high cost of antibody production, but the development process often impairs scFv functions such as binding affinity and pharmacokinetics. Multimerization is one strategy for recovering or enhancing these lost functions. Previously, we constructed several antiepidermal growth factor receptor (EGFR) scFv multimers by modifying linker length and domain order. Antitumor effects comparable with those of the currently approved anti-EGFR therapeutic antibodies were observed for scFv trimers. In the present study, we fractionated an anti-EGFR scFv tetramer from the intracellular soluble fraction of an Escherichia coli transformant. Compared with the trimer, the tetramer showed higher affinity, greater cancer cell growth inhibition, and prolonged blood retention time. Furthermore, the tetramer did not dissociate into the trimer or other smaller species during long-term storage (up to 33 weeks). Thus, our developed scFv tetramer is an attractive candidate next-generation anti-EGFR therapeutic antibody that can be produced via a low-cost bacterial expression system. PMID:27419062

  18. Measuring Multivalent Binding Interactions by Isothermal Titration Calorimetry.

    PubMed

    Dam, Tarun K; Talaga, Melanie L; Fan, Ni; Brewer, Curtis F

    2016-01-01

    Multivalent glycoconjugate-protein interactions are central to many important biological processes. Isothermal titration calorimetry (ITC) can potentially reveal the molecular and thermodynamic basis of such interactions. However, calorimetric investigation of multivalency is challenging. Binding of multivalent glycoconjugates to proteins (lectins) often leads to a stoichiometry-dependent precipitation process due to noncovalent cross-linking between the reactants. Precipitation during ITC titration severely affects the quality of the baseline as well as the signals. Hence, the resulting thermodynamic data are not dependable. We have made some modifications to address this problem and successfully studied multivalent glycoconjugate binding to lectins. We have also modified the Hill plot equation to analyze high quality ITC raw data obtained from multivalent binding. As described in this chapter, ITC-driven thermodynamic parameters and Hill plot analysis of ITC raw data can provide valuable information about the molecular mechanism of multivalent lectin-glycoconjugate interactions. The methods described herein revealed (i) the importance of functional valence of multivalent glycoconjugates, (ii) that favorable entropic effects contribute to the enhanced affinities associated with multivalent binding, (iii) that with the progression of lectin binding, the microscopic affinities of the glycan epitopes of a multivalent glycoconjugate decrease (negative cooperativity), (iv) that lectin binding to multivalent glycoconjugates, especially to mucins, involves internal diffusion jumps, (bind and jump) and (v) that scaffolds of glycoconjugates influence their entropy of binding.

  19. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    PubMed

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An; Chang, Ya-Chun

    2015-10-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.

  20. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    PubMed Central

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  1. Refolding single-chain antibody (scFv) using lauroyl-L-glutamate as a solubilization detergent and arginine as a refolding additive.

    PubMed

    Kudou, Motonori; Ejima, Daisuke; Sato, Haruna; Yumioka, Ryosuke; Arakawa, Tsutomu; Tsumoto, Kouhei

    2011-05-01

    Therapeutic potential of immunoconjugates has opened a new window for antibody-based biopharmaceuticals. Greater tissue penetration and hence enhanced cell toxicity are obtained with a smaller version of antibodies. While the whole antibody can be readily produced via mammalian expression system, antibody fragments often require refolding of insoluble proteins. Here we report a new refolding method for antibody fragments using a novel amino acid-based detergent as a solubilizing agent and arginine-assisted refolding. Inclusion bodies of antibody fragments were solubilized by 2.5% lauroyl-L-Glu (C12-L-Glu) and successfully refolded by multi-step dilution into a buffer solution containing arginine hydrochloride and thiol/disulfide-exchange reagents. Adjustment of temperature was found to be critical for increase in the refolding yield. Although each protein requires appropriate optimization, solubilization by C12-L-Glu and dilution refolding assisted by arginine can generate the native, functional antibody fragments. The procedure should enable us to utilize bacterial expression systems for the large-scale manufacturing.

  2. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    PubMed Central

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers. PMID:25993332

  3. Biotinylated multivalent glycoconjugates for surface coating.

    PubMed

    Chinarev, Alexander A; Galanina, Oxana E; Bovin, Nicolai V

    2010-01-01

    Systematic studying of biological processes driven by multipoint high-cooperative carbohydrate recognition requires application of multivalent carbohydrates as tools. In this regard polyacrylamides with various pendant carbohydrate residues and labels are probably the most well advanced class of carbohydrate multimerics. Here we describe a synthetic approach to polyacrylamide-based glycoconjugates with biotin tag, with special emphasis to development of carbohydrate biosensors and arrays.

  4. Biotinylated multivalent glycoconjugates for surface coating.

    PubMed

    Chinarev, Alexander A; Galanina, Oxana E; Bovin, Nicolai V

    2010-01-01

    Systematic studying of biological processes driven by multipoint high-cooperative carbohydrate recognition requires application of multivalent carbohydrates as tools. In this regard polyacrylamides with various pendant carbohydrate residues and labels are probably the most well advanced class of carbohydrate multimerics. Here we describe a synthetic approach to polyacrylamide-based glycoconjugates with biotin tag, with special emphasis to development of carbohydrate biosensors and arrays. PMID:19882121

  5. Development of specific scFv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis.

    PubMed

    Ribeiro, Vanessa da Silva; Araújo, Thaise Gonçalves; Gonzaga, Henrique Tomaz; Nascimento, Rafael; Goulart, Luiz Ricardo; Costa-Cruz, Julia Maria

    2013-01-01

    We have shown previously that detection of circulating antibodies against mimotopes selected by phage display were useful in neurocysticercosis diagnosis. However, circulating antigens may also be useful in patients' clinical follow-up. Therefore, we aimed to select novel combinatorial antibodies, single-chain variable fragment (scFv), which can be used for specific antigens with pre-defined affinity and specificity without prior immunization. A phage scFv antibody library was selected against Taenia solium mimotopes displayed on phages coupled in beads and total saline extract of T. solium metacestodes (S) immobilized on microtiter plate wells. After two rounds of selection, 96 phage clones were evolved and validated against each target by enzyme linked immunosorbent assay (ELISA), and dot-blot, and three specific antibodies (B6, G10 and A4) were further characterized by sequencing and indirect immunofluorescence (IFI) assays. IFI revealed tegument staining for the B6, while the others showed a non-uniform staining in the whole parasite. The selected scFvs were used to capture their antigen targets that were elucidated through mass spectrometry, and used for antibody detection in NC patients' sera by ELISA, which achieved sensitivities greater than 97% and specificities above 95%. We have successfully developed scFv antibodies against important mimotopes used in NC diagnosis, and can be further explored to detect circulating antigens for clinical follow-up of patients with NC. Our strategy also highlighted the possibility of using this combinatorial approach to select, capture and characterize specific antigens to better understand this intriguing parasite infection and disease evolution.

  6. Targeting of Adenovirus Serotype 5 Pseudotyped with Short Fiber from Serotype 41 to c-erbB2-Positive Cells Using Bispecific Single-Chain Diabody

    PubMed Central

    Kashentseva, Elena A.; Douglas, Joanne T.; Zinn, Kurt R.; Curiel, David T.; Dmitriev, Igor P.

    2009-01-01

    Summary The purpose of the current study was to alter the broad native tropism of human adenovirus for virus targeting to c-erbB2-positive cancer cells. First, we engineered a single-chain antibody (scFv) against the c-erbB2 oncoprotein into minor capsid protein IX (pIX) of adenovirus serotype 5 (Ad5) in a manner commensurate with virion integrity and binding to the soluble extracellular c-erbB2 domain. To ablate native viral tropism and facilitate binding of the pIX-incorporated scFv to cellular c-erbB2 we replaced the Ad5 fiber with the Ad41 short (41s) fiber devoid of all known cell-binding determinants. The resultant Ad5F41sIX6.5 vector demonstrated increased cell binding and gene transfer as compared to the Ad5F41s control, however, this augmentation of virus infectivity was not c-erbB2-specific. Incorporation of a six histidine (His6) peptide into the C-terminus of the 41s fiber protein resulted in markedly increased Ad5F41s6H infectivity in 293AR cells, which express a membrane-anchored scFv against the C-terminal oligo-histidine tag, as compared to the Ad5F41s vector and the parental 293 cells. These data suggested that a 41s fiber-incorporated His6 tag could serve for attachment of an adapter protein designed to guide Ad5F41s6H infection in a c-erbB2-specific manner. We therefore engineered a bispecific scFv diabody (scDb) combining affinities for both c-erbB2 and the His6 tag and showed its ability to provide up to 25-fold increase of Ad5F41s6H infectivity in c-erbB2-positive cells. Thus, Ad5 fiber replacement by a His6–tagged 41s fiber coupled with virus targeting mediated by an scDb adapter represents a promising strategy to confer Ad5 vector tropism for c-erbB2-positive cancer cells. PMID:19285990

  7. Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment.

    PubMed

    Tao, Xiaoqi; Chen, Min; Jiang, Haiyang; Shen, Jianzhong; Wang, Zhanhui; Wang, Xia; Wu, Xiaoping; Wen, Kai

    2013-09-01

    A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin-ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC50) at 0.12 μg kg(-1) for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 μg kg(-1) and the LODs for CIP and ENR were all <0.2 μg kg(-1). Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 μg kg(-1) for PEF to 2.1 μg kg(-1) for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 μg kg(-1)) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool.

  8. Quantitatively Resolving Multivalent Interactions on Macroscopic Scale Using Force Spectroscopy

    PubMed Central

    Hu, Qiongzheng; Yang, Haopeng; Wang, Yuhong; Xu, Shoujun

    2016-01-01

    Multivalent interactions remain difficult to be characterized and consequently controlled, particularly on a macroscopic scale. Using force-induced remnant magnetization spectroscopy (FIRMS), we have resolved the single-, double-, and triple- biotin—streptavidin interactions, multivalent DNA interactions and CXCL12-CXCR4 interactions, on millimetre-scale surfaces. Our results establish FIRMS as a viable method for systematic resolution and controlled formation of multivalent interactions. PMID:26864087

  9. Synthesis of carbohydrate-scaffolded thymine glycoconjugates to organize multivalency

    PubMed Central

    Ciuk, Anna K

    2015-01-01

    Summary Multivalency effects are essential in carbohydrate recognition processes as occurring on the cell surface. Thus many synthetic multivalent glycoconjugates have been developed as important tools for glycobiological research. We are expanding this collection of molecules by the introduction of carbohydrate-scaffolded divalent glycothymine derivatives that can be intramolecularily dimerized by [2 + 2] photocycloaddition. Thus, thymine functions as a control element that allows to restrict the conformational flexibility of the scaffolded sugar ligands and thus to “organize” multivalency. With this work we add a parameter to multivalency studies additional to valency. PMID:26124869

  10. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

    PubMed Central

    Tillotson, Benjamin J.; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  11. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

    PubMed

    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.

  12. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

    PubMed

    Tillotson, Benjamin J; Goulatis, Loukas I; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  13. Supramolecular Nanoparticles via Single-Chain Folding Driven by Ferrous Ions.

    PubMed

    Wang, Fei; Pu, Hongting; Jin, Ming; Wan, Decheng

    2016-02-01

    Single-chain nanoparticles can be obtained via single-chain folding assisted by intramolecular crosslinking reversibly or irreversibly. Single-chain folding is also an efficient route to simulate biomacromolecules. In present study, poly(N-hydroxyethylacrylamide-co-4'-(propoxy urethane ethyl acrylate)-2,2':6',2''-terpyridine) (P(HEAm-co-EMA-Tpy)) is synthesized via reversible addition fragmentation chain transfer polymerization. Single-chain folding and intramolecular crosslinking of P(HEAm-co-EMA-Tpy) are achieved via metal coordination chemistry. The intramolecular interaction is characterized on ultraviolet/visible spectrophotometer (UV-vis spectroscopy), proton nuclear magnetic resonance ((1)H NMR), and differential scanning calorimetry (DSC). The supramolecular crosslinking mediated by Fe(2+) plays an important role in the intramolecular collapsing of the single-chain and the formation of the nanoparticles. The size and morphology of the nanoparticles can be controlled reversibly via metal coordination chemistry, which can be characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and atomic force microscope (AFM).

  14. Production and Purification of a Novel Anti-TNF-α Single Chain Fragment Variable Antibody

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. Methods: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. Results: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. Conclusion: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality. PMID:26793614

  15. Identification of Novel Single Chain Fragment Variable Antibodies Against TNF-α Using Phage Display Technology

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. Methods: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. Results: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. Conclusion: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α. PMID:26793613

  16. Structural and Functional Characterization of a Single-Chain Form of the Recognition Domain of Complement Protein C1q

    PubMed Central

    Moreau, Christophe; Bally, Isabelle; Chouquet, Anne; Bottazzi, Barbara; Ghebrehiwet, Berhane; Gaboriaud, Christine; Thielens, Nicole

    2016-01-01

    Complement C1q is a soluble pattern recognition molecule comprising six heterotrimeric subunits assembled from three polypeptide chains (A–C). Each heterotrimer forms a collagen-like stem prolonged by a globular recognition domain. These recognition domains sense a wide variety of ligands, including pathogens and altered-self components. Ligand recognition is either direct or mediated by immunoglobulins or pentraxins. Multivalent binding of C1q to its targets triggers immune effector mechanisms mediated via its collagen-like stems. The induced immune response includes activation of the classical complement pathway and enhancement of the phagocytosis of the recognized target. We report here, the first production of a single-chain recombinant form of human C1q globular region (C1q-scGR). The three monomers have been linked in tandem to generate a single continuous polypeptide, based on a strategy previously used for adiponectin, a protein structurally related to C1q. The resulting C1q-scGR protein was produced at high yield in stably transfected 293-F mammalian cells. Recombinant C1q-scGR was correctly folded, as demonstrated by its X-ray crystal structure solved at a resolution of 1.35 Å. Its interaction properties were assessed by surface plasmon resonance analysis using the following physiological C1q ligands: the receptor for C1q globular heads, the long pentraxin PTX3, calreticulin, and heparin. The 3D structure and the binding properties of C1q-scGR were similar to those of the three-chain fragment generated by collagenase digestion of serum-derived C1q. Comparison of the interaction properties of the fragments with those of native C1q provided insights into the avidity component associated with the hexameric assembly of C1q. The interest of this functional recombinant form of the recognition domains of C1q in basic research and its potential biomedical applications are discussed. PMID:26973654

  17. Multivalent supramolecular dendrimer-based drugs.

    PubMed

    Galeazzi, Simone; Hermans, Thomas M; Paolino, Marco; Anzini, Maurizio; Mennuni, Laura; Giordani, Antonio; Caselli, Gianfranco; Makovec, Francesco; Meijer, E W; Vomero, Salvatore; Cappelli, Andrea

    2010-01-11

    Supramolecular complexes consisting of a hydrophobic dendrimer host [DAB-dendr-(NHCONH-Ad)(64)] as well as solubilizing and bioactive guest molecules have been synthesized using a noncovalent approach. The guest-host supramolecular assembly is first preassembled in chloroform and transferred via the neat phase to aqueous solution. The bioactive guest molecules can bind to a natural (serotonin 5-HT(3)) receptor with nanomolar affinity as well as to the synthetic dendrimer receptor in aqueous solution, going toward a dynamic multivalent supramolecular construct capable of adapting itself to a multimeric receptor motif.

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

    PubMed

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

    2004-02-01

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

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

    PubMed

    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.

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

    PubMed

    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

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

    PubMed Central

    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

  2. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    PubMed

    Hosking, Christopher G; McWilliam, Hamish E G; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P; Ilag, Leodevico L; Meeusen, Els N T; Veer, Michael J de

    2015-12-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  3. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    PubMed Central

    Hosking, Christopher G.; McWilliam, Hamish E. G.; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P.; Ilag, Leodevico L.; Meeusen, Els N. T.; de Veer, Michael J.

    2015-01-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  4. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    PubMed

    Hosking, Christopher G; McWilliam, Hamish E G; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P; Ilag, Leodevico L; Meeusen, Els N T; Veer, Michael J de

    2015-12-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

  5. Evaluation of anti-HER2 scFv-conjugated PLGA-PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells

    NASA Astrophysics Data System (ADS)

    Thuy Duong Le, Thi; Pham, Thu Hong; Nghia Nguyen, Trong; Giang Ngo, Thi Hong; Nhung Hoang, Thi My; Huan Le, Quang

    2016-06-01

    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv-Doc-PLGA-PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv-Doc-PLGA-PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv-Doc-PLGA-PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies.

  6. Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.

    PubMed

    Lv, Xinxin; Zhang, Jian; Xu, Rui; Dong, Yuguo; Sun, Aiyou; Shen, Yaling; Wei, Dongzhi

    2016-07-01

    Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells. PMID:27063011

  7. Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

    PubMed Central

    Sina, Mohammad; Farajzadeh, Davoud; Dastmalchi, Siavoush

    2015-01-01

    Purpose: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. Methods: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. Results: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. Conclusion: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein. PMID:26819916

  8. The story of a unique molecule in hemophilia A: recombinant single-chain factor VIII.

    PubMed

    Pabinger-Fasching, Ingrid

    2016-05-01

    For patients with hemophilia A, replacement of deficient factor VIII (FVIII) using plasma-derived or recombinant FVIII (rFVIII) products to restore hemostatic control can reduce bleeding complications and preserve musculoskeletal function. Despite the clinical availability of several of these products, challenges remain in the treatment of hemophilia A, the most notable of which are the risk of inhibitor development and the limited half-life of existing FVIII concentrates, which can make prophylaxis burdensome for patients. The use of recombinant protein technology may lead to novel FVIII products with improved properties. This article describes the story of a unique recombinant FVIII protein, rVIII-SingleChain, which is currently in development. In contrast to native FVIII and other commercially available rFVIII preparations, rVIII-SingleChain uses a strong, covalent bond to connect the light and heavy chains, thereby creating a stable, single-chain rFVIII. It has enhanced intrinsic stability, better integrity after reconstitution, and a higher binding affinity to von Willebrand factor. The physicochemical profile of rVIII-SingleChain and preclinical data on its activity and phamacokinetics strengthened the rationale for its clinical investigation. Available data from the AFFINITY clinical trial program are promising; indicating that it has good hemostatic efficacy when used on demand, for prophylaxis, and in the surgical setting, and is also very well tolerated. A pediatric study and an extension study are ongoing as part of the AFFINITY program.

  9. The story of a unique molecule in hemophilia A: recombinant single-chain factor VIII.

    PubMed

    Pabinger-Fasching, Ingrid

    2016-05-01

    For patients with hemophilia A, replacement of deficient factor VIII (FVIII) using plasma-derived or recombinant FVIII (rFVIII) products to restore hemostatic control can reduce bleeding complications and preserve musculoskeletal function. Despite the clinical availability of several of these products, challenges remain in the treatment of hemophilia A, the most notable of which are the risk of inhibitor development and the limited half-life of existing FVIII concentrates, which can make prophylaxis burdensome for patients. The use of recombinant protein technology may lead to novel FVIII products with improved properties. This article describes the story of a unique recombinant FVIII protein, rVIII-SingleChain, which is currently in development. In contrast to native FVIII and other commercially available rFVIII preparations, rVIII-SingleChain uses a strong, covalent bond to connect the light and heavy chains, thereby creating a stable, single-chain rFVIII. It has enhanced intrinsic stability, better integrity after reconstitution, and a higher binding affinity to von Willebrand factor. The physicochemical profile of rVIII-SingleChain and preclinical data on its activity and phamacokinetics strengthened the rationale for its clinical investigation. Available data from the AFFINITY clinical trial program are promising; indicating that it has good hemostatic efficacy when used on demand, for prophylaxis, and in the surgical setting, and is also very well tolerated. A pediatric study and an extension study are ongoing as part of the AFFINITY program. PMID:27288063

  10. Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

    PubMed Central

    Richman, Sarah A.; Aggen, David H.; Dossett, Michelle L.; Donermeyer, David L.; Allen, Paul M.; Greenberg, Philip D.; Kranz, David M.

    2009-01-01

    The variable (V) domains of antibodies and T cell receptors (TCRs) share sequence homology and striking structural similarity. Single-chain antibody V domain constructs (scFv) are routinely expressed in a variety of heterologous systems, both for production of soluble protein as well as for in vitro engineering. In contrast, single-chain T cell receptor V domain constructs (scTCR) are prone to aggregation and misfolding and are refractory to display on phage or yeast in their wild-type form. However, through random mutagenesis and yeast display engineering, it has been possible to isolate scTCR mutants that are properly folded and displayed on the yeast surface. These displayed mutants can serve not only as a scaffold for further engineering but also as scTCR variants that exhibit favorable biophysical properties in E. coli expression. Thus, a more comprehensive understanding of the V domain mutations that allowed display would be beneficial. Our goal here was to identify generalizable patterns of important mutations that can be applied to different TCRs. We compared five different scTCRs, four from mice and one from a human, for yeast surface display. Analysis of a collection of mutants revealed four distinct regions of TCR V domains that were most important for enabling surface expression: the Vα-Vβ interface, the HV4 of Vβ, and the region of the Vα and Vβ domains normally apposed against the constant (C) domains. Consistent with the role of the V-C interface in surface display, reconstitution of this interface, by including the constant domains of each chain, allowed V domain display and αβ chain association on the yeast surface, thus providing an alternative TCR scaffold. However, the surface levels of TCR achieved with engineered scTCR mutants were superior to that of the VαCα/VβCβ constructs. Therefore, we describe further optimization of the current strategy for surface display of the single-chain format in order to facilitate yeast display

  11. Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use.

    PubMed

    Hagemeyer, Christoph E; Alt, Karen; Johnston, Angus P R; Such, Georgina K; Ta, Hang T; Leung, Melissa K M; Prabhu, Sandeep; Wang, Xiaowei; Caruso, Frank; Peter, Karlheinz

    2015-01-01

    Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d. PMID:25502886

  12. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

    PubMed Central

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  13. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

    PubMed

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

  14. Single-chain Variable Fragment Albumin Fusions Bind the Neonatal Fc Receptor (FcRn) in a Species-dependent Manner

    PubMed Central

    Andersen, Jan Terje; Cameron, Jason; Plumridge, Andrew; Evans, Leslie; Sleep, Darrell; Sandlie, Inger

    2013-01-01

    Albumin has a serum half-life of 3 weeks in humans. This has been utilized to extend the serum persistence of biopharmaceuticals that are fused to albumin. In light of the fact that the neonatal Fc receptor (FcRn) is a key regulator of albumin homeostasis, it is crucial to address how fusion of therapeutics to albumin impacts binding to FcRn. Here, we report on a detailed molecular investigation on how genetic fusion of a short peptide or an single-chain variable fragment (scFv) fragment to human serum albumin (HSA) influences pH-dependent binding to FcRn from mouse, rat, monkey, and human. We have found that fusion to the N- or C-terminal end of HSA only slightly reduces receptor binding, where the most noticeable effect is seen after fusion to the C-terminal end. Furthermore, in contrast to the observed strong binding to human and monkey FcRn, HSA and all HSA fusions bound very poorly to mouse and rat versions of the receptor. Thus, we demonstrate that conventional rodents are limited as preclinical models for analysis of serum half-life of HSA-based biopharmaceuticals. This finding is explained by cross-species differences mainly found within domain III (DIII) of albumin. Our data demonstrate that although fusion, particularly to the C-terminal end, may slightly reduce the affinity for FcRn, HSA is versatile as a carrier of biopharmaceuticals. PMID:23818524

  15. Blood Clotting-Inspired Control of Single-Chain Molecules in Flows

    NASA Astrophysics Data System (ADS)

    Sing, Charles; Alexander-Katz, Alfredo

    2011-03-01

    Recent experimental evidence has demonstrated a clear link between mechanical stimuli and the activation of von Willebrand Factor (vWF), a protein that plays a critical role in the blood clotting cascade. This protein exhibits counter-intuitive conformational and adsorption responses that suggest novel ways of controlling the single-chain dynamics of polymer chains. Specifically, we are using simulation and theoretical approaches to elucidate the fundamental physics that govern globule-stretch transitions in collapsed polymers due to the effect of fluid flows. We begin to extend this general approach to the case of globule adsorption-desorption transitions in the presence of fluid flows, and demonstrate how kinetic considerations must be taken into account to describe the basic features of these transitions. We expect that these results will both allow the development of novel techniques for single-chain targeting and assembly and offer insight into the physiological behavior of vWF.

  16. Stepwise Unfolding of Single-Chain Nanoparticles by Chemically Triggered Gates.

    PubMed

    Fischer, Tobias S; Schulze-Sünninghausen, David; Luy, Burkhard; Altintas, Ozcan; Barner-Kowollik, Christopher

    2016-09-01

    The orthogonal, stepwise, and order-independent unfolding of single-chain nanoparticles (SCNPs) is introduced as a key step towards actively controlling the folding dynamics of SCNPs. The SCNPs are compacted by multiple hydrogen bonds and host-guest interactions. Well-defined diblock (AB) and tetrablock (ABCD) copolymers are equipped with orthogonal recognition motifs via modular ligation along the lateral chain. Initially, single-chain folding of the diblock copolymer was induced by the host-guest complexation of benzo-21-crown-7 (B21C7, host) and a secondary ammonium salt (AS, guest), representing an efficient avenue for single-chain collapse. Next, both orthogonal Hamilton wedge (HW) and cyanuric acid (CA) as well as B21C7-AS motifs were employed to generate SCNPs based on the ABCD polymer system. Subsequently, the stepwise dual-gated and order-independent unfolding of the SCNPs was investigated by the addition of external stimuli. The folding and unfolding were explored by 1D (1) H NMR spectroscopy, dynamic light scattering (DLS), and diffusion-ordered NMR spectroscopy (DOSY). PMID:27357944

  17. Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo.

    PubMed

    Rülker, Torsten; Voß, Luzie; Thullier, Philippe; O' Brien, Lyn M; Pelat, Thibaut; Perkins, Stuart D; Langermann, Claudia; Schirrmann, Thomas; Dübel, Stefan; Marschall, Hans-Jürgen; Hust, Michael; Hülseweh, Birgit

    2012-01-01

    Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE. PMID:22666347

  18. Identification of human single-chain antibodies with broad reactivity for noroviruses

    PubMed Central

    Huang, Wanzhi; Samanta, Moumita; Crawford, Sue E.; Estes, Mary K.; Neill, Frederick H.; Atmar, Robert L.; Palzkill, Timothy

    2014-01-01

    Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose norovirus infections are needed. The goal of this study was to identify antibodies that have broad reactivity of binding to various genogroups of norovirus. A human scFv phage display library was used to identify two antibodies, HJT-R3-A9 and HJT-R3-F7, which bind to both genogroups I and II norovirus virus-like particles (VLPs). Mapping experiments indicated that the HJT-R3-A9 clone binds to the S-domain while the HJT-R3-F7 clone binds the P-domain of the VP1 capsid protein. In addition, a family of scFv antibodies was identified by elution of phage libraries from the GII.4 VLP target using a carbohydrate that serves as an attachment factor for norovirus on human cells. These antibodies were also found to recognize both GI and GII VLPs in enzyme-linked immunosorbent assay (ELISA) experiments. The HJT-R3-A9, HJT-R3-F7 and scFv antibodies identified with carbohydrate elution were shown to detect antigen from a clinical sample known to contain GII.4 norovirus but not a negative control sample. Finally, phages displaying the HJT-R3-A9 scFv can be used directly to detect both GI.1 and GII.4 norovirus from stool samples, which has the potential to simplify and reduce the cost of diagnostics based on antibody-based ELISA methods. PMID:24946948

  19. Cloning, bacterial expression and crystallization of Fv antibody fragments

    NASA Astrophysics Data System (ADS)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  20. Construction and Validation of SRA-FV Need Assessment.

    PubMed

    Thornton, David; Knight, Raymond A

    2015-08-01

    This article describes the construction and testing of a newly designed instrument to assess psychological factors associated with increased rates of sexual recidivism. The new instrument (Structured Risk Assessment-Forensic Version or SRA-FV) was based on previous research using the SRA framework. This article describes the results of testing SRA-FV with a large sample (N = 566) of sexual offenders being evaluated for an early civil commitment program. SRA-FV was found to significantly predict sexual recidivism for both child molesters and rapists and to have incremental predictive value relative to two widely used static actuarial instruments (Static-99R; Risk Matrix 2000/S).

  1. Clinical applications of phage-derived sFvs and sFv fusion proteins.

    PubMed

    Chester, K A; Bhatia, J; Boxer, G; Cooke, S P; Flynn, A A; Huhalov, A; Mayer, A; Pedley, R B; Robson, L; Sharma, S K; Spencer, D I; Begent, R H

    2000-01-01

    Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNF alpha aims to reduce sequestration and increase tumor concentrations of systemically administered TNF alpha.

  2. Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

    PubMed Central

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Jittavisutthikul, Surasak; Seesuay, Watee; Chulanetra, Monrat; Sakolvaree, Yuwaporn; Srimanote, Potjanee; Chaicumpa, Wanpen

    2014-01-01

    A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. PMID:25517317

  3. Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase.

    PubMed

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Jittavisutthikul, Surasak; Seesuay, Watee; Chulanetra, Monrat; Sakolvaree, Yuwaporn; Srimanote, Potjanee; Chaicumpa, Wanpen

    2014-01-01

    A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. PMID:25517317

  4. Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE).

    PubMed

    Song, Yong-Hong; Sun, Xue-Wen; Jiang, Bo; Liu, Ji-En; Su, Xian-Hui

    2015-12-01

    Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings of traditional one-factor-at-a-time (OFAT) approach for protein purification optimization. In this study, a DoE approach was applied for optimizing purification of a recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. In first capture step using Capto L, a 2-level fractional factorial analysis and successively a central composite circumscribed (CCC) design were used to identify the optimal elution conditions. Two main effects, pH and trehalose, were identified, and high recovery (above 95%) and low aggregates ratio (below 10%) were achieved at the pH range from 2.9 to 3.0 with 32-35% (w/v) trehalose added. In the second step using cation exchange chromatography, an initial screening of media and elution pH and a following CCC design were performed, whereby the optimal selectivity of the scFv was obtained on Capto S at pH near 6.0, and the optimal conditions for fulfilling high DBC and purity were identified as pH range of 5.9-6.1 and loading conductivity range of 5-12.5 mS/cm. Upon a further gel filtration, the final purified scFv with a purity of 98% was obtained. Finally, the optimized conditions were verified by a 20-fold scale-up experiment. The purities and yields of intermediate and final products all fell within the regions predicted by DoE approach, suggesting the robustness of the optimized conditions. We proposed that the DoE approach described here is also applicable in production of other recombinant antibody constructs.

  5. A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells

    PubMed Central

    Lim, Ki Jung; Sung, Bong Hyun; Shin, Ju Ri; Lee, Young Woong; Kim, Da Jung; Yang, Kyung Seok; Kim, Sun Chang

    2013-01-01

    Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity. PMID:23776609

  6. The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F.

    PubMed

    Woitok, Mira; Klose, Diana; Niesen, Judith; Richter, Wolfgang; Abbas, Muhammad; Stein, Christoph; Fendel, Rolf; Bialon, Magdalena; Püttmann, Christiane; Fischer, Rainer; Barth, Stefan; Kolberg, Katharina

    2016-10-28

    Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs.

  7. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

  8. The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F.

    PubMed

    Woitok, Mira; Klose, Diana; Niesen, Judith; Richter, Wolfgang; Abbas, Muhammad; Stein, Christoph; Fendel, Rolf; Bialon, Magdalena; Püttmann, Christiane; Fischer, Rainer; Barth, Stefan; Kolberg, Katharina

    2016-10-28

    Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs. PMID:27502168

  9. Chiral single-chain magnet: helically stacked [Mn(III)2Cu(II)] triangles.

    PubMed

    Shiga, Takuya; Maruyama, Kazuya; Newton, Graham N; Inglis, Ross; Brechin, Euan K; Oshio, Hiroki

    2014-05-01

    The one-dimensional complex [Mn(III)2Cu(II)(μ3-O)(Cl-sao)3(EtOH)2]·EtOH (Mn2Cu) was obtained by the metal replacement reaction of the trinuclear manganese complex (Et3NH)[Mn(III)3(μ3-O)Cl2(Cl-sao)3(MeOH)2(H2O)2] with [Cu(acac)2]. The Mn2Cu chain exhibits single-chain-magnet behavior with finite-size effects due to its large magnetic anisotropy.

  10. Crystal engineering to control the magnetic interaction between weak ferromagnetic single-chain magnets assembled in a 3D framework.

    PubMed

    Su, Lei; Song, Wei-Chao; Zhao, Jiong-Peng; Liu, Fu-Chen

    2016-07-01

    A new single-chain-magnet (SCM), [Co4(OMe)3(HCO2)2(L)3·DMF]n, (L = 4-(pyridin-4-yl)benzolate) (2), was constructed by changing the spacers of a weak ferromagnetic single-chain magnet [Co8(OMe)6(HCO2)4(isonic)6·H2O]n (1). By contrasting the magnetism of the two complexes, it is found that the longer the linker the stronger the magnetic properties.

  11. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    PubMed

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

  12. Preparative crystallization of a single chain antibody using an aqueous two-phase system.

    PubMed

    Huettmann, Hauke; Berkemeyer, Matthias; Buchinger, Wolfgang; Jungbauer, Alois

    2014-11-01

    A simultaneous crystallization and aqueous two-phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two-phase system, and to favor crystallization, using sodium sulfate and PEG-2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol-rich phase. The crystal nucleation took place in the sodium sulfate-rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol-rich phase. The crystals in the polyethylene glycol-rich phase grew to a size of >50 µm. Additionally, polyethylene glycol acted as an anti-solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63-87%, respectively. Our results show that crystallization in aqueous two-phase systems is an opportunity to foster process integration.

  13. Properties and Applications of Single-Chain Major Histocompatibility Complex Class I Molecules

    PubMed Central

    Kotsiou, Eleni; Brzostek, Joanna

    2011-01-01

    Abstract Stable major histocompatibility complex (MHC) class I molecules at the cell surface consist of three separate, noncovalently associated components: the class I heavy chain, the β2-microglobulin light chain, and a presented peptide. These three components are assembled inside cells via complex pathways involving many other proteins that have been studied extensively. Correct formation of disulfide bonds in the endoplasmic reticulum is central to this process of MHC class I assembly. For a single specific peptide to be presented at the cell surface for possible immune recognition, between hundreds and thousands of peptide-containing precursor polypeptides are required, so the overall process is relatively inefficient. To increase the efficiency of antigen presentation by MHC class I molecules, and for possible therapeutic purposes, single-chain molecules have been developed in which the three, normally separate components have been joined together via flexible linker sequences in a single polypeptide chain. Remarkably, these single-chain MHC class I molecules fold up correctly, as judged by functional recognition by cells of the immune system, and more recently by X-ray crystallographic structural data. This review focuses on the interesting properties and potential of this new type of engineered MHC class I molecule. Antioxid. Redox Signal. 15, 645–655. PMID:21126187

  14. Spatiotemporal control and superselectivity in supramolecular polymers using multivalency

    PubMed Central

    Albertazzi, Lorenzo; Martinez-Veracoechea, Francisco J.; Leenders, Christianus M. A.; Voets, Ilja K.; Frenkel, Daan; Meijer, E. W.

    2013-01-01

    Multivalency has an important but poorly understood role in molecular self-organization. We present the noncovalent synthesis of a multicomponent supramolecular polymer in which chemically distinct monomers spontaneously coassemble into a dynamic, functional structure. We show that a multivalent recruiter is able to bind selectively to one subset of monomers (receptors) and trigger their clustering along the self-assembled polymer, behavior that mimics raft formation in cell membranes. This phenomenon is reversible and affords spatiotemporal control over the monomer distribution inside the supramolecular polymer by superselective binding of single-strand DNA to positively charged receptors. Our findings reveal the pivotal role of multivalency in enabling structural order and nonlinear recognition in water-soluble supramolecular polymers, and it offers a design principle for functional, structurally defined supramolecular architectures. PMID:23836666

  15. Spatiotemporal control and superselectivity in supramolecular polymers using multivalency.

    PubMed

    Albertazzi, Lorenzo; Martinez-Veracoechea, Francisco J; Leenders, Christianus M A; Voets, Ilja K; Frenkel, Daan; Meijer, E W

    2013-07-23

    Multivalency has an important but poorly understood role in molecular self-organization. We present the noncovalent synthesis of a multicomponent supramolecular polymer in which chemically distinct monomers spontaneously coassemble into a dynamic, functional structure. We show that a multivalent recruiter is able to bind selectively to one subset of monomers (receptors) and trigger their clustering along the self-assembled polymer, behavior that mimics raft formation in cell membranes. This phenomenon is reversible and affords spatiotemporal control over the monomer distribution inside the supramolecular polymer by superselective binding of single-strand DNA to positively charged receptors. Our findings reveal the pivotal role of multivalency in enabling structural order and nonlinear recognition in water-soluble supramolecular polymers, and it offers a design principle for functional, structurally defined supramolecular architectures.

  16. Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera.

    PubMed

    Stoyanova, Vishnya; Aleksandrov, Radoslav; Lukarska, Maria; Duhalov, Deyan; Atanasov, Vasil; Petrova, Svetla

    2012-10-01

    Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits--basic and toxic phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones--D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially vipoxin's acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of vipoxin and its pure PLA₂ subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A₂ activity for the vipoxin toxicity and its effective neutralization.

  17. Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2.

    PubMed

    Pecora, A; Malacari, D A; Perez Aguirreburualde, M S; Bellido, D; Nuñez, M C; Dus Santos, M J; Escribano, J M; Wigdorovitz, A

    2015-09-22

    The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the β-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds. PMID:26279338

  18. Purification and characterization of mouse single-chain antibody against polycyclic aromatic hydrocarbons.

    PubMed

    Ustinov, Valentin A; Averjanov, Anton V; Glushkov, Andrey N

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyren mainly induce lung cancer in humans. We characterized the mouse single chain antibody against benzo[a]pyren (pSh). pSh was expressed and purified as cellulose binding domain fusion (pSh-CBD). The pSh-CBD bound five different PAH with high affinity. The 18 amino acid linker connected pSh-CBD heavy and light chains provided correct protein folding. The KDs for pSh-CBD and polycyclic aromatic hydrocarbons were similar to KDs for monoclonal antibody, approximately 10(-8). Separately heavy and light chains of pSh-CBD did not interact with benzo[a]pyren. Previously defined eleven pSh-CBD aa involved to benzo[a]pyren binding were confirmed by mutagenesis.

  19. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles. PMID:26949165

  20. Induced crystallization of single-chain polyethylene on a graphite surface: molecular dynamics simulation.

    PubMed

    Yang, Hua; Zhao, Xiao Jun; Sun, Miao

    2011-07-01

    Molecular dynamics (MD) simulations have been carried out on the crystallization of single-chain polyethylene (PE) which was adsorbed on a graphite (001) surface on one side and exposed to vacuum on the other at different temperatures. The MD simulation data have been analyzed to provide information about the crystallization process of polymer adsorbed on the solid substrate. The isothermal crystallization of PE proceeds in two steps: (1) adsorption and (2) orientation. The results detail the radial density distribution function, ordered parameters, local bond-orientational order parameters, and the local properties displayed in layers of the polymer parallel to the graphite and vacuum interfaces. It was also shown that the film thickness affected the critical crystallization temperature of the adsorbed polymer on the substrate surface. Furthermore, the influence of the graphite surface area on the crystallization of PE is discussed by comparing the crystallinity evolution of PE on graphite with different coverage.

  1. Magnetic Relaxation and Coercivity of Finite-size Single Chain Magnets

    NASA Astrophysics Data System (ADS)

    Gredig, Thomas; Byrne, Matthew; Vindigni, Alessandro

    2015-03-01

    The magnetic coercivity of hysteresis loops for iron phthalocyanine thin films depends on the iron chain length and the measurement sweep speed below 5 K. The average one-dimensional (1D) iron chain length in samples is controlled during deposition. These 1D iron chains can be tuned over one order of magnitude with the shortest chain having 100 elements. We show that the coercivity strongly increases with the average length of the iron chains, which self-assemble parallel to the substrate surface. Magnetic relaxation and sweep speed data suggest spin dynamics play an important role. Implementing Glauber dynamics with a finite-sized 1D Ising model provides qualitative agreement with experimental data. This suggests that iron phthalocyanine thin films act as single chain magnets and provide a solid test system for tunable finite-sized magnetic chains. This research has been supported with the NSF-DMR 0847552 grant.

  2. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles.

  3. A modular approach to introduce function into single-chain polymeric nanoparticles.

    PubMed

    Huerta, Elisa; van Genabeek, Bas; Stals, Patrick J M; Meijer, E W; Palmans, Anja R A

    2014-08-01

    Here, a modular approach is reported to introduce a specific function into single-chain polymeric nanoparticles (SCPNs). Hereto, an amphiphilic polymer with pendant benzene-1,3,5-tricarboxamide (BTA) units is mixed with a "free" BTA that contains a functional group, either a fluorescent naphthalimide or a catalytically active l-proline. Taking advantage of hydrophobic interactions and self-recognition properties of the BTA units, the "free" BTAs are captured into the interior of the SCPN in water as evidenced by fluorescence studies. To illustrate that function can be readily introduced using a modular approach, l-proline-based BTAs are incorporated to procure a catalytically active SCPN in water. The aldol reaction between p-nitrobenzaldehyde and cyclohexanone shows good conversions at low catalyst loadings and substrate concentrations, and high stereoselectivities are obtained (de = 91% and ee = 98%). PMID:24962087

  4. Effect of coil-globule transition on the single-chain crystallization.

    PubMed

    Wang, Mao-Xiang

    2013-05-30

    The folding process of a single chain including coil-globule transition and crystallization has been investigated through dynamic Monte Carlo simulations. The results based upon ensemble averaging illustrated three distinct states: coil, molten globule, and globule states. Furthermore, the crystallization process from these collapsed states demonstrated various characteristics and it also verified the thermodynamic partitions. The isothermal crystallization in the three states showed the folding rates, and the final crystallite morphologies strongly depended on the collapsed states. Especially, the onset temperature of crystallization in the intermediate molten globule state demonstrated the strongest sensitivity to the solvent qualities in the three different states. Moreover, the crystallization in this intermediate state illustrated a two-step folding mechanism with the prior dense core serving as a precursor to induce the subsequent crystallization. Our observations would help in understanding the thermodynamics and kinetics of phase transition of a single macromolecule. Possible relations to the protein folding were also discussed. PMID:23646890

  5. An improved single-chain Fab platform for efficient display and recombinant expression.

    PubMed

    Koerber, James T; Hornsby, Michael J; Wells, James A

    2015-01-30

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

  6. An improved single-chain Fab platform for efficient display and recombinant expression.

    PubMed

    Koerber, James T; Hornsby, Michael J; Wells, James A

    2015-01-30

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies. PMID:25481745

  7. A Novel Single-Chain Antibody Fragment for Detection of Mannose 6-Phosphate-Containing Proteins

    PubMed Central

    Müller-Loennies, Sven; Galliciotti, Giovanna; Kollmann, Katrin; Glatzel, Markus; Braulke, Thomas

    2010-01-01

    Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III. PMID:20472886

  8. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

    PubMed

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which

  9. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

    PubMed Central

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2–7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2–7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2–7 and BLC3) are close to the human germline sequences

  10. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

    PubMed

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which

  11. Architectures of Multivalent Glycomimetics for Probing Carbohydrate-Lectin Interactions

    NASA Astrophysics Data System (ADS)

    Lahmann, Martina

    Well-defined multivalent glycoconjugates are valued tools in glycoscience and they are particularly valuable for the investigation of carbohydrate-lectin interactions. In addition to the relatively globularly shaped glycodendrimers many other designs have been realized. This chapter gives an overview on the common different architectures and their chemical synthesis by focussing on the achievements made since 2001.

  12. Mono and Multivalency In Tethered Protein-Carbohydrate Bonds

    SciTech Connect

    Ratto, T V; Langry, K C; Rudd, R E; Balhorn, R L; McElfresh, M W

    2004-01-29

    Molecular recognition in biological systems typically involves large numbers of interactions simultaneously. By using a multivalent approach, weak interactions with fairly low specificity can become strong highly specific interactions. Additionally, this allows an organism to control the strength and specificity of an interaction simply by controlling the number of binding molecules (or binding sites), which in turn can be controlled through transcriptional regulation.

  13. Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

    PubMed

    Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

    2016-03-15

    Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. PMID:26829652

  14. Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

    PubMed

    Paul, S; Snary, D; Hoebeke, J; Allen, D; Balloul, J M; Bizouarne, N; Dott, K; Geist, M; Hilgers, J; Kieny, M P; Burchell, J; Taylor-Papadimitriou, J; Acres, R B

    2000-07-01

    Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1. PMID:10910139

  15. Crystal engineering to control the magnetic interaction between weak ferromagnetic single-chain magnets assembled in a 3D framework.

    PubMed

    Su, Lei; Song, Wei-Chao; Zhao, Jiong-Peng; Liu, Fu-Chen

    2016-07-01

    A new single-chain-magnet (SCM), [Co4(OMe)3(HCO2)2(L)3·DMF]n, (L = 4-(pyridin-4-yl)benzolate) (2), was constructed by changing the spacers of a weak ferromagnetic single-chain magnet [Co8(OMe)6(HCO2)4(isonic)6·H2O]n (1). By contrasting the magnetism of the two complexes, it is found that the longer the linker the stronger the magnetic properties. PMID:27333437

  16. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    PubMed Central

    2012-01-01

    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 libraries was highly

  17. Influence of backbone rigidness on single chain conformation of thiophene-based conjugated polymers.

    PubMed

    Hu, Zhongjian; Liu, Jianhua; Simón-Bower, Lauren; Zhai, Lei; Gesquiere, Andre J

    2013-04-25

    Structural order of conjugated polymers at different length scales directs the optoelectronic properties of the corresponding materials; thus it is of critical importance to understand and control conjugated polymer morphology for successful application of these materials in organic optoelectronics. Herein, with the aim of probing the dependence of single chain folding properties on the chemical structure and rigidness of the polymer backbones, single molecule fluorescence spectroscopy was applied to four thiophene-based conjugated polymers. These include regioregular poly(3-hexylthiophene) (RR-P3HT), poly(2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT-14), poly(2,5-bis(3-tetradecylthiophen-2-yl)thiophene-2-yl)thiophen-2-ylthiazolo[5,4-d]thiazole) (PTzQT-12), and poly(3,3-didodecylquaterthiophene)] (PQT-12). Our previous work has shown that RR-P3HT and PBTTT-14 polymer chains fold in their nanostructures, whereas PQT-12 and PTzQT-12 do not fold in their nanostructures. At the single molecule level, it was found that RR-P3HT single chains almost exclusively fold into loosely and strongly aggregated conformations, analogous to the folding properties in nanostructures. PQT-12 displays significant chain folding as well, but only into loosely aggregated conformations, showing an absence of strongly aggregated polymer chains. PBTTT-14 exhibits a significant fraction of rigid polymer chain. The findings made for single molecules of PQT-12 and PBTTT-14 are thus in contrast with the observations made in their corresponding nanostructures. PTzQT-12 appears to be the most rigid and planar conjugated polymer of these four polymers. However, although the presumably nonfolding polymers PQT-12 and PTzQT-12 exhibit less folding than RR-P3HT, there is still a significant occurrence of chain folding for these polymers at the single molecule level. These results suggest that the folding properties of conjugated polymers can be influenced by the architecture of the

  18. Effects of Water on the Single-Chain Elasticity of Poly(U) RNA.

    PubMed

    Luo, Zhonglong; Cheng, Bo; Cui, Shuxun

    2015-06-01

    Water, the dominant component under the physiological condition, is a complicated solvent which greatly affects the properties of solute molecules. Here, we utilize atomic force microscope-based single-molecule force spectroscopy to study the influence of water on the single-molecule elasticity of an unstructured single-stranded RNA (poly(U)). In nonpolar solvents, RNA presents its inherent elasticity, which is consistent with the theoretical single-chain elasticity calculated by quantum mechanics calculations. In aqueous buffers, however, an additional energy of 1.88 kJ/mol·base is needed for the stretching of the ssRNA chain. This energy is consumed by the bound water rearrangement (Ew) during chain elongation. Further experimental results indicate that the Ew value is uncorrelated to the salt concentrations and stretching velocity. The results obtained in an 8 M guanidine·HCl solution provide more evidence that the bound water molecules around RNA give rise to the observed deviation between aqueous and nonaqueous environments. Compared to synthetic water-soluble polymers, the value of Ew of RNA is much lower. The weak interference of water is supposed to be the precondition for the RNA secondary structure to exist in aqueous solution. PMID:25989243

  19. New approach for designing single-chain magnets: organization of chains via hydrogen bonding between nucleobases.

    PubMed

    Zhang, Wei-Xiong; Shiga, Takuya; Miyasaka, Hitoshi; Yamashita, Masahiro

    2012-04-25

    Two one-dimensional (1D) manganese complexes, [Mn(2)(naphtmen)(2)(L)](ClO(4))·2Et(2)O·2MeOH·H(2)O (1) and [Mn(2)(naphtmen)(2)(HL)](ClO(4))(2)·MeOH (2), were synthesized by using a bridging ligand with a nucleobase moiety, 6-amino-9-β-carboxyethylpurine, and a salen-type manganese(III) dinuclear complex, [Mn(2)(naphtmen)(2)(H(2)O)(2)](ClO(4))(2) (naphtmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(naphthylideneiminato) dianion). In 1 and 2, the carboxylate-bridged Mn(III) dinuclear units are alternately linked by two kinds of weak Mn···O interactions into 1D chains. As a result, canted antiferromagnetic and ferromagnetic interactions are alternately present along the chains, leading to a 1D chain with non-cancellation of anisotropic spins. Since the chains connected via H-bonds between nucleobase moieties are magnetically isolated, both 1 and 2 act as single-chain magnets (SCMs). More importantly, this result shows the smaller canting angles hinder long-range ordering in favor of SCM dynamics.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25494541

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

    PubMed

    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.

  2. Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design

    PubMed Central

    Leone, Serena; Pica, Andrea; Merlino, Antonello; Sannino, Filomena; Temussi, Piero Andrea; Picone, Delia

    2016-01-01

    Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction. PMID:27658853

  3. Slow dynamics of the magnetization in one-dimensional coordination polymers: single-chain magnets.

    PubMed

    Miyasaka, Hitoshi; Julve, Miguel; Yamashita, Masahiro; Clérac, Rodolphe

    2009-04-20

    Slow relaxation of the magnetization (i.e., "magnet-like" behavior) in materials composed of magnetically isolated chains was observed for the first time in 2001. This type of behavior was predicted in the 1960s by Glauber in a chain of ferromagnetically coupled Ising spins (the so-called Glauber dynamics). In 2002, this new class of nanomagnets was named single-chain magnets (SCMs) by analogy to single-molecule magnets that are isolated molecules displaying related superparamagnetic properties. A long-range order occurs only at T = 0 K in any pure one-dimensional (1D) system, and thus such systems remain in their paramagnetic state at any finite temperature. Nevertheless, the combined action of large uniaxial anisotropy and intrachain magnetic interactions between high-spin magnetic units of the 1D arrangement promotes long relaxation times for the magnetization reversal with decreasing temperature, and finally at significantly low temperatures, the material can behave as a magnet. In this Forum Article, we summarize simple theoretical approaches used for understanding typical SCM behavior and some rational synthetic strategies to obtain SCM materials together with representative examples of SCMs previously reported.

  4. Single-Chain Magnets Based on Octacyanotungstate with the Highest Energy Barriers for Cyanide Compounds

    PubMed Central

    Wei, Rong-Min; Cao, Fan; Li, Jing; Yang, Li; Han, Yuan; Zhang, Xiu-Ling; Zhang, Zaichao; Wang, Xin-Yi; Song, You

    2016-01-01

    By introducing large counter cations as the spacer, two isolated 3, 3-ladder compounds, (Ph4P)[CoII(3-Mepy)2.7(H2O)0.3WV(CN)8]·0.6H2O (1) and (Ph4As)[CoII(3-Mepy)3WV(CN)8] (2, 3-Mepy = 3-methylpyridine), were synthesized and characterized. Static and dynamic magnetic characterizations reveal that compounds 1 and 2 both behave as the single-chain magnets (SCMs) with very high energy barriers: 252(9) K for 1 and 224(7) K for 2, respectively. These two compounds display the highest relaxation barriers for cyano-bridged SCMs and are preceded only by two cobalt(II)-radical compounds among all SCMs. Meanwhile, a large coercive field of 26.2 kOe (1) and 22.6 kOe (2) were observed at 1.8 K. PMID:27071451

  5. Antibodies and intrabodies against huntingtin: production and screening of monoclonals and single-chain recombinant forms.

    PubMed

    Khoshnan, Ali; Ou, Susan; Ko, Jan; Patterson, Paul H

    2013-01-01

    Antibodies can be extremely useful tools for the field of triplet repeats diseases. These reagents are important for localizing proteins in tissues and they can be used in the isolation and characterization of the components of protein complexes. In the context of huntingtin (Htt), antibodies can distinguish Htt with normal or an expanded polyglutamine (polyQ) repeats, and they can identify distinct conformations of Htt. Htt is the protein that, when mutated to contain an expanded polyQ motif, causes Huntington's disease (HD). Our group has produced monoclonal and recombinant single-chain antibodies (intrabodies) that can be used for these purposes and to perturb the function of Htt in living cells. Studies with anti-Htt intrabodies have led to identification of novel pathogenic epitopes. Moreover, some of the isolated intrabodies can reduce the neurotoxicity of mutant Htt in cell culture and animal models of HD. Thus, the production of antibodies and intrabodies has made a significant contribution to the understanding of HD pathogenesis and has introduced a novel strategy to treat this debilitating neurodegenerative disorder.

  6. Single-Chain Magnets Based on Octacyanotungstate with the Highest Energy Barriers for Cyanide Compounds

    NASA Astrophysics Data System (ADS)

    Wei, Rong-Min; Cao, Fan; Li, Jing; Yang, Li; Han, Yuan; Zhang, Xiu-Ling; Zhang, Zaichao; Wang, Xin-Yi; Song, You

    2016-04-01

    By introducing large counter cations as the spacer, two isolated 3, 3-ladder compounds, (Ph4P)[CoII(3-Mepy)2.7(H2O)0.3WV(CN)8]·0.6H2O (1) and (Ph4As)[CoII(3-Mepy)3WV(CN)8] (2, 3-Mepy = 3-methylpyridine), were synthesized and characterized. Static and dynamic magnetic characterizations reveal that compounds 1 and 2 both behave as the single-chain magnets (SCMs) with very high energy barriers: 252(9) K for 1 and 224(7) K for 2, respectively. These two compounds display the highest relaxation barriers for cyano-bridged SCMs and are preceded only by two cobalt(II)-radical compounds among all SCMs. Meanwhile, a large coercive field of 26.2 kOe (1) and 22.6 kOe (2) were observed at 1.8 K.

  7. IL-8 single-chain homodimers and heterodimers: interactions with chemokine receptors CXCR1, CXCR2, and DARC.

    PubMed Central

    Leong, S. R.; Lowman, H. B.; Liu, J.; Shire, S.; Deforge, L. E.; Gillece-Castro, B. L.; McDowell, R.; Hébert, C. A.

    1997-01-01

    Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants. PMID:9070443

  8. Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach

    PubMed Central

    Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

    2015-01-01

    Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability. PMID:26337947

  9. Integrated optical biosensor for detection of multivalent proteins

    SciTech Connect

    Kelly, Dan; Grace, Karen M.; Song, Xuedong; Swanson, Basil I.; Frayer, Daniel; Mendes, Sergio B.; Peyghambarian, Nasser

    1999-12-01

    We have developed a simple, highly sensitive and specific optical waveguide sensor for the detection of multivalent proteins. The optical biosensor is based on optically tagged glycolipid receptors embedded within a fluid phospholipid bilayer membrane formed upon the surface of a planar optical waveguide. Binding of multivalent cholera toxin triggers a fluorescence resonance energy transfer that results in a two-color optical change that is monitored by measurement of emitted luminescence above the waveguide surface. The sensor approach is highly sensitive and specific and requires no additional reagents and washing steps. Demonstration of protein-receptor recognition by use of planar optical waveguides provides a path forward for the development of fieldable miniaturized biosensor arrays. (c) 1999 Optical Society of America.

  10. Glycodendrimers: tools to explore multivalent galectin-1 interactions.

    PubMed

    Cousin, Jonathan M; Cloninger, Mary J

    2015-01-01

    Four generations of lactose-functionalized polyamidoamine (PAMAM) were employed to further the understanding of multivalent galectin-1 mediated interactions. Dynamic light scattering and fluorescence microscopy were used to study the multivalent interaction of galectin-1 with the glycodendrimers in solution, and glycodendrimers were observed to organize galectin-1 into nanoparticles. In the presence of a large excess of galectin-1, glycodendrimers nucleated galectin-1 into nanoparticles that were remarkably homologous in size (400-500 nm). To understand augmentation of oncologic cellular aggregation by galectin-1, glycodendrimers were used in cell-based assays with human prostate carcinoma cells (DU145). The results revealed that glycodendrimers provided competitive binding sites for galectin-1, which diverted galectin-1 from its typical function in cellular aggregation of DU145 cells.

  11. Switch-like surface binding of competing multivalent particles

    NASA Astrophysics Data System (ADS)

    Tito, N. B.; Frenkel, D.

    2016-07-01

    Multivalent particles competing for binding on the same surface can exhibit switch-like behaviour, depending on the concentration of receptors on the surface. When the receptor concentration is low, energy dominates the free energy of binding, and particles having a small number of strongly-binding ligands preferentially bind to the surface. At higher receptor concentrations, multivalent effects become significant, and entropy dominates the binding free energy; particles having many weakly-binding ligands preferentially bind to the surface. Between these two regimes there is a "switch-point", at which the surface binds the two species of particles equally strongly. We demonstrate that a simple theory can account for this switch-like behaviour and present numerical calculations that support the theoretical predictions. We argue that binding selectivity based on receptor density, rather than identity, may have practical applications.

  12. Convolution properties for certain classes of multivalent functions

    NASA Astrophysics Data System (ADS)

    Sokól, Janusz; Trojnar-Spelina, Lucyna

    2008-01-01

    Recently N.E. Cho, O.S. Kwon and H.M. Srivastava [Nak Eun Cho, Oh Sang Kwon, H.M. Srivastava, Inclusion relationships and argument properties for certain subclasses of multivalent functions associated with a family of linear operators, J. Math. Anal. Appl. 292 (2004) 470-483] have introduced the class of multivalent analytic functions and have given a number of results. This class has been defined by means of a special linear operator associated with the Gaussian hypergeometric function. In this paper we have extended some of the previous results and have given other properties of this class. We have made use of differential subordinations and properties of convolution in geometric function theory.

  13. Multivalent Protein Assembly Using Monovalent Self-Assembling Building Blocks

    PubMed Central

    Petkau-Milroy, Katja; Sonntag, Michael H.; Colditz, Alexander; Brunsveld, Luc

    2013-01-01

    Discotic molecules, which self-assemble in water into columnar supramolecular polymers, emerged as an alternative platform for the organization of proteins. Here, a monovalent discotic decorated with one single biotin was synthesized to study the self-assembling multivalency of this system in regard to streptavidin. Next to tetravalent streptavidin, monovalent streptavidin was used to study the protein assembly along the supramolecular polymer in detail without the interference of cross-linking. Upon self-assembly of the monovalent biotinylated discotics, multivalent proteins can be assembled along the supramolecular polymer. The concentration of discotics, which influences the length of the final polymers at the same time dictates the amount of assembled proteins. PMID:24152447

  14. Rod-like polyelectrolyte brushes with mono- and multivalent counterions

    NASA Astrophysics Data System (ADS)

    Fazli, H.; Golestanian, R.; Hansen, P. L.; Kolahchi, M. R.

    2006-02-01

    A model of rod-like polyelectrolyte brushes in the presence of monovalent and multivalent counterions but with no added salt is studied using Monte Carlo simulation. The average height of the brush, the histogram of rod conformations, and the counterion density profile are obtained for different values of the grafting density of the charge-neutral wall. For a domain of grafting densities, the brush height is found to be relatively insensitive to the density due to a competition between counterion condensation and inter-rod repulsion. In this regime, multivalent counterions collapse the brush in the form of linked clusters. Nematic order emerges at high grafting densities, resulting in an abrupt increase of the brush height.

  15. Trigonal scaffolds for multivalent targeting of melanocortin receptors.

    PubMed

    Elshan, N G R Dayan; Jayasundera, Thanuja; Anglin, Bobbi L; Weber, Craig S; Lynch, Ronald M; Mash, Eugene A

    2015-02-14

    Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (∼2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed. PMID:25502141

  16. Design and synthesis of multivalent neoglycoconjugates by click conjugations

    PubMed Central

    Ding, Feiqing; Ji, Li; William, Ronny; Chai, Hua

    2014-01-01

    Summary A highly stereoselective BF3∙OEt2-promoted tandem hydroamination/glycosylation on glycal scaffolds has been developed to form propargyl 3-tosylamino-2,3-dideoxysugars in a one-pot manner. Subsequent construction of multivalent 3-tosylamino-2,3-dideoxyneoglycoconjugates with potential biochemical applications was presented herein involving click conjugations as the key reaction step. The copper-catalyzed regioselective click reaction was tremendously accelerated with assistance of microwave irradiation. PMID:24991285

  17. Glycodendrimers and Modified ELISAs: Tools to Elucidate Multivalent Interactions of Galectins 1 and 3

    PubMed Central

    Wolfenden, Mark; Cousin, Jonathan; Nangia-Makker, Pratima; Raz, Avraham; Cloninger, Mary

    2015-01-01

    Multivalent protein-carbohydrate interactions that are mediated by sugar-binding proteins, i.e., lectins, have been implicated in a myriad of intercellular recognition processes associated with tumor progression such as galectin-mediated cancer cellular migration/metastatic processes. Here, using a modified ELISA, we show that glycodendrimers bearing mixtures of galactosides, lactosides, and N-acetylgalactosaminosides, galectin-3 ligands, multivalently affect galectin-3 functions. We further demonstrate that lactose functionalized glycodendrimers multivalently bind a different member of the galectin family, i.e., galectin-1. In a modified ELISA, galectin-3 recruitment by glycodendrimers was shown to directly depend on the ratio of low to high affinity ligands on the dendrimers, with lactose-functionalized dendrimers having the highest activity and also binding well to galectin-1. The results depicted here indicate that synthetic multivalent systems and upfront assay formats will improve the understanding of the multivalent function of galectins during multivalent protein carbohydrate recognition/interaction. PMID:25903363

  18. Secondary batteries with multivalent ions for energy storage.

    PubMed

    Xu, Chengjun; Chen, Yanyi; Shi, Shan; Li, Jia; Kang, Feiyu; Su, Dangsheng

    2015-01-01

    The use of electricity generated from clean and renewable sources, such as water, wind, or sunlight, requires efficiently distributed electrical energy storage by high-power and high-energy secondary batteries using abundant, low-cost materials in sustainable processes. American Science Policy Reports state that the next-generation "beyond-lithium" battery chemistry is one feasible solution for such goals. Here we discover new "multivalent ion" battery chemistry beyond lithium battery chemistry. Through theoretic calculation and experiment confirmation, stable thermodynamics and fast kinetics are presented during the storage of multivalent ions (Ni(2+), Zn(2+), Mg(2+), Ca(2+), Ba(2+), or La(3+) ions) in alpha type manganese dioxide. Apart from zinc ion battery, we further use multivalent Ni(2+) ion to invent another rechargeable battery, named as nickel ion battery for the first time. The nickel ion battery generally uses an alpha type manganese dioxide cathode, an electrolyte containing Ni(2+) ions, and Ni anode. The nickel ion battery delivers a high energy density (340 Wh kg(-1), close to lithium ion batteries), fast charge ability (1 minute), and long cycle life (over 2200 times).

  19. Resonant energy transfer based biosensor for detection of multivalent proteins.

    SciTech Connect

    Song, X.; Swanson, Basil I.

    2001-01-01

    We have developed a new fluorescence-based biosensor for sensitive detection of species involved in a multivslent interaction. The biosensor system utilizes specific interactions between proteins and cell surface receptors, which trigger a receptor aggregation process. Distance-dependent fluorescence self-quenching and resonant energy transfer mechanisms were coupled with a multivalent interaction to probe the receptor aggregation process, providing a sensitive and specific signal transduction method for such a binding event. The fluorescence change induced by the aggregation process can be monitored by different instrument platforms, e.g. fluorimetry and flow cytometry. In this article, a sensitive detection of pentavalent cholera toxin which recognizes ganglioside GM1 has been demonstrated through the resonant energy transfer scheme, which can achieve a double color change simultaneously. A detection sensitivity as high as 10 pM has been achieved within a few minutes (c.a. 5 minutes). The simultaneous double color change (an increase of acceptor fluorescence and a decrease of donor fluorescence intensity) of two similar fluorescent probes provides particularly high detection reliability owing to the fact that they act as each other's internal reference. Any external perturbation such as environmental temperature change causes no significant change in signal generation. Besides the application for biological sensing, the method also provides a useful tool for investigation of kinetics and thermodynamics of a multivalent interaction. Keywords: Biosensor, Fluorescence resonant energy transfer, Multivalent interaction, Cholera Toxin, Ganglioside GM1, Signal Transduction

  20. Multivalent counterions inhibit DNA ejection from viral capsid

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan

    2008-03-01

    Viral DNA packaged inside a bacteriophage is tighly bent. This stored bending energy of DNA is believed to be the main driving force to eject viral DNA into host cell upon capsid binding. One can control the amount of ejected DNA by subjecting the virus to a solution of PEG8000 molecules. The molecules cannot penetrate the viral capsid, therefore, they exert an osmotic pressure on the virus preventing DNA ejection. Experiments showed that for a given osmotic pressure, the degree of ejection also depends on the concentration of small ions in solution. Interestingly, for multivalent ions (such as Mg2+, Spd3+ or HexCo3+), this dependence is non-monotonic. We propose a simple electrostatic theory to explain this non-monotonic behavior. This is based on the fact that DNA molecules can invert its net charge at high enough multivalent counterion concentration. In other words, as multivalent counterion concentration is increased from zero, charge of DNA molecules change from negative to positive. At the concentration where DNA net charge is zero, the DNA molecules experience an attraction between different segments and DNA ejected amount is reduced. At low or high counterion concentration, DNA segments are charged (negatively or positively), repel each other and DNA ejected amount is increased. Fitting the result of the theory to experimental data, we obtain a numerical value for Mg2+ mediated DNA - DNA attraction energy to be -0.008kT per base.

  1. Secondary batteries with multivalent ions for energy storage

    NASA Astrophysics Data System (ADS)

    Xu, Chengjun; Chen, Yanyi; Shi, Shan; Li, Jia; Kang, Feiyu; Su, Dangsheng

    2015-09-01

    The use of electricity generated from clean and renewable sources, such as water, wind, or sunlight, requires efficiently distributed electrical energy storage by high-power and high-energy secondary batteries using abundant, low-cost materials in sustainable processes. American Science Policy Reports state that the next-generation “beyond-lithium” battery chemistry is one feasible solution for such goals. Here we discover new “multivalent ion” battery chemistry beyond lithium battery chemistry. Through theoretic calculation and experiment confirmation, stable thermodynamics and fast kinetics are presented during the storage of multivalent ions (Ni2+, Zn2+, Mg2+, Ca2+, Ba2+, or La3+ ions) in alpha type manganese dioxide. Apart from zinc ion battery, we further use multivalent Ni2+ ion to invent another rechargeable battery, named as nickel ion battery for the first time. The nickel ion battery generally uses an alpha type manganese dioxide cathode, an electrolyte containing Ni2+ ions, and Ni anode. The nickel ion battery delivers a high energy density (340 Wh kg-1, close to lithium ion batteries), fast charge ability (1 minute), and long cycle life (over 2200 times).

  2. Multivalent Ligand-Receptor Binding on Supported Lipid Bilayers

    PubMed Central

    Jung, Hyunsook; Robison, Aaron D.; Cremer, Paul S.

    2009-01-01

    Fluid supported lipid bilayers provide an excellent platform for studying multivalent protein-ligand interactions because the two-dimensional fluidity of the membrane allows for lateral rearrangement of ligands in order to optimize binding. Our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy (TIRFM) to obtain equilibrium dissociation constant (KD) data for these systems. This high throughput, on-chip approach provides highly accurate thermodynamic information about multivalent binding events while requiring only very small sample volumes. Herein, we review some of the most salient findings from these studies. In particular, increasing ligand density on the membrane surface can provide a modest enhancement or attenuation of ligand-receptor binding depending upon whether the surface ligands interact strongly with each other. Such effects, however, lead to little more than one order of magnitude change in the apparent KD values. On the other hand, the lipophilicity and presentation of lipid bilayer-conjugated ligands can have a much greater impact. Indeed, changing the way a particular ligand is conjugated to the membrane can alter the apparent KD value by at least three orders of magnitude. Such a result speaks strongly to the role of ligand availability for multivalent ligand-receptor binding. PMID:19508894

  3. Fouling mitigation in membrane bioreactors using multivalent cations.

    PubMed

    Mehrnia, Mohammad Reza; Azami, Hamed; Sarrafzadeh, Mohammad Hossein

    2013-09-01

    Several cations have been used to study the effect of mineral coagulants on activated sludge properties and membrane fouling in submerged membrane bioreactors (MBRs). The flocculability and settling properties of activated sludge were studied in various concentrations of sodium, potassium, magnesium, calcium, ferrum (in Fe(3+) form), and aluminum. Significant effect of cations and their concentrations on different parameters have been analyzed by 2-way ANOVA. Results showed that multivalent cations induce flocculation in bioreactors while monovalent cations have a detrimental effect on flocculability of activated sludge. The ratio of tight bound extracellular polymeric substances (TB-EPSs) to loosely bound (LB-EPSs) increased with concentration of multivalent cations that was in accordance with enhancement of biosorption in high concentration of multivalent cations. Moreover, these cations improved the settleability and dewatering properties of activated sludge. They also mitigated membrane fouling in the MBRs so that calcium and aluminum reduced membrane fouling to nearly 30%. Simple but useful correlations were developed for description of activated sludge properties based on easy measurable parameters that could be used also for estimation of membrane fouling. It was found that the ratio of TB-EPS to LB-EPS and also flocs size distribution are the main parameters affecting membrane fouling.

  4. Secondary batteries with multivalent ions for energy storage

    PubMed Central

    Xu, Chengjun; Chen, Yanyi; Shi, Shan; Li, Jia; Kang, Feiyu; Su, Dangsheng

    2015-01-01

    The use of electricity generated from clean and renewable sources, such as water, wind, or sunlight, requires efficiently distributed electrical energy storage by high-power and high-energy secondary batteries using abundant, low-cost materials in sustainable processes. American Science Policy Reports state that the next-generation “beyond-lithium” battery chemistry is one feasible solution for such goals. Here we discover new “multivalent ion” battery chemistry beyond lithium battery chemistry. Through theoretic calculation and experiment confirmation, stable thermodynamics and fast kinetics are presented during the storage of multivalent ions (Ni2+, Zn2+, Mg2+, Ca2+, Ba2+, or La3+ ions) in alpha type manganese dioxide. Apart from zinc ion battery, we further use multivalent Ni2+ ion to invent another rechargeable battery, named as nickel ion battery for the first time. The nickel ion battery generally uses an alpha type manganese dioxide cathode, an electrolyte containing Ni2+ ions, and Ni anode. The nickel ion battery delivers a high energy density (340 Wh kg−1, close to lithium ion batteries), fast charge ability (1 minute), and long cycle life (over 2200 times). PMID:26365600

  5. General model of phospholipid bilayers in fluid phase within the single chain mean field theory.

    PubMed

    Guo, Yachong; Pogodin, Sergey; Baulin, Vladimir A

    2014-05-01

    Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

  6. Single chain structure in thin polymer films: corrections to Flory's and Silberberg's hypotheses

    NASA Astrophysics Data System (ADS)

    Cavallo, A.; Müller, M.; Wittmer, J. P.; Johner, A.; Binder, K.

    2005-05-01

    Conformational properties of polymer melts confined between two hard structureless walls are investigated by Monte Carlo simulation of the bond fluctuation model. Parallel and perpendicular components of chain extension, bond-bond correlation function and structure factor are computed and compared with recent theoretical approaches attempting to go beyond Flory's and Silberberg's hypotheses. We demonstrate that for ultrathin films where the thickness, H, is smaller than the excluded volume screening length (blob size), ξ, the chain size parallel to the walls diverges logarithmically, R2/2Napb2+clog(N) with c~1/H. The corresponding bond-bond correlation function decreases like a power law, C(s) = d/sω with s being the curvilinear distance between bonds and ω = 1. Upon increasing the film thickness, H, we find—in contrast to Flory's hypothesis—the bulk exponent ω = 3/2 and, more importantly, a decreasing d(H) that gives direct evidence for an enhanced self-interaction of chain segments reflected at the walls. Systematic deviations from the Kratky plateau as a function of H are found for the single chain form factor parallel to the walls in agreement with the non-monotonic behaviour predicted by theory. This structure in the Kratky plateau might give rise to an erroneous estimation of the chain extension from scattering experiments. For large H the deviations are linear with the wavevector, q, but are very weak. In contrast, for ultrathin films, H<ξ, very strong corrections (albeit logarithmic in q) are found suggesting a possible experimental verification of our results.

  7. Targeting of HIV-1 Tat traffic and function by transduction-competent single chain antibodies.

    PubMed

    Theisen, Dietmar M; Pongratz, Carola; Wiegmann, Katja; Rivero, Francisco; Krut, Oleg; Krönke, Martin

    2006-04-12

    Human immunodeficiency virus type 1-encoded Tat protein is a transactivating factor essentially required for viral replication. Tat binds specifically to the transactivation response RNA stem loop, which is formed at the 5' end of all viral transcripts. The TAR binding motif of Tat also contains a protein transduction domain, PTD that mediates not only nuclear localization of Tat but is also capable of transducing cargo across cellular membranes. In order to target a Tat antagonist directly to the TAR binding site in the nucleus, we engineered a chimeric protein consisting of the Tat-derived PTD fused to the anti-Tat single chain antibody scFvtat1 that binds intracellularly to Tat. Recombinant scFvtat1-PTD(TAT) fusion antibody retained both, anti-Tat specificity and PTD(TAT)-mediated transduction-competence leading to its nuclear accumulation within living cells. Incubation of Jurkat T cells with scFvtat1-PTD(TAT) suppressed Tat-dependent transcription of a HIV-1 reporter gene by >80%. Transfection of a scFvtat1-PTD(TAT) expression plasmid in HEK293 cells resulted in diffuse cytoplasmic and nuclear expression. ScFvtat1-PTD(TAT) did not inhibit HIV-1 Tat translocation to the nucleus, yet showed increased inhibition of 78%, indicating a nuclear site of scFvtat1-PTD(TAT) action. Strikingly, the PTD(TAT) alone showed 55% inhibition in the HIV-1 luciferase reporter assay, indicating competition with HIV-1 Tat binding to the TAR element. The results of this study suggest that Tat traffic can only marginally be affected by anti-Tat antibodies and that effective inhibition of Tat function requires both competition with HIV Tat for TAR binding mediated by PTD(TAT) and steric hindrance mediated by the scFvtat1 moiety.

  8. Molecular dynamics simulation of secondary sorption behavior of montmorillonite modified by single chain quaternary ammonium cations.

    PubMed

    Zhao, Qian; Burns, Susan E

    2012-04-01

    Organoclays synthesized from single chain quaternary ammonium cations (QAC) ((CH(3))(3)NR(+)) exhibit different mechanisms for the sorption of nonpolar organic compounds as the length of the carbon chain is increased. The interaction between a nonpolar sorbate and an organoclay intercalated with small QACs has been demonstrated to be surface adsorption, while partitioning is the dominant mechanism in clays intercalated with long chain surfactants. This study presents the results of a molecular dynamics (MD) simulation performed to examine the sorption mechanisms of benzene in the interlayer of three organoclays with chain lengths ranging from 1 to 16 carbons: tetramethylammonium (TMA) clay; decyltrimethylammonium (DTMA) clay; and hexadecyltrimethylammonium (HDTMA) clay. The basis of the overall simulation was a combined force field of ClayFF and CVFF. In the simulations, organic cations were intercalated and benzene molecules were introduced to the interlayer, followed by whole system NPT and NVT time integration. Trajectories of all the species were recorded after the system reached equilibrium and subsequently analyzed. Simulation results confirmed that the arrangement of the surfactants controlled the sorption mechanism of organoclays. Benzene molecules were observed to interact directly with the clay surface in the presence of TMA cations, but tended to interact with the aliphatic chain of the HDTMA cation in the interlayer. The simulation provided insight into the nature of the adsorption/partitioning mechanisms in organoclays, and explained experimental observations of decreased versus increased uptake capacities as a function of increasing total organic carbon (TOC) for TMA clay and HDTMA clay, respectively. The transition of sorption mechanisms was also quantified with simulation of DTMA clay, with a chain length between that of TMA and HDTMA. Furthermore, this study suggested that at the molecular level, the controlling factor for the ultimate sorption

  9. The single chain limit of structural relaxation in a polyolefin blend

    NASA Astrophysics Data System (ADS)

    May, Andrew F.; Maranas, Janna K.

    2006-07-01

    The influence of composition on component dynamics and relevant static properties in a miscible polymer blend is investigated using molecular dynamics simulation. Emphasis is placed on dynamics in the single chain dilution limit, as this limit isolates the role of inherent component mobility in the polymer's dynamic behavior when placed in a blend. For our systems, a biased local concentration affecting dynamics must arise primarily from chain connectivity, which is quantified by the self-concentration, because concentration fluctuations are minimized due to restraints on chain lengths arising from simulation considerations. The polyolefins simulated [poly(ethylene-propylene) (PEP) and poly(ethylene-butene) (PEB)] have similar structures and glass transition temperatures, and all interactions are dispersive in nature. We find that the dependence of dynamics upon composition differs between the two materials. Specifically, PEB (slower component) is more influenced by the environment than PEP. This is linked to a smaller self-concentration for PEB than PEP. We examine the accuracy of the Lodge-McLeish model (which is based on chain connectivity acting over the Kuhn segment length) in predicting simulation results for effective concentration. The model predicts the simulation results with high accuracy when the model's single parameter, the self-concentration, is calculated from simulation data. However, when utilizing the theoretical prediction of the self-concentration the model is not quantitatively accurate. The ability of the model to link the simulated self-concentration with biased local compositions at the Kuhn segment length provides strong support for the claim that chain connectivity is the leading cause of distinct mobility in polymer blends. Additionally, the direct link between the willingness of a polymer to be influenced by the environment and the value of the self-concentration emphasizes the importance of the chain connectivity. Furthermore, these

  10. HER2-siRNA delivered by EGFR-specific single chain antibody inhibits NSCLC cell proliferation and tumor growth

    PubMed Central

    Liu, Li; Li, Fakai; Zhang, Jian; Ye, Mingxiang; Zhao, Hu; Zhao, Jing; Yan, Bo; Yang, Angang; Zhang, Rui; Li, Xia; Ren, Xinling

    2016-01-01

    Overexpression of human epidermal growth factor receptor type2 (HER2) is closely associated with aggressive progression and poor prognosis in non-small cell lung cancer (NSCLC). Here, we generated an EGFR-scFv-arginine nonamer peptide fusion protein (scFv-9R) as a cargo to deliver HER2 specific siRNA into HER2-positive NSCLC cells both in vitro and in vivo. HER2-siRNAs delivered by scFv-9R effeciently silenced HER2 expression in EGFR-positive NSCLC cells, and consequently resulted in G1 arrest and cell growth inhibition. Importantly, intravenous injection of scFv-9R/HER2-siRNA complex markedly suppressed growth of EGFR-positive NSCLC xenograft in nude mice, resulting from downregulated HER2 expression, reduced cell proliferation and enhanced cell apoptosis. Collectively, our study provides a novel therapeutic strategy for the treatment of EGFR-positive, HER2-overexpressed NSCLC. PMID:26988752

  11. Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

    PubMed

    Medecigo, M; Manoutcharian, K; Vasilevko, V; Govezensky, T; Munguia, M E; Becerril, B; Luz-Madrigal, A; Vaca, L; Cribbs, D H; Gevorkian, G

    2010-06-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

  12. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display.

    PubMed

    Abou El-Magd, Rabab M; Vozza, Nicolas F; Tuszynski, Jack A; Wishart, David S

    2016-01-01

    Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important. PMID:26608419

  13. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    PubMed Central

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  14. Formal Verification of the AAMP-FV Microcode

    NASA Technical Reports Server (NTRS)

    Miller, Steven P.; Greve, David A.; Wilding, Matthew M.; Srivas, Mandayam

    1999-01-01

    This report describes the experiences of Collins Avionics & Communications and SRI International in formally specifying and verifying the microcode in a Rockwell proprietary microprocessor, the AAMP-FV, using the PVS verification system. This project built extensively on earlier experiences using PVS to verify the microcode in the AAMP5, a complex, pipelined microprocessor designed for use in avionics displays and global positioning systems. While the AAMP5 experiment demonstrated the technical feasibility of formal verification of microcode, the steep learning curve encountered left unanswered the question of whether it could be performed at reasonable cost. The AAMP-FV project was conducted to determine whether the experience gained on the AAMP5 project could be used to make formal verification of microcode cost effective for safety-critical and high volume devices.

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

    PubMed

    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

  16. Multivalent bifunctional chelator scaffolds for gallium-68 based positron emission tomography imaging probe design: signal amplification via multivalency.

    PubMed

    Singh, Ajay N; Liu, Wei; Hao, Guiyang; Kumar, Amit; Gupta, Anjali; Öz, Orhan K; Hsieh, Jer-Tsong; Sun, Xiankai

    2011-08-17

    The role of the multivalent effect has been well recognized in the design of molecular imaging probes toward the desired imaging signal amplification. Recently, we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds ((t)Bu(3)-1-COOH, (t)Bu(3)-2-(COOH)(2), and (t)Bu(3)-3-(COOH)(3)) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for (68)Ga-based PET probe design and signal amplification via the multivalent effect. For proof of principle, a known integrin α(v)β(3) specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H(3)1, H(3)2, and H(3)3), which present 1-3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin α(v)β(3) binding affinities (IC(50) values), enhanced specific binding was observed for multivalent conjugates (H(3)2: 43.9 ± 16.1 nM; H(3)3: 14.7 ± 5.0 nM) as compared to their monovalent counterpart (H(3)1: 171 ± 60 nM) and the intact c(RGDyK) peptide (204 ± 76 nM). The obtained conjugates were efficiently labeled with (68)Ga(3+) within 30 min at room temperature in high radiochemical yields (>95%). The in vivo evaluation of the labeled conjugates, (68)Ga-1, (68)Ga-2, and (68)Ga-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin α(v)β(3) positive PC-3 tumor xenografts (n = 3). All (68)Ga-labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h postinjection (p.i.). The PET signal amplification in PC-3 tumor by the multivalent effect was clearly displayed by the tumor uptake of the (68)Ga-labeled conjugates ((68)Ga-3: 2.55 ± 0.50%ID/g; (68)Ga-2: 1.90 ± 0.10%ID/g; (68)Ga-1: 1.66 ± 0.15%ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal

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

    NASA Astrophysics Data System (ADS)

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

    1994-05-01

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

  18. Passive immunotherapy for anthrax toxin mediated by an adenovirus expressing an anti-protective antigen single-chain antibody.

    PubMed

    Kasuya, Kazuhiko; Boyer, Julie L; Tan, Yadi; Alipui, D Olivier; Hackett, Neil R; Crystal, Ronald G

    2005-02-01

    In the 2001 U.S. bioterror attacks, 33,000 individuals required postexposure prophylaxis, 18 subjects contracted anthrax (11 inhalation, 7 cutaneous), and despite optimal medical therapy, 5 deaths resulted. Rapid protection against anthrax is required in a bioterrorism scenario; this study describes an in vivo gene transfer-based therapy that uses a human adenovirus (Ad)-based vector (AdalphaPAscAb) encoding a single-chain antibody directed against protective antigen (PA), a critical component of Bacillus anthracis lethal toxin. Following AdalphaPAscAb administration to mice, anti-PA single-chain antibody and anti-PA neutralizing activity were detected in serum over a 2-week period. Substantial survival advantage from anthrax lethal toxin was conferred by AdalphaPAscAb following administration from 1 to 14 days prior to toxin challenge, compared to no survival associated with an Ad vector expressing a control single-chain antibody. Passive immunotherapy with an Ad-based vector may be a rapid, convenient approach for protecting a susceptible population against anthrax, including use as an adjunct to antibiotic therapy.

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

    PubMed

    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

  20. Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies.

    PubMed Central

    Mhashilkar, A M; Bagley, J; Chen, S Y; Szilvay, A M; Helland, D G; Marasco, W A

    1995-01-01

    Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N-terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS. Images PMID:7537216

  1. Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.

    PubMed Central

    Taremi, S. S.; Beyer, B.; Maher, M.; Yao, N.; Prosise, W.; Weber, P. C.; Malcolm, B. A.

    1998-01-01

    Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401). PMID:9792101

  2. Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123.

    PubMed

    Moradi-Kalbolandi, Shima; Habibi-Anbouhi, Mahdi; Golkar, Majid; Behdani, Mahdi; Rezaei, Gashin; Ghazizadeh, Leila; Abolhassani, Mohsen; Shokrgozar, Mohammad Ali

    2016-09-01

    Current therapies for acute myeloid leukemia (AML), are associated with high relapse rates. Hence, development of new therapeutic strategies is crucial to circumvent this problem. Bivalent antibody technology has been used to engineer novel antibody fragments with increased avidity, by assembling two scFv in a single molecule. Here, we present accompanying data from construction and characterization experiments of a biscFv antibody targeting CD123, the most important biomarker of leukemic cancer stem cells which play a key role in relapsed AML after chemotherapy. Data in this article are related to the research paper "Development of a novel engineered antibody targeting human CD123" Moradi-Kalbolandi S. et al. (2016) [1]. PMID:27536714

  3. Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli.

    PubMed

    Lindner, Robert; Moosmann, Anna; Dietrich, Alexander; Böttinger, Heiner; Kontermann, Roland; Siemann-Herzberg, Martin

    2014-12-20

    Prokaryotic production systems have been widely used to manufacture recombinant therapeutic proteins. Economically, the prokaryotic production – especially of small therapeutic molecules – is advantageous compared to eukaryotic production strategies. However, due to the potential endotoxin and host cell protein contamination, the requirements for the purification process are disproportionately higher and therefore more expensive and elaborate to circumvent. For this reason, the goal of this work was to develop and establish a rapid, simple, inexpensive and ‘up-scalable’ production and purification process, using the therapeutic relevant protein anti-EGFR scFv hu225 as model molecule. Configuring high cell density cultivation of Escherichia coli – using the rha-BAD expression system as production platform – a specific product concentration up to 20 mgscFv/gCDW was obtained. By combining freeze-and-thaw, osmotic shock and pH induced host cell protein precipitation, almost 70% of the product was extracted from the biomass. In a novel approach a mixed mode chromatography was implemented as a capturing and desalting step, which allowed the direct application of further ion exchange chromatography steps for purification up to pharmaceutical grade. Thereby, 50% of the produced scFv could be purified within 10 h while maintaining the biological activity. PMID:25450642

  4. Programmable Multivalent Display of Receptor Ligands using Peptide Nucleic Acid Nanoscaffolds

    PubMed Central

    Englund, Ethan A.; Wang, Deyun; Fujigaki, Hidetsugu; Sakai, Hiroyasu; Micklitsch, Christopher M.; Ghirlando, Rodolfo; Martin-Manso, Gema; Pendrak, Michael L.; Roberts, David D.; Durell, Stewart R.; Appella, Daniel H.

    2012-01-01

    Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models. PMID:22233624

  5. Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

    PubMed

    Moricoli, Diego; Muller, William Anthony; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Watson, Richard; Fiori, Valentina; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2014-06-01

    Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

  6. Is a multivalent hand, foot, and mouth disease vaccine feasible?

    PubMed Central

    Klein, Michel; Chong, Pele

    2015-01-01

    Enterovirus A infections are the primary cause of hand, foot and mouth disease (HFMD) in infants and young children. Although enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the predominant causes of HFMD epidemics worldwide, EV-A71 has emerged as a major neurovirulent virus responsible for severe neurological complications and fatal outcomes. HFMD is a serious health threat and economic burden across the Asia-Pacific region. Inactivated EV-A71 vaccines have elicited protection against EV-A71 but not against CV-A16 infections in large efficacy trials. The current development of a bivalent inactivated EV-A71/CV-A16 vaccine is the next step toward that of multivalent HFMD vaccines. These vaccines should ultimately include other prevalent pathogenic coxsackieviruses A (CV-A6 and CV-A10), coxsackieviruses B (B3 and B5) and echovirus 30 that often co-circulate during HFMD epidemics and can cause severe HFMD, aseptic meningitis and acute viral myocarditis. The prospect and challenges for the development of such multivalent vaccines are discussed. PMID:26009802

  7. Adding Mono- and Multivalent Ions to Lyotropic Chromonic Liquid Crystals

    NASA Astrophysics Data System (ADS)

    Tortora, Luana; Park, Heung-Shik; Antion, Kelly; Woolwerton, Chris; Finotello, Daniele; Lavrentovich, Oleg

    2006-03-01

    Lyotropic Chromonic Liquid Crystals (LCLCs) are a distinct class of liquid crystals formed in aqueous solutions by molecules with rigid polyaromatic cores and ionic groups at the periphery [1-4]. The phase diagrams of these materials should depend on entropic factors (as in the Onsager model) and electrostatic interactions. Using optical polarizing microscopy, we studied the effects of mono- and multivalent ions on the phase diagrams of Blue 27 [3] and Sunset Yellow [2]. The monovalent ions change the temperatures of phase transitions, as described in [4], while the effect of multivalent ions is more dramatic and, in addition to the changed temperatures of phase transitions by tens of degrees, it often involves condensation of LCLC aggregates into domains with birefringence much higher than that in a normal nematic phase. Work supported by OBR B-7844. [1]J. Lydon, Current Opin. Colloid & Interface Sci. 3, 458 (1998);8, 480-489 (2004); [2]V. R. Horowitz, L. A. Janowitz, A. L. Modic, P. J. Heiney, and P. J. Collings, 2005, Phys. Rew. E 72, 041710; [3]Yu. A. Nastishin, H. Liu, T. Schneider, T., V. Nazarenko, R. Vasyuta, S. V. Shiyanovskii, and O. D. Lavrentovich, 2005, Phys. Rev. E 72, 041711; [4]A.F. Kostko, B. H. Cipriano, O. A. Pinchuk, L. Ziserman, M. A. Anisimov, D. Danino, and S. R. Raghavan. J. Phys. Chem. B 109, 19126-19133 (2005)

  8. Superdiffusive transport by multivalent molecular walkers moving under load.

    PubMed

    Olah, Mark J; Stefanovic, Darko

    2013-06-01

    We introduce a model for translational molecular motors to demonstrate that a multivalent catalytic walker with flexible, uncoordinated legs can transform the free energy of surface-bound substrate sites into mechanical work and undergo biased, superdiffusive motion, even in opposition to an external load force. The walker in the model lacks any inherent orientation of body or track, and its legs have no chemomechanical coupling other than the passive constraint imposed by their connection to a common body. Yet, under appropriate kinetic conditions, the walker's motion is biased in the direction of unvisited sites, which allows the walker to move nearly ballistically away from the origin as long as a local supply of unmodified substrate sites is available. The multivalent random walker model is mathematically formulated as a continuous-time Markov process and is studied numerically. We use Monte Carlo simulations to generate ensemble estimates of the mean squared displacement and mean work done for this nonergodic system. Our results show that a residence time bias between visited and unvisited sites leads to superdiffusive motion over significant times and distances. This mechanism can be used to adapt any enzyme-substrate system with appropriate kinetics for use as a functional chemical implementation of a molecular motor, without the need for structural anisotropy or conformationally mediated chemomechanical coordination.

  9. Phase transitions in the assembly of multivalent signalling proteins

    SciTech Connect

    Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun; Kim, Soyeon; Chen, Baoyu; Guo, Liang; Llaguno, Marc; Hollingsworth, Javoris V.; King, David S.; Banani, Salman F.; Russo, Paul S.; Jiang, Qiu-Xing; Nixon, B. Tracy; Rosen, Michael K.

    2013-04-08

    Cells are organized on length scales ranging from angstrom to micrometers. However, the mechanisms by which angstrom-scale molecular properties are translated to micrometer-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometer-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

  10. DNA-DNA interaction inside bacteriophage modulated by multivalent counterions

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan; Lee, Seil; Le, Tung

    2010-03-01

    The problem of inhibiting viral DNA ejection from bacteriophages by multivalent counterions, especially Mg^+2 counterions, is studied. Experimentally, it is known that MgSO4 salt has a strong and non-monotonic effect on the amount of DNA ejected. There exists an optimal concentration at which the least DNA is ejected from the virus. At lower or higher concentrations, more DNA is ejected from the capsid. We propose that this phenomenon is the result of DNA overcharging by Mg^+2 multivalent counterions. As Mg^+2 concentration increases from zero, DNA net charge changes from negative to positive. The optimal inhibition corresponds to the Mg^+2 concentration where DNA is neutral. At lower/higher concentrations, DNA genome is charged. It prefers to be in solution to lower its electrostatic self-energy, which consequently leads to an increase in DNA ejection. Our theory fits experimental data well. The strength of DNA - DNA short range attraction, mediated by Mg^+2, is found to be - 0.003 kBT per nucleotide base. Results from expanded ensemble Monte-Carlo simulation of hexagonal DNA bundles are discussed and are shown to be in good agreement with theoretical results.

  11. Superdiffusive transport by multivalent molecular walkers moving under load

    NASA Astrophysics Data System (ADS)

    Olah, Mark J.; Stefanovic, Darko

    2013-06-01

    We introduce a model for translational molecular motors to demonstrate that a multivalent catalytic walker with flexible, uncoordinated legs can transform the free energy of surface-bound substrate sites into mechanical work and undergo biased, superdiffusive motion, even in opposition to an external load force. The walker in the model lacks any inherent orientation of body or track, and its legs have no chemomechanical coupling other than the passive constraint imposed by their connection to a common body. Yet, under appropriate kinetic conditions, the walker's motion is biased in the direction of unvisited sites, which allows the walker to move nearly ballistically away from the origin as long as a local supply of unmodified substrate sites is available. The multivalent random walker model is mathematically formulated as a continuous-time Markov process and is studied numerically. We use Monte Carlo simulations to generate ensemble estimates of the mean squared displacement and mean work done for this nonergodic system. Our results show that a residence time bias between visited and unvisited sites leads to superdiffusive motion over significant times and distances. This mechanism can be used to adapt any enzyme-substrate system with appropriate kinetics for use as a functional chemical implementation of a molecular motor, without the need for structural anisotropy or conformationally mediated chemomechanical coordination.

  12. A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity.

    PubMed Central

    Howe, A. Y.; Chase, R.; Taremi, S. S.; Risano, C.; Beyer, B.; Malcolm, B.; Lau, J. Y.

    1999-01-01

    Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 (a.a. 3-631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity. PMID:10386883

  13. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  14. Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein.

    PubMed

    Bhatia, J; Sharma, S K; Chester, K A; Pedley, R B; Boden, R W; Read, D A; Boxer, G M; Michael, N P; Begent, R H

    2000-02-15

    Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme selectively to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Previous clinical work using antibody-enzyme chemical conjugates has been limited by the moderate efficiency of tumor targeting of these molecules. To address this a recombinant fusion protein composed of MFE-23, an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody, fused to the amino-terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA-producing tumors. MFE-23::CPG2 fusion protein was overexpressed in Escherichia coli and purified using CEA affinity chromatography. Efficacy of MFE-23::CPG2 delivery to tumors in vivo was assessed by measuring catalytic activity after intravenous injection of purified MFE-23::CPG2 into nude mice bearing CEA-positive LS174T human colon adenocarcinoma xenografts. Recombinant MFE-23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to plasma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr). (125)I-MFE-23::CPG2 was retained in kidney, liver and spleen but MFE-23::CPG2 catalytic activity was not, resulting in excellent tumor to normal tissue enzyme ratios 48 hr after injection. These were 371:1 (tumor to liver), 450:1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1,618:1 (tumor to spleen). Favorable tumor : normal tissue ratios occurred at early time points when there was still 21% (24 hr) and 9.5% (48 hr) of the injected activity present per gram of tumor tissue. The high tumor concentrations and selective tumor retention of active enzyme delivered by MFE-23::CPG2 establish that this recombinant fusion protein has potential to give improved clinical efficiency for ADEPT.

  15. Mn(III)(tetra-biphenyl-porphyrin)-TCNE single-chain magnet via suppression of the interchain interactions.

    PubMed

    Ishikawa, Ryuta; Katoh, Keiichi; Breedlove, Brian K; Yamashita, Masahiro

    2012-08-20

    A single-chain magnet (SCM) of [Mn(TBPP)(TCNE)]·4m-PhCl(2) (1), where TBPP(2-) = meso-tetra(4-biphenyl)porphyrinate; TCNE(•-) = tetracyanoethenide radical anion; m-PhCl(2) = meta-dichlorobenzene, was prepared via suppression of interchain interactions. 1 has a one-dimensional alternating Mn(III)(porphrin)-TCNE(•-)chain structure similar to those of a family of complexes reported by Miller and co-workers. From a comparison of the static magnetic properties of 1 with other Mn(III)(porphyrin)-TCNE(•-) chains, a magneto-structural correlation between the intrachain magnetic exchange and both the dihedral angle between the mean plane on [Mn(TBPP)(TCNE)] and Mn-N≡C was observed. The ac magnetic susceptibility data of 1 could be fit with the Arrhenius law, indicating that slow magnetic relaxation and ruling out three-dimensional long-range ordering and spin-glass-like behavior. The Cole-Cole plot for 1 was semicircular, verifying that it is an SCM. Therefore, 1 is an ideal single-chain magnet with significantly strong intrachain magnetic exchange interactions beyond the Ising limit.

  16. 77 FR 35850 - Safety Zone; F/V Deep Sea, Penn Cove, WA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-15

    ... the Fishing Vessel (F/V) Deep Sea, located in Penn Cove, WA. This action is necessary to ensure the... Fishing Vessel Deep Sea located at approximately 48 13'18'' N, 122 47'42'' W, Penn Cove, WA. (b... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone; F/V Deep Sea, Penn Cove, WA AGENCY:...

  17. FvSO regulates vegetative hyphal fusion, asexual growth, fumonisin B1 production, and virulence in Fusarium verticillioides.

    PubMed

    Guo, Li; Wenner, Nancy; Kuldau, Gretchen A

    2015-12-01

    Hyphal anastomosis is a hallmark of filamentous fungi and plays vital roles including cellular homoeostasis, interhyphal communication and nutrient translocation. Here we identify a gene, FvSO, in Fusarium verticillioides, a filamentous ascomycete causing maize ear and stalk rot and producing fumonisin mycotoxins. FvSO, like its Neurospora crassa homologue SO, is required for vegetative hyphal fusion. It is also essential for normal vegetative growth, sporulation, and pathogenesis. FvSO encodes a predicted WW domain protein and shares 70 % protein sequence identity with N. crassa SO. FvSO deletion mutants (ΔFvSO) had abnormal distribution of conidia size, and conidia of ΔFvSO germinated much later and slower than wild type. ΔFvSO was deficient in hyphal anastomosis, had slower radial growth and produced less fungal biomass than wild type. ΔFvSO were unable to perform anastomosis, a key feature of filamentous fungi. Interestingly, production of fumonisin B1 by ΔFvSO was significantly reduced compared to wild type. Additionally, ΔFvSO was nonpathogenic to corn ears, stalks and seedlings, likely due to defective growth and development. In conclusion, FvSO is essential for vegetative hyphal fusion and is required for normal vegetative growth and sporulation, normal levels of fumonisin production and pathogenicity in F. verticillioides. The pleiotropic nature of ΔFvSO phenotypes suggests that FvSO is likely involved in certain signalling pathways that regulate multiple cellular functions.

  18. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    PubMed Central

    Pansri, Potjamas; Jaruseranee, Nanthnit; Rangnoi, Kuntalee; Kristensen, Peter; Yamabhai, Montarop

    2009-01-01

    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. PMID:19175944

  19. Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.

    PubMed

    Berry, M J; Wattam, T A; Willets, J; Lindner, N; de Graaf, T; Hunt, T; Gani, M; Davis, P J; Porter, P

    1994-01-01

    Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.

  20. Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.

    PubMed

    Berry, M J; Wattam, T A; Willets, J; Lindner, N; de Graaf, T; Hunt, T; Gani, M; Davis, P J; Porter, P

    1994-01-01

    Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules. PMID:7508476

  1. Multivalent protein polymers with controlled chemical and physical properties.

    PubMed

    Top, Ayben; Kiick, Kristi L

    2010-12-30

    In this review, we describe our work on the design, characterization, and modification of a series of alanine-rich helical polypeptides with novel functions. Glycosylation of the polypeptides has permitted investigation of polymer architecture effects on multivalent interactions. One of the members of this polypeptide family exhibits polymorphological behavior that is easily manipulated via simple changes in solution pH and temperature. Polypeptide-based fibrils formed at acidic pH and high temperature were shown to direct the one-dimensional organization of gold nanoparticles via electrostatic interactions. As a precursor to fibrils, aggregates likely comprising alanine-rich cores form at low temperatures and acidic pH and reversibly dissociate into monomers upon deprotonation. PEGylation of these polypeptides does not alter the self-association or conformational behavior of the polypeptide, suggesting potential applications in the development of assembled delivery vehicles, as modification of the polypeptides should be a useful strategy for controlling assembly. PMID:20562016

  2. Evaluating the Thickness of Multivalent Glycopolymer Brushes for Lectin Binding.

    PubMed

    Lazar, Jaroslav; Park, Hyunji; Rosencrantz, Ruben R; Böker, Alexander; Elling, Lothar; Schnakenberg, Uwe

    2015-08-01

    Electrochemical impedance spectroscopy (EIS) is applied for investigating binding of lectins to multivalent glycopolymer brushes grafted from interdigital gold microelectrodes. By variation of the measuring frequency, EIS allows simultaneous analysis of binding at different subnanometer distances from the sensor surfaces. In this way, the binding dynamics along the brushes are quantified, giving an idea about the motion of the lectin through the brush layer. Two different brush lengths are investigated, revealing distinct dynamics of lectin binding due to changing topology of the brushes. Moreover, very low K D values in the nanomolar range are obtained. This unique platform may be used as sophisticated biosensor for detailed investigation of high-affinity protein binding to poly-mer layers. PMID:26096302

  3. A highly versatile convergent/divergent "onion peel" synthetic strategy toward potent multivalent glycodendrimers.

    PubMed

    Sharma, Rishi; Kottari, Naresh; Chabre, Yoann M; Abbassi, Leïla; Shiao, Tze Chieh; Roy, René

    2014-11-11

    Both convergent and divergent strategies for the synthesis of "onion peel" glycodendrimers are reported which resulted in one of the best multivalent ligands known against the virulent factor from a bacterial lectin isolated from Pseudomonas aeruginosa. PMID:25227948

  4. RB virus: a strain of Friend virus that produces a 'Friend virus-like' disease in Fv-2rr mice.

    PubMed

    Geib, R W; Seaward, M B; Stevens, M L; Cho, C L; Majumdar, M

    1989-10-01

    RB virus is a newly derived strain of Friend virus that was adapted to produce a 'Friend-like' disease in mice that are genetically resistant to wild-type Friend virus. RB virus was produced by passing high titers of the wild-type Friend virus (Lilly-Steeves polycythemia-producing strain) through adult Fv-2rr mice. Titration of the defective spleen focus-forming virus indicated RB virus infected similar numbers of Fv-2ss or Fv-2rr target cells. Analysis of the spleens from mice infected with RB virus indicated that RB induced the early stage of Friend disease (erythroid proliferation) in both Fv-2rr and Fv-2ss mice. Fv-2ss mice infected with RB virus developed the classical Friend disease within 3 weeks. In contrast, the percentage of Fv-2rr mice developing the 'Friend-like' disease after infection with RB virus never exceeded 60%. The latency period of RBV in Fv-2rr mice was strain dependent. D2.R16 (Fv-2rr) developed the syndrome more rapidly than C57BL/6 (Fv-2rr). RB virus retained the capacity to transform erythroprogenitor cells from both Fv-2ss and Fv-2rr animals. Cells infected with RB virus consistently produced a modified SFFV envelope protein, gp48.

  5. Two water-bridged cobalt(II) chains with isomeric naphthoate spacers: from metamagnetic to single-chain magnetic behaviour.

    PubMed

    Liu, Zhong-Yi; Xia, Yan-Fei; Jiao, Jiao; Yang, En-Cui; Zhao, Xiao-Jun

    2015-12-14

    The crystal structures and magnetic behaviours of two water-bridged one-dimensional (1D) cobalt(II) chains with different isomeric naphthoate (na(-)) terminals, [Co(H2O)3(2-na)2]n (1) and {[Co(H2O)3(1-na)2]·2H2O}n (2), were reported to investigate the effect of interchain distance on their magnetic properties. Complex 1 with trans-2-na(-) blocks and dense interchain separation exhibits a metamagnetic transition from antiferromagnetic ordering to a saturated paramagnetic phase. By contrast, complex 2 possessing cis-arranged 1-na(-) spacers and good interchain isolation shows unusual single-chain magnetic behavior under a zero dc field. Thus, completely different interchain packing by isomeric naphthoate ligands governs the ratio of intra- to inter-chain magnetic interactions and further results in different magnetic phenomena, which provide significant magnetostructural information on 1D magnetic systems. PMID:26514974

  6. Self-assembly of different single-chain bolaphospholipids and their miscibility with phospholipids or classical amphiphiles.

    PubMed

    Blume, Alfred; Drescher, Simon; Graf, Gesche; Köhler, Karen; Meister, Annette

    2014-06-01

    A variety of bolalipids with a single long alkyl chain and two identical headgroups self-assemble in aqueous solutions into helical entangled nanofibers leading to the formation of a hydrogel. An increase in temperature usually leads to the break-up of the fiber structure into micellar aggregates. In this paper the question is addressed whether bolalipids of different lengths or different headgroup structures can form mixed fibers. Also, the stability of the fiber aggregation of bolalipids in mixtures with phospholipids forming lamellar bilayers is discussed. Here, the question whether single-chain bolalipids can be incorporated into phospholipid bilayers to stabilize bilayer membranes is important, as possibly lipid vesicles used for drug delivery can be improved. Finally, the stability of the fiber aggregate against solubilisation by common surfactants was studied. The paper addresses the question which type of aggregate structure dominates the self-assembly of bipolar and monopolar amphiphiles in aqueous suspension.

  7. Detection of the single-chain precursor in the production and purification process of recombinant human insulin.

    PubMed

    Leng, Chunsheng; Li, Qingwei; Wu, Fenfang; Chen, Liyong; Su, Peng

    2013-08-01

    High quality recombinant insulin requires being free of single-chain precursor (proinsulin), a task that depends on the selectivity and sensitivity of the monitoring process for detecting proinsulin. In this study we developed an enzyme-linked immunosorbent assay (ELISA) system that was specifically tailored to detect recombinant proinsulin. The proinsulin consists of six components: an initiating methionine, 48 amino acids from human growth hormones (HGH, used as the protection peptide), first connecting Arg-residue, B-chain of insulin, and second connecting Arg-peptide and A-chain of insulin. This form of proinsulin is more stable and can be efficiently expressed by E. coli than insulin. Herein, we evaluated the specificity, precision, recovery, sensitivity, and detection range of the proinsulin ELISA kit. The results showed that the ELISA kit is a very useful tool for monitoring the proinsulin yield in early stages of insulin production as well as the residual proinsulin in the final product, insulin.

  8. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    PubMed

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-01-01

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M. PMID:27294959

  9. Anti-Human Endoglin (hCD105) Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    PubMed Central

    Barriuso, Begoña; Antolín, Pilar; Arias, F. Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-01-01

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M. PMID:27294959

  10. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter

    PubMed Central

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  11. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter.

    PubMed

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  12. Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

    PubMed Central

    Jiang, W; Venugopal, K; Gould, E A

    1995-01-01

    A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus. PMID:7815482

  13. EXPERIMENTAL CHALLENGE STUDY OF FV3-LIKE RANAVIRUS INFECTION IN PREVIOUSLY FV3-LIKE RANAVIRUS INFECTED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) TO ASSESS INFECTION AND SURVIVAL.

    PubMed

    Hausmann, Jennifer C; Wack, Allison N; Allender, Matthew C; Cranfield, Mike R; Murphy, Kevin J; Barrett, Kevin; Romero, Jennell L; Wellehan, James F X; Blum, Stella A; Zink, M Christine; Bronson, Ellen

    2015-12-01

    The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

  14. Potent inhibition of tau fibrillization with a multivalent ligand

    SciTech Connect

    Honson, Nicolette S.; Jensen, Jordan R.; Darby, Michael V.; Kuret, Jeff

    2007-11-09

    Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy. Results showed that the inhibitory activity of the bis-thiacarbocyanine was qualitatively similar to a monomeric cyanine dye, but was more potent with 50% inhibition achieved at {approx}80 nM concentration. At all concentrations tested in aqueous solution, the bis-thiacarbocyanine collapsed to form a closed clamshell structure. However, the presence of tau protein selectively stabilized the open conformation. These results suggest that the inhibitory activity of bis-thiacarbocyanine results from multivalency, and reveal a route to more potent tau aggregation inhibitors.

  15. Multivalent anthelminthic vaccine to prevent hookworm and schistosomiasis.

    PubMed

    Hotez, Peter J; Bethony, Jeffrey M; Oliveira, Sergio Costa; Brindley, Paul J; Loukas, Alex

    2008-08-01

    Hookworm infection and schistosomiasis are two of the world's most important human parasitic infections, affecting hundreds of millions of people in developing countries. Measured together in disability-adjusted life years, hookworm infection and schistosomiasis rank closely behind malaria as the most prevalent human parasitic diseases. A major approach for the control of these two helminth infections relies on periodic, mass chemotherapy with anthelminthics. However, high rates of post-treatment reinfection, the declining efficacy with repeated treatment, rebound morbidity (in the case of schistosomiasis) and the potential for the emergence of anthelminthic drug resistance threaten the sustainability of mass drug administration as the only form of control. Hence, there is a strong rationale for developing a vaccine that simultaneously targets both hookworms and schistosomes because of similarities in the pathobiology of both parasites, the ability of both helminths to cause anemia and their coendemicity in sub-Saharan Africa, Brazil and East Asia. A multivalent anthelminthic vaccine for hookworm infection and schistosomiasis would represent an important new tool for combating disease and poverty.

  16. Multivalent chelators for spatially and temporally controlled protein functionalization.

    PubMed

    You, Changjiang; Piehler, Jacob

    2014-05-01

    Site-specific protein modification-e.g. for immobilization or labelling-is a key prerequisite for numerous bioanalytical applications. Although modification by use of short peptide tags is particularly attractive, efficient and bio-orthogonal systems are still lacking. Here, we review the application of multivalent chelators (MCH) for high-affinity yet reversible recognition of oligohistidine (His)-tagged proteins. MCH are based on multiple nitrilotriacetic acid (NTA) moieties grafted on to molecular scaffolds suitable for conjugation to surfaces, probes or other biomolecules. Reversible interaction with the His-tag is mediated via transition metal ions chelated by the NTA moieties. The small size and biochemical compatibility of these recognition units and the possibility of rapid dissociation of the interaction with His-tagged proteins despite sub-nanomolar binding affinity, enable distinct and versatile handling and modification of recombinant proteins. In this review, we briefly introduce the key principles and features of MCH-His-tag interactions and recapitulate the broad spectrum of bioanalytical applications with a focus on quantitative protein interaction analysis on micro or nano-patterned solid surfaces and specific protein labelling in living cells. PMID:24770786

  17. Multivalent Antigens for Promoting B and T Cell Activation

    PubMed Central

    Bennett, Nitasha R.; Zwick, Daniel B.; Courtney, Adam H.; Kiessling, Laura L.

    2015-01-01

    Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines. PMID:25970017

  18. Radioiodination and biodistribution of the monoclonal antibody TU-20 and its scFv fragment

    NASA Astrophysics Data System (ADS)

    Kubaštová, H.; Kleinova, V.; Seifert, D.; Fišer, M.; Kranda, K.

    2006-01-01

    The ability of the monoclonal antibody TU-20 and its scFv fragment to specifically bind to the C-end of the class III beta-tubulin makes these preparations useful as potential diagnostics for in vivo determination of neurodegenerative diseases that entail degradation of neuronal cytoskeleton. To examine this hypothesis, TU-20 and its scFv were labelled with 125I and their properties were extensively investigated. TU-20 and its scFv were labelled via chloramine-T with the yield 90 95% and 64 78%, respectively. Their quality control, performed by an ELISA and gel electrophoresis, determined adequate properties for further studies. The in vitro experiment, involving autoradiography and immunohistochemistry of mice’ brain slices, enabled confirmation of preserved immunospecificity of the radiolabelled substances. Finally, the in vivo biodistribution proved differences in elimination of either TU-20, scFv TU-20, or iodide from the mice.

  19. Expression of recombinant human anti-TNF-α scFv-Fc in Arabidopsis thaliana seeds.

    PubMed

    Yao, N; Ai, L; Dong, Y Y; Liu, X M; Wang, D Z; Wang, N; Li, X W; Wang, F W; Li, Xk; Li, H Y; Jiang, C

    2016-01-01

    Recombinant human anti-tumor necrosis factor (TNF)-α scFv-Fc was expressed in TKO mutant Arabidopsis thaliana seeds using plant-specific codons. Immunoblotting using a human IgG1 antibody detected the expression of anti-TNF-α proteins in plants. Results from qRT-PCR analysis demonstrated that the time of harvest significantly affected the protein yield and quality. Our results indicate that the Phaseolus vulgaris β-phaseolin promoter directed anti-TNF-α scFv-Fc expression in A. thaliana seeds, with a maximum yield obtained at 20-days of development. Although the yield of anti-TNF-α scFv-Fc protein was not very high, accumulation of recombinant proteins in seeds is an attractive and simple method that can be used to purify biologically active anti-TNF-α scFv-Fc. PMID:27420937

  20. Multivalent ligands control stem cell behaviour in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Conway, Anthony; Vazin, Tandis; Spelke, Dawn P.; Rode, Nikhil A.; Healy, Kevin E.; Kane, Ravi S.; Schaffer, David V.

    2013-11-01

    There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.

  1. Direct production of Fv-fragments from a family of monoclonal IgGs papain digestion.

    PubMed

    Ornatowska, M; Glasel, J A

    1991-01-01

    Fv fragments of four monoclonal antibodies specific for morphine binding have been produced from their divalent IgG forms by papain digestion using the classic procedure for Fab formation. The binding characteristics of one of the Fv fragments have been determined relative to the intact antibody by equilibrium dialysis. Its dissociation constant is a factor of five lower than the IgG. Previous work had resulted in the sequences of each the chains for the four Fv fragments. The light chains are all from the highly homologous lambda subclass while the gamma heavy chains are closely related except for their CDR regions. In this work optical molar extinction coefficients are predicted from amino acid sequences for each of the fragments. It is found that they differ significantly from each other and from the commonly used value for intact IgG. Detailed comparisons between our results and those reported previously on the molecular masses of Fv-derived light and heavy chains and hapten-Fv dissociation constants are given based on analytical gel electrophoresis and electroblotting experiments using dye and immunovisualization techniques. Isoelectric focusing experiments have been performed and the pIs obtained are compared to those predicted theoretically from the chain sequences. Gel filtration column chromatography, acrylamide gel electrophoresis and equilibrium dialysis experiments are consistent with significant aggregation of the Fv fragments in neutral solution with accompanying inactivation of the binding site. Comparison of sequences for the Fv light and heavy chains are made with those which have been proposed to be important for chain dimer association and for canonical hypervariable regions. This methods of Fv production is not regarded as a general one. However, it may be an approach which is general to lambda chain containing antibodies.

  2. Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

    PubMed Central

    Goldschmidt, Jürgen; Pethe, Annette; Hagemeyer, Christoph E.; Neudorfer, Irene; Zirlik, Andreas; Weber, Wolfgang A.; Bode, Christoph; Meyer, Philipp T.

    2011-01-01

    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/mm2, 4010±630 DLU/mm2 and 4520±293 DLU/mm2) produced a significantly higher ligand uptake compared to 111In-control (2101±76 DLU/mm2, 1181±96 DLU/mm2 and 1866±246 DLU/mm2) 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/mm2 vs. 17390±7470 DLU/mm2; 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 activated platelets in plaque pathology and atherosclerosis

  3. Effect of the Fv-1 locus on the titration of murine leukemia viruses.

    PubMed

    Jolicoeur, P; Baltimore, D

    1975-12-01

    Titration of N- and B-tropic murine leukemia viruses on sensitive and resistant cell lines has been studied by direct XC plaque assay and infective center assay. The titration of cloned B-tropic virus by infective center assay on BALB/3T3 (Fv-1b/b) and NIH/3T3 (Fv-1n/n) cells gave one-hit patterns, with 100-fold less infected NIH/3T3 cells than BALB/3T3 cells. The titration of B-tropic virus on DBA/2 cells (Fv-1n/n) was also a one-hit. The titration of a one-hit curve, and there were about 100-fold less infected BALB/3T3 cells than NIH/3T3 cells. Comparable results were obtained by titrating the cloned N-tropic virus on congenic SIM (Fv-1n/n) and SIM.R (Fv-1b/b) cells or the Gross N-tropic virus on BALB/3T3 cells. Therefore, our data indicate that the multiple-hit phenomenon described previously may not be an essential part of the Fv-1 gene restriction.

  4. Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats

    PubMed Central

    Zhan, Xinhua; Ander, Bradley P; Liao, Isaac H; Hansen, James E; Kim, Chester; Clements, Douglas; Weisbart, Richard H; Nishimura, Robert N; Sharp, Frank R

    2010-01-01

    Background and Purpose This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior following experimental ischemic stroke. Methods Focal cerebral ischemia was produced by occluding the middle cerebral artery (MCA) using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 h. At 2 ¼ h and 3 h after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg / kg) or saline was injected via the tail vein. Sensory-motor function and infarction volume were assessed at 24 h following ischemia. Results Administration of Fv-Hsp70 following focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensory-motor function compared to the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. Conclusion Fv-Hsp70 protects ischemic brain in this experimental stroke model. PMID:20075343

  5. Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

    PubMed Central

    Song, Hyung-Nam; Jang, Jun-Hyuck; Kim, Young-Wan; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2014-01-01

    Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VH–VL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. PMID:25530617

  6. Controlled multivalent interactions in the inhibition of toxins via well-designed glycopolypeptides

    NASA Astrophysics Data System (ADS)

    Maheshwari, Ronak

    Many critical recognition events in biology are mediated via multivalent interactions between multiple saccharide ligands and their protein receptors. These proteincarbohydrate interactions are therefore important and being extensively investigated as they play a crucial role in several processes including pathogen recognition, inflammation, cell signaling, differentiation, and adhesion of various bacterial toxins. Multiple research groups have investigated these interactions by developing multivalent polymeric antagonists for carbohydrate binding proteins. In our work, we have selected cholera toxin (CT) as a model example to study these multivalent bindings by developing multivalent inhibitors. Various investigations have employed diverse guidelines that are believed to govern multivalency in the design of inhibitors for CT-GM1 interactions. Although successful in many respects, they are limited by certain architectural features such as a lack of synthetic versatility, significant polydispersity, and uncontrolled density and arrangement of saccharide ligands. Thus the mechanism by which multivalency is functioning in these systems is impractical to analyze and control. A more detailed understanding of multivalent binding by polymeric materials therefore requires the development of well-designed glycopolymers in which architectural features are well defined and controlled. Our approach aims to develop polymers via protein engineering methods and to equip these polypeptides with multivalent sugar ligands via chemical methods, to competitively bind with such toxins and neutralize them. This method allows control over architectural features such as number and spacing of saccharide ligands on the polymer, precise placement of charges and conformation of the polymer backbone. Such control over the architectural features allows for more purposeful design of polymers for inhibition of the multivalent binding event. Polypeptides with chemically reactive natural or non

  7. Cyclic Peptide-Decorated Self-Assembled Nanohybrids for Selective Recognition and Detection of Multivalent RNAs.

    PubMed

    Choi, Jun Shik; Han, So-hee; Kim, Hyoseok; Lim, Yong-Beom

    2016-03-16

    Although there has been substantial advancement in the development of nanostructures, the development of self-assembled nanostructures that can selectively recognize multivalent targets has been very difficult. Here we show the proof of concept that topology-controlled peptide nanoassemblies can selectively recognize and detect a multivalent RNA target. We compared the differential behaviors of peptides in a linear or cyclic topology in terms of peptide-gold nanoparticle hybrid nanostructure formation, conformational stabilization, monovalent and multivalent RNA binding in vitro, and multivalent RNA recognition in live cells. When the topology-dependent selectivity amplification of the cyclic peptide hybrids is combined with the noninvasive nature of dark-field microscopy, the cellular localization of the viral Rev response element (RRE) RNA can be monitored in situ. Because intracellular interactions are often mediated by overlapping binding partners with weak affinity, the topology-controlled peptide assemblies can provide a versatile means to convert weak ligands into multivalent ligands with high affinity and selectivity.

  8. A Uranium-Based UO2+–Mn2+ Single-Chain Magnet Assembled trough Cation–Cation Interactions**

    PubMed Central

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; de Geyer, Arnaud; Pécaut, Jacques; Mazzanti, Marinella

    2014-01-01

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f–3d heterometallic 1D chains [{[UO2(salen)(py)][M(py)4](NO3)}]n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation–cation interaction from the reaction of the uranyl(V) complex [UO2(salen)py][Cp*2Co] (Cp*=pentamethylcyclopentadienyl) with Cd(NO3)2 or Mn(NO3)2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134±0.8 K (93±0.5 cm−1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T<6 K, with an impressive coercive field of 3.4 T at 2 K. PMID:24311434

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

    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

  11. The O-P-O bridged Mn2(salen)2 chains showing coexistence of single chain magnet and metamagnet behaviour.

    PubMed

    Wang, Ting-Ting; Ren, Min; Bao, Song-Song; Cai, Zhong-Sheng; Liu, Bin; Zheng, Ze-Hua; Xu, Zhong-Li; Zheng, Li-Min

    2015-03-01

    Three new chain compounds in which the Mn2(salen)2 dimers are bridged by O-P-O units are reported, namely, [Mn2(salen)2(C6H9PO3H)](ClO4) (1), [Mn2(salen)2(C6H5PO3H)](ClO4) (2) and [Mn2(salen)2(C6H5PHO2)](ClO4) (3). The phosphonate or phosphinate ligands adopt a syn-anti bidentate bridging mode in 1, while a syn-syn bidentate bridging mode in 2 and 3, thus leading to a difference in the Mn-O···O-Mn torsion angle over the O-P-O bridge. Compound 1 shows a paramagnetic behavior with dominant antiferromagnetic interactions. In compounds 2 and 3, the antiferromagnetic interactions through the O-P-O bridges are considerably stronger than those in 1. They display coexistence of single chain magnet (SCM) behaviour with a spin canted structure and metamagnetism at low temperature. The results demonstrate that the magnetic dynamics of the O-P-O bridged Mn2(salen)2 chains may be modulated by selecting suitable phosphonate or phosphinate ligands.

  12. Molecular characterization and systemic induction of single-chain ribosome-inactivating proteins (RIPs) in sugar beet (Beta vulgaris) leaves.

    PubMed

    Iglesias, Rosario; Pérez, Yolanda; de Torre, Carlos; Ferreras, J Miguel; Antolín, Pilar; Jiménez, Pilar; Rojo, M Angeles; Méndez, Enrique; Girbés, Tomás

    2005-06-01

    Sugar beet (Beta vulgaris L.) leaves contain virus-inducible type 1 (single chain) ribosome-inactivating proteins that have been named beetins. The structural and functional characterization, the cellular location, and the potential role of beetins as antiviral agents are reported here. Beetins are formed of a single polypeptide chain with a varying degree of glycosylation and strongly inhibited in vitro protein synthesis in rabbit reticulocyte lysates (IC50=1.15 ng ml(-1)) and a Vicia sativa L. cell-free system (IC50=68 ng ml(-1)) through the single depurination of the large rRNA. Beetins trigger the multidepurination of tobacco mosaic virus (TMV) genomic RNA which underwent extensive degradation upon treatment with acid aniline. Beetins are extracellular proteins that were recovered from the apoplastic fluid. Induction of sugar beet RIPs with either H2O2 or artichoke mottled crinkle virus (AMCV) was observed in leaves distant from the site of application of such elicitors. The external application of purified beetin to sugar leaves prevented infection by AMCV which supports the preliminary hypothesis that beetins could be involved in plant systemic acquired resistance subjected to induction by phytopathogens.

  13. Amino-Terminal Extended Peptide Single-Chain Trimers are Potent Synthetic Agonists for Memory Human CD8+ T cells

    PubMed Central

    Carreno, Beatriz M.; Becker-Hapak, Michelle; Chan, Megan; Lie, Wen-Rong; Wang, Xiaoli; Hansen, Ted H.; Linette, Gerald P.

    2012-01-01

    Upon antigen exposure, most memory T cells undergo re-stimulation induced cell death. Here we describe a novel synthetic agonist, an amino-terminal extended decamer peptide expressed as a single chain trimer, the AT-SCT, that preferentially promotes the growth of memory human CD8+ T cells with minimal re-stimulation-induced cell death. Using the CMV pp65 and melanoma gp100 antigens, we observe the in vitro numerical expansion of a clonally diverse poly-functional population of antigen-specific CD8+ T cells from normal individuals and vaccinated melanoma patients, respectively. Memory CD8+ T cells stimulated with AT-SCT presented on MHC class I/II null cells show reduced cytokine production, slower kinetics of TCR down-regulation and decreased cell death when compared to native nonamer SCT-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC (p-MHC) complexes using fluorochrome-conjugated TCR multimers suggest that nonamer and decamer-linked peptides may be anchored differently to HLA-A2 peptide binding groove. Our findings demonstrate that modified p-MHC structures such as AT-SCT can be engineered as T cell agonists to promote the growth and expansion of memory human CD8+ T cells. PMID:22573808

  14. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase

    PubMed Central

    Tran, Timothy H.; Hsiao, Yu-Shan; Jo, Jeanyoung; Chou, Chi-Yuan; Dietrich, Lars E.P.; Walz, Thomas; Tong, Liang

    2014-01-01

    Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds 1–6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase (ACC) is a target for drug discovery against diabetes, cancer and other diseases 7–9. We report here the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kD), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active toward short- and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase (LCC). The holoenzyme is a homo-hexamer with molecular weight of 720 kD. The 3.0 Å crystal structure of Mycobacterium avium subspecies paratuberculosis LCC (MapLCC) holoenzyme revealed an architecture that is strikingly different compared to those of related biotin-dependent carboxylases 10,11. In addition, the domains of each monomer have no direct contacts with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources. PMID:25383525

  15. Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue

    NASA Astrophysics Data System (ADS)

    Lee, Hyun Chul; Kim, Su-Jin; Kim, Kyung-Sup; Shin, Hang-Cheol; Yoon, Ji-Won

    2000-11-01

    A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of β cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic β cells by autoimmune responses specific to β cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic β cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

  16. Crystal structures of ricin toxin's enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies.

    PubMed

    Rudolph, Michael J; Vance, David J; Cheung, Jonah; Franklin, Matthew C; Burshteyn, Fiana; Cassidy, Michael S; Gary, Ebony N; Herrera, Cristina; Shoemaker, Charles B; Mantis, Nicholas J

    2014-08-26

    Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines. PMID:24907552

  17. The Significance of Multivalent Bonding Motifs and "Bond Order" in DNA-Directed Nanoparticle Crystallization.

    PubMed

    Thaner, Ryan V; Eryazici, Ibrahim; Macfarlane, Robert J; Brown, Keith A; Lee, Byeongdu; Nguyen, SonBinh T; Mirkin, Chad A

    2016-05-18

    Multivalent oligonucleotide-based bonding elements have been synthesized and studied for the assembly and crystallization of gold nanoparticles. Through the use of organic branching points, divalent and trivalent DNA linkers were readily incorporated into the oligonucleotide shells that define DNA-nanoparticles and compared to monovalent linker systems. These multivalent bonding motifs enable the change of "bond strength" between particles and therefore modulate the effective "bond order." In addition, the improved accessibility of strands between neighboring particles, either due to multivalency or modifications to increase strand flexibility, gives rise to superlattices with less strain in the crystallites compared to traditional designs. Furthermore, the increased availability and number of binding modes also provide a new variable that allows previously unobserved crystal structures to be synthesized, as evidenced by the formation of a thorium phosphide superlattice. PMID:27148838

  18. Opposing Effects of Multivalent Ions on the Flexibility of DNA and RNA

    NASA Astrophysics Data System (ADS)

    Drozdetski, Aleksander V.; Tolokh, Igor S.; Pollack, Lois; Baker, Nathan; Onufriev, Alexey V.

    2016-07-01

    Increasing the concentration of counterions (salt) is known to reduce the bending persistence length of DNA. Here we use atomistic molecular dynamics simulations to predict that multivalent counterions have the opposite effect on double-stranded RNA, increasing its bending rigidity by at least 30%. This counterintuitive effect is observed for various tri- and tetravalent ions alike, and is robust to methodological details and the RNA sequence. In contrast to DNA, multivalent counterions bind inside the RNA major groove, causing significant contraction of the molecule along its helical axis—as a result, its further deformation due to bending becomes energetically more expensive compared to bending without bound multivalent ions. Thus, the relationship between mechanical properties of a charged polymer and its ionic atmosphere may be richer than previously thought.

  19. Opposing Effects of Multivalent Ions on the Flexibility of DNA and RNA.

    PubMed

    Drozdetski, Aleksander V; Tolokh, Igor S; Pollack, Lois; Baker, Nathan; Onufriev, Alexey V

    2016-07-01

    Increasing the concentration of counterions (salt) is known to reduce the bending persistence length of DNA. Here we use atomistic molecular dynamics simulations to predict that multivalent counterions have the opposite effect on double-stranded RNA, increasing its bending rigidity by at least 30%. This counterintuitive effect is observed for various tri- and tetravalent ions alike, and is robust to methodological details and the RNA sequence. In contrast to DNA, multivalent counterions bind inside the RNA major groove, causing significant contraction of the molecule along its helical axis-as a result, its further deformation due to bending becomes energetically more expensive compared to bending without bound multivalent ions. Thus, the relationship between mechanical properties of a charged polymer and its ionic atmosphere may be richer than previously thought. PMID:27447528

  20. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    PubMed Central

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  1. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

    PubMed

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I; Fischer, Kathelijn; Gill, Joan C; P'Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St Ledger, Katie; Pabinger, Ingrid

    2016-08-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

  2. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

    PubMed

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I; Fischer, Kathelijn; Gill, Joan C; P'Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St Ledger, Katie; Pabinger, Ingrid

    2016-08-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  3. Expression, purification and characterization of B72.3 Fv fragments.

    PubMed Central

    King, D J; Byron, O D; Mountain, A; Weir, N; Harvey, A; Lawson, A D; Proudfoot, K A; Baldock, D; Harding, S E; Yarranton, G T

    1993-01-01

    The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv. Images Figure 1 Figure 2 PMID:8457200

  4. Spinel compounds as multivalent battery cathodes: A systematic evaluation based on ab initio calculations

    DOE PAGES

    Liu, Miao; Rong, Ziqin; Malik, Rahul; Canepa, Pieremanuele; Jain, Anubhav; Ceder, Gerbrand; Persson, Kristin A.

    2014-12-16

    In this study, batteries that shuttle multivalent ions such as Mg2+ and Ca2+ ions are promising candidates for achieving higher energy density than available with current Li-ion technology. Finding electrode materials that reversibly store and release these multivalent cations is considered a major challenge for enabling such multivalent battery technology. In this paper, we use recent advances in high-throughput first-principles calculations to systematically evaluate the performance of compounds with the spinel structure as multivalent intercalation cathode materials, spanning a matrix of five different intercalating ions and seven transition metal redox active cations. We estimate the insertion voltage, capacity, thermodynamic stabilitymore » of charged and discharged states, as well as the intercalating ion mobility and use these properties to evaluate promising directions. Our calculations indicate that the Mn2O4 spinel phase based on Mg and Ca are feasible cathode materials. In general, we find that multivalent cathodes exhibit lower voltages compared to Li cathodes; the voltages of Ca spinels are ~0.2 V higher than those of Mg compounds (versus their corresponding metals), and the voltages of Mg compounds are ~1.4 V higher than Zn compounds; consequently, Ca and Mg spinels exhibit the highest energy densities amongst all the multivalent cation species. The activation barrier for the Al³⁺ ion migration in the Mn₂O₄ spinel is very high (~1400 meV for Al3+ in the dilute limit); thus, the use of an Al based Mn spinel intercalation cathode is unlikely. Amongst the choice of transition metals, Mn-based spinel structures rank highest when balancing all the considered properties.« less

  5. Conformational transitions of flexible hydrophobic polyelectrolytes in solutions of monovalent and multivalent salts and their mixtures.

    PubMed

    Trotsenko, Oleksandr; Roiter, Yuri; Minko, Sergiy

    2012-04-10

    Conformations of cationic polyelectrolytes (PEs), a weak poly(2-vinylpyridine) (P2VP) and a strong poly(N-methyl-2-vinylpyridinium iodide) (qP2VP), adsorbed on mica from saline solutions in the presence of counterions of different valences are studied using in situ atomic force microscopy (AFM). Quantitative characteristics of chain conformations are analyzed using AFM images of the adsorbed molecules. The results of the statistical analysis of the chain contour reveal collapse of the PE coils when ionic strength is in a range from tens to hundreds of millimoles per kilogram and re-expansion of the coils with a further increase of ionic strength up to a region of the saturated saline solutions. The competition between monovalent and multivalent counterions simultaneously present in solutions strongly affects conformations of PE chains even at a very small fraction of multivalent counterions. Shrinkage of PE coils is steeper for multivalent counterions than for monovalent counterions. However, the re-expansion is only incremental in the presence of multivalent counterions. Extended adsorbed coils at low salt concentrations and at very high concentrations of monovalent salt exhibit conformation corresponding to a 2D coil with 0.95 fraction of bound segments (segments in "trains") in the regime of diluted surface concentration of the PE. Shrunken coils in the intermediate range of ionic strength resemble 3D-globules with 0.8 fraction of trains. The incrementally re-expanded PE coils at a high ionic strength remain unchanged at higher multivalent salt concentrations up to the solubility limit of the salt. The formation of a strong PE complex with multivalent counterions at high ionic strength is not well understood yet. A speculative explanation of the observed experimental result is based on possible stabilization of the complex due to hydrophobic interactions of the backbone.

  6. Tunable Graphitic Carbon Nano-Onions Development in Carbon Nanofibers for Multivalent Energy Storage

    SciTech Connect

    Schwarz, Haiqing L.

    2016-01-01

    We developed a novel porous graphitic carbon nanofiber material using a synthesis strategy combining electrospinning and catalytic graphitization. RF hydrogel was used as carbon precursors, transition metal ions were successfully introduced into the carbon matrix by binding to the carboxylate groups of a resorcinol derivative. Transition metal particles were homogeneously distributed throughout the carbon matrix, which are used as in-situ catalysts to produce graphitic fullerene-like nanostructures surrounding the metals. The success design of graphitic carbons with enlarged interlayer spacing will enable the multivalent ion intercalation for the development of multivalent rechargeable batteries.

  7. Iminosugar-Cyclopeptoid Conjugates Raise Multivalent Effect in Glycosidase Inhibition at Unprecedented High Levels.

    PubMed

    Lepage, Mathieu L; Schneider, Jérémy P; Bodlenner, Anne; Meli, Alessandra; De Riccardis, Francesco; Schmitt, Marjorie; Tarnus, Céline; Nguyen-Huynh, Nha-Thi; Francois, Yannis-Nicolas; Leize-Wagner, Emmanuelle; Birck, Catherine; Cousido-Siah, Alexandra; Podjarny, Alberto; Izzo, Irene; Compain, Philippe

    2016-04-01

    A series of cyclopeptoid-based iminosugar clusters has been evaluated to finely probe the ligand content-dependent increase in α-mannosidase inhibition. This study led to the largest binding enhancement ever reported for an enzyme inhibitor (up to 4700-fold on a valency-corrected basis), which represents a substantial advance over the multivalent glycosidase inhibitors previously reported. Electron microscopy imaging and analytical data support, for the best multivalent effects, the formation of a strong chelate complex in which two mannosidase molecules are cross-linked by one inhibitor. PMID:26917097

  8. Incorporation of a calixarene-based glucose functionalised bolaamphiphile into lipid bilayers for multivalent lectin recognition.

    PubMed

    Aleandri, S; Casnati, A; Fantuzzi, L; Mancini, G; Rispoli, G; Sansone, F

    2013-08-01

    The synthesis, characterization and inclusion in liposomes of a glucosylated bolaamphiphile built on a calix[4]arene scaffold are described. The new glucocalixarene bolaamphiphile destabilizes bilayers of saturated lipids whereas it rigidifies those of unsaturated lipids, thus reducing leakage of calcein from the liposome internal aqueous compartment. Moreover, from fluorescence and turbidimetry experiments it was found that the glucose units of bolaamphiphile 1 functionalised liposomes allow a specific multivalent interaction with the tetrameric glucose binding protein Concanavalin A. These results therefore represent a novel strategy to functionalise liposomes with saccharides, exploiting multivalent glycosylated ligands to be used in the preparation of drug delivery systems potentially able to target specific lectins. PMID:23771589

  9. Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin.

    PubMed

    Sahasrabuddhe, Anagh A; Gaikwad, Sushama M; Krishnasastry, M V; Khan, M Islam

    2004-12-01

    Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl-alpha-galactose (Ka: 2.48 x 10(2)) in comparison to nJacalin (Ka: 1.58 x 10(4)), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galbeta1-3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Gal

  10. Annual parallax and a dimming event of a Mira variable star, FV Bootis

    NASA Astrophysics Data System (ADS)

    Kamezaki, Tatsuya; Nakagawa, Akiharu; Omodaka, Toshihiro; Inoue, Kan-ichiro; Chibueze, James O.; Nagayama, Takumi; Ueno, Yuji; Matsunaga, Noriyuki

    2016-10-01

    We present the first measurement of the trigonometric parallax of water masers associated with a Mira star, FV Bootis (FV Boo) using VLBI Exploration of Radio Astrometry (VERA). Based on our multi-epoch VERA observations, we derived the parallax to be 0.97 ± 0.06 mas, which corresponds to a distance of 1.03^{+0.07}_{-0.06} kpc. The water masers around FV Boo were spatially distributed over an area of 41 au × 41 au, and their internal motions indicate the presence of an outflow. Using the Kagoshima University 1 m optical/infrared telescope, we determined the period to be 305.6 d and the mean apparent magnitude to be +2.91 mag in the K'-band. On the period-luminosity plane, the obtained period and K'-band magnitude puts FV Boo slightly below the sequence of Miras, possibly due to circumstellar reddening. Combining our photometric data with COBE and 2MASS datasets spanning over 20 years, we found in the near infrared that FV Boo was significantly fainter in 2005 compared with preceding and later phases. Its color, however, did not show a large variation through this change. We infer that the dimming could be caused by an eclipse due to a cloud in a binary system.

  11. Annual parallax and a dimming event of a Mira variable star, FV Bootis

    NASA Astrophysics Data System (ADS)

    Kamezaki, Tatsuya; Nakagawa, Akiharu; Omodaka, Toshihiro; Inoue, Kan-ichiro; Chibueze, James O.; Nagayama, Takumi; Ueno, Yuji; Matsunaga, Noriyuki

    2016-08-01

    We present the first measurement of the trigonometric parallax of water masers associated with a Mira star, FV Bootis (FV Boo) using VLBI Exploration of Radio Astrometry (VERA). Based on our multi-epoch VERA observations, we derived the parallax to be 0.97 ± 0.06 mas, which corresponds to a distance of 1.03^{+0.07}_{-0.06} kpc. The water masers around FV Boo were spatially distributed over an area of 41 au × 41 au, and their internal motions indicate the presence of an outflow. Using the Kagoshima University 1 m optical/infrared telescope, we determined the period to be 305.6 d and the mean apparent magnitude to be +2.91 mag in the K'-band. On the period-luminosity plane, the obtained period and K'-band magnitude puts FV Boo slightly below the sequence of Miras, possibly due to circumstellar reddening. Combining our photometric data with COBE and 2MASS datasets spanning over 20 years, we found in the near infrared that FV Boo was significantly fainter in 2005 compared with preceding and later phases. Its color, however, did not show a large variation through this change. We infer that the dimming could be caused by an eclipse due to a cloud in a binary system.

  12. Isolation of anti-T cell receptor scFv mutants by yeast surface display.

    PubMed

    Kieke, M C; Cho, B K; Boder, E T; Kranz, D M; Wittrup, K D

    1997-11-01

    Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

  13. A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.

    PubMed

    Kramer, Karl; Fiedler, Markus; Skerra, Arne; Hock, Bertold

    2002-04-01

    Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

  14. Clinical Applications of Gamma Delta T Cells with Multivalent Immunity

    PubMed Central

    Deniger, Drew C.; Moyes, Judy S.; Cooper, Laurence J. N.

    2014-01-01

    γδ T cells hold promise for adoptive immunotherapy because of their reactivity to bacteria, viruses, and tumors. However, these cells represent a small fraction (1–5%) of the peripheral T-cell pool and require activation and propagation to achieve clinical benefit. Aminobisphosphonates specifically expand the Vγ9Vδ2 subset of γδ T cells and have been used in clinical trials of cancer where objective responses were detected. The Vγ9Vδ2 T cell receptor (TCR) heterodimer binds multiple ligands and results in a multivalent attack by a monoclonal T cell population. Alternatively, populations of γδ T cells with oligoclonal or polyclonal TCR repertoire could be infused for broad-range specificity. However, this goal has been restricted by a lack of applicable expansion protocols for non-Vγ9Vδ2 cells. Recent advances using immobilized antigens, agonistic monoclonal antibodies (mAbs), tumor-derived artificial antigen presenting cells (aAPC), or combinations of activating mAbs and aAPC have been successful in expanding gamma delta T cells with oligoclonal or polyclonal TCR repertoires. Immobilized major histocompatibility complex Class-I chain-related A was a stimulus for γδ T cells expressing TCRδ1 isotypes, and plate-bound activating antibodies have expanded Vδ1 and Vδ2 cells ex vivo. Clinically sufficient quantities of TCRδ1, TCRδ2, and TCRδ1negTCRδ2neg have been produced following co-culture on aAPC, and these subsets displayed differences in memory phenotype and reactivity to tumors in vitro and in vivo. Gamma delta T cells are also amenable to genetic modification as evidenced by introduction of αβ TCRs, chimeric antigen receptors, and drug-resistance genes. This represents a promising future for the clinical application of oligoclonal or polyclonal γδ T cells in autologous and allogeneic settings that builds on current trials testing the safety and efficacy of Vγ9Vδ2 T cells. PMID:25566249

  15. Clinical applications of gamma delta T cells with multivalent immunity.

    PubMed

    Deniger, Drew C; Moyes, Judy S; Cooper, Laurence J N

    2014-01-01

    γδ T cells hold promise for adoptive immunotherapy because of their reactivity to bacteria, viruses, and tumors. However, these cells represent a small fraction (1-5%) of the peripheral T-cell pool and require activation and propagation to achieve clinical benefit. Aminobisphosphonates specifically expand the Vγ9Vδ2 subset of γδ T cells and have been used in clinical trials of cancer where objective responses were detected. The Vγ9Vδ2 T cell receptor (TCR) heterodimer binds multiple ligands and results in a multivalent attack by a monoclonal T cell population. Alternatively, populations of γδ T cells with oligoclonal or polyclonal TCR repertoire could be infused for broad-range specificity. However, this goal has been restricted by a lack of applicable expansion protocols for non-Vγ9Vδ2 cells. Recent advances using immobilized antigens, agonistic monoclonal antibodies (mAbs), tumor-derived artificial antigen presenting cells (aAPC), or combinations of activating mAbs and aAPC have been successful in expanding gamma delta T cells with oligoclonal or polyclonal TCR repertoires. Immobilized major histocompatibility complex Class-I chain-related A was a stimulus for γδ T cells expressing TCRδ1 isotypes, and plate-bound activating antibodies have expanded Vδ1 and Vδ2 cells ex vivo. Clinically sufficient quantities of TCRδ1, TCRδ2, and TCRδ1(neg)TCRδ2(neg) have been produced following co-culture on aAPC, and these subsets displayed differences in memory phenotype and reactivity to tumors in vitro and in vivo. Gamma delta T cells are also amenable to genetic modification as evidenced by introduction of αβ TCRs, chimeric antigen receptors, and drug-resistance genes. This represents a promising future for the clinical application of oligoclonal or polyclonal γδ T cells in autologous and allogeneic settings that builds on current trials testing the safety and efficacy of Vγ9Vδ2 T cells. PMID:25566249

  16. Biodistribution of the Radiolabeled Anti III {beta}-Tubulin scFv Fragment in Mice

    SciTech Connect

    Kleinova, Veronika; Svecova, H.; Chaloupkova, H.; Kranda, K.; Fiser, M.

    2007-11-26

    For studies of new potential radiopharmaceutical such as radiolabeled compound, the biodistribution exoeriments are essential to describe behavior of the substance in organism. The specific binding of the scFv fragment of the monoclonal antibody TU-20 to the C-end of the class III {beta}-tubulin makes this substance useful as a potential diagnostics for in vivo neurodegenerative diseases determination. To examine this hypothesis, scFv was radio-labeled with {sup 125}I and {sup 123}I, and its biochemical properties were studied. The in vivo bio-distribution confirmed the expected elimination behavior of the radio-labeled scFv TU-20 in mice. The bi-exponential model for two-phase clearance to determine short phase half-life t{sub 1/2{alpha}} and long phase half-life t{sub 1/2{beta}} values was used to evaluate the experimental data.

  17. Multivalency governs HP1α association dynamics with the silent chromatin state

    PubMed Central

    Kilic, Sinan; Bachmann, Andreas L.; Bryan, Louise C.; Fierz, Beat

    2015-01-01

    Multivalent interactions between effector proteins and histone post-translational modifications are an elementary mechanism of dynamic chromatin signalling. Here we elucidate the mechanism how heterochromatin protein 1α (HP1α), a multivalent effector, is efficiently recruited to the silent chromatin state (marked by trimethylated H3 at Lys9, H3K9me3) while remaining highly dynamic. Employing chemically defined nucleosome arrays together with single-molecule total internal reflection fluorescence microscopy (smTIRFM), we demonstrate that the HP1α residence time on chromatin depends on the density of H3K9me3, as dissociated factors can rapidly rebind at neighbouring sites. Moreover, by chemically controlling HP1α dimerization we find that effector multivalency prolongs chromatin retention and, importantly, accelerates the association rate. This effect results from increased avidity together with strengthened nonspecific chromatin interactions of dimeric HP1α. We propose that accelerated chromatin binding is a key feature of effector multivalency, allowing for fast and efficient competition for binding sites in the crowded nuclear compartment. PMID:26084584

  18. Multivalent drug design and inhibition of cholera toxin by specific and transient protein-ligand interactions.

    PubMed

    Liu, Jiyun; Begley, Darren; Mitchell, Daniel D; Verlinde, Christophe L M J; Varani, Gabriele; Fan, Erkang

    2008-05-01

    Multivalent inhibitors of the cholera toxin B pentamer are potential therapeutic drugs for treating cholera and serve as models for demonstrating multivalent ligand effects through a structure-based approach. A crucial yet often overlooked aspect of multivalent drug design is the length, rigidity and chemical composition of the linker used to connect multiple binding moieties. To specifically study the role of chemical linkers in multivalent ligand design, we have synthesized a series of compounds with one and two binding motifs connected by several different linkers. These compounds have affinity for and potency against the cholera toxin B pentamer despite the fact that none can simultaneously bind two toxin receptor sites. Results from saturation transfer difference NMR reveal transient, non-specific interactions between the cholera toxin and linker groups contribute significantly to overall binding affinity of monovalent compounds. However, the same random protein-ligand interactions do not appear to affect binding of bivalent molecules. Moreover, the binding affinities and potencies of these 'non-spanning' bivalent ligands appear to be wholly independent of linker length. Our detailed analysis identifies multiple effects that account for the improved inhibitory potencies of bivalent ligands and suggest approaches to further improve the activity of this class of compounds.

  19. Multivalent inhibitors for carbohydrate-processing enzymes: beyond the "lock-and-key" concept.

    PubMed

    Gouin, Sébastien G

    2014-09-01

    During the last decades, tremendous chemical efforts have been dedicated to design monovalent inhibitors of carbohydrate-processing enzymes, with comparatively few rewards in terms of marketed drugs. Recently, an alternative to the traditional "lock and key" approach has emerged. Multivalency, a widely used strategy for lectin inhibition, has been successfully applied to specific glycosidases and glycosyltransferases.

  20. The influence of multivalent cations on the flocculation of activated sludge with different sludge retention times.

    PubMed

    Li, Haisong; Wen, Yue; Cao, Asheng; Huang, Jingshui; Zhou, Qi

    2014-05-15

    The mechanism governing the flocculation of activated sludge (AS) with different sludge retention times (SRTs) was studied in this paper. AS samples were cultivated in 8 lab-scale reactors with SRTs of 5 d, 7.5 d, 10 d, 12.5 d, 15 d, 20 d, 30 d, and 40 d. The bulk solution, loosely bound extracellular polymeric substances (LB-EPS), tightly bound EPS (TB-EPS), and pellet were extracted for all 8 AS samples. There was a clear trend that the effluent turbidity decreased as the SRT increased, and we deduced that this is because AS samples with longer SRTs have lower interaction energy barriers and lower LB-EPS content. Furthermore, the concentrations of multivalent cations (especially trivalent cations) in the pellets were found to be closely correlated to the AS flocculability, total interaction energy (Wtot), and LB-EPS content. The multivalent (especially trivalent) cations possess greater binding ability, and this ability to bind tightly to AS in large quantities is responsible for the superior flocculability of AS samples with longer SRTs. Hence, the concentrations of multivalent cations in the pellets are an important indicator of AS flocculability. We deduced that variations in the quantities of multivalent cations that tightly bind with the AS rather than remaining in the influent are the core reason behind observed fluctuations in the AS flocculability with different SRTs.

  1. Evaluating Goddard Multi-Scale Modeling Framework at Different fv-GCM Grid Spacing

    NASA Astrophysics Data System (ADS)

    Chern, J.; Matsui, T.; Shen, B.; Tao, W.

    2009-12-01

    The Goddard Multi-scale Modeling Framework (MMF) is based on the coupling of the two-dimensional Goddard Cumulus Ensemble (GCE) model and the finite-volume GCM (fv-GCM). Thus MMF enables explicit resolution of stochastic moist convection process by embedded GCE simulations, unlike traditional GCMs that rely on convection parameterization. At each fv-GCM column, the fv-GCM provides mean atmospheric conditions and large-scale temperature and moisture advection to drive the 2D GCE models, which feedback the tendencies of thermodynamic parameter and cloud statistics to the fv-GCM. Earlier investigations show that the Goddard MMF simulates better cloudiness (high and low), single ITCZ and a more realistic diurnal variation of rainfall than traditional GCMs. Another advantages of using the Goddard MMF is that the resolution of GCE-simulated clouds is compatible to satellite observations, while traditional GCM requires disaggregation of grid-volume feature to compare with high-resolution satellite observations. Thus, satellite instrumental simulator can be directly applied to translate MMF simulations into the satellite instrumental signals in straightforward way. In this year, we examine the sensitivity of the Goddard MMF simulation at different fv-GCM grid spacing, and evaluated performances against the TRMM satellite. Previously, fv-GCM was run at 2x2.5 degree horizontal lat-lon grid spacing, and we are currently running fv-GCM at 1x1.25 degree. We examine the performance of the Goddard MMF at different fv-GCM grid spacing with respect to rainfall frequency, rain structure, and microphysics using multi-sensor radiance-based evaluation method, known as the TRMM Triple-Sensor Three-step Evaluation Framework (T3EF). T3EF utilizes multi-sensor satellite simulators, Goddard Satellite Data Simulation Unit, and novel statistics of multi-sensor radiance and backscattering signals observed from the TRMM satellite. Specifically, T3EF compares GCE and satellite observations in

  2. Internalization of exogenous cystatin F supresses cysteine proteases and induces the accumulation of single-chain cathepsin L by multiple mechanisms.

    PubMed

    Colbert, Jeff D; Matthews, Stephen P; Kos, Janko; Watts, Colin

    2011-12-01

    Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169-179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.

  3. Single chain morphology and nanofiber-like aggregates of branched β-(1 → 3)-D-glucan in water/dimethylsulfoxide solution.

    PubMed

    Chen, Cong; Meng, Yan; Li, Sheng; Wu, Wenhua; Liu, Chuanjun; Xu, Xiaojuan; Zhang, Lina

    2016-02-10

    A polysaccharide coded as PR-CA was extracted from Polyporus rhinoceros and determined to be a β-(1 → 3)-D-glucan with multiple branches. The weight-average molecular weights (Mw) of PR-CA in dimethylsulfoxide (DMSO) and in water were determined with static light scattering (SLS) to be 3.57 × 10(5) and 1.79 × 10(7), indicating existence of the single chains in DMSO and co-existence of single chains and aggregates in water. Moreover, the stiffness of single chains of PR-CA in water was directly visualized by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The hollow structure of PR-CA nanofibers with width of 30-40 nm and length of ∼ 350 nm formed in the water/DMSO (9:1, v:v) was demonstrated by a fluorescent probe tetraphenylethylene (TPE) via aggregation-induced emission (AIE). The formation of PR-CA nanofibers was ascribed to the parallel aggregation of the extended PR-CA chains due to the hydrogen bonding and hydrophobic interaction. This work offered valuable results for promising applications of natural branched β-glucans in the biological fields of drug inclusion, delivery and disease diagnosis. PMID:26686132

  4. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody.

    PubMed Central

    Marasco, W A; Haseltine, W A; Chen, S Y

    1993-01-01

    A single-chain antibody, derived from a human monoclonal antibody that recognizes the CD4 binding region of the human immunodeficiency virus type 1 (HIV-1) envelope protein, has been designed for intracellular expression in eukaryotic cells. The single-chain antibody is composed of an immunoglobulin heavy-chain leader sequence and heavy- and light-chain variable regions that are joined by an interchain linker. The antibody is stably expressed and retained in the endoplasmic reticulum and is not toxic to the cells. The antibody binds to the envelope protein within the cell and inhibits processing of the envelope precursor and syncytia formation. The infectivity of the HIV-1 particles produced by cells that express the single-chain antibody is substantially reduced. These studies illustrate the feasibility of designing antibodies that bind and inactivate molecules intracellularly. Antibodies that act on target molecules within cells should provide a useful tool for research as well as for control of infectious and other diseases. Images Fig. 1 Fig. 2 Fig. 3 PMID:8356098

  5. Genetic evidence for a product of the Fv-1 locus that transfers resistance to mouse leukemia viruses.

    PubMed Central

    Tennant, R W; Schluter, B; Myer, F E; Otten, J A; Yang, W K; Brown, A

    1976-01-01

    Extracts of mouse cells have been shown to transfer to N- or B-trophic host range types of mouse leukemia viruses. The genetic specificity of the inhibition was tested in two ways: (i) by correlating the Fv-1 genotype of a number of mouse strains with the restriction-transferring activity of extracts of the respective embryo cell cultures, and (ii) by correlating the Fv-1 genotype of BLC3F2 (C57BL/6 female [Fv-1bb] by C3H male [Fv-1nn] parental strains) mouse embryos, which segregate the Fv-1 alleles in a 12:1 ratio, with the inhibitor activity of extracts of the cells from each embryo. Five independent matings, totaling 45 individual embryos, were tested. Each embryo was cultured, and the Fv-1 genotype was determined independently by titration of N- and B-tropic viruses; the extracts of replicate secondary cultures were tested for their effect on infection of permissive cells by N- and B-tropic viruses. The specific-restriction-transferring activity of the embryos was found to segregate with the appropriate Fv-1 genotype. These res-lts confirm the suggestion that the inhibitor of the leukemia virus host range types in the cellular extracts is a product of the Fv-1 locus. PMID:186636

  6. Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites

    PubMed Central

    Hartmann, Ulrike; Forsgren, Eva; Charrière, Jean-Daniel; Neumann, Peter; Gauthier, Laurent

    2015-01-01

    Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies. PMID:26008705

  7. Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites.

    PubMed

    Hartmann, Ulrike; Forsgren, Eva; Charrière, Jean-Daniel; Neumann, Peter; Gauthier, Laurent

    2015-05-22

    Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

  8. Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma.

    PubMed

    Krenciute, Giedre; Krebs, Simone; Torres, David; Wu, Meng-Fen; Liu, Hao; Dotti, Gianpietro; Li, Xiao-Nan; Lesniak, Maciej S; Balyasnikova, Irina V; Gottschalk, Stephen

    2016-02-01

    Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies. PMID:26514825

  9. Increased stability and DNA site discrimination of "single chain" variants of the dimeric beta-barrel DNA binding domain of the human papillomavirus E2 transcriptional regulator.

    PubMed

    Dellarole, Mariano; Sánchez, Ignacio E; Freire, Eleonora; de Prat-Gay, Gonzalo

    2007-10-30

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric beta-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  10. Increased Stability and DNA Site Discrimination of Single Chain Variants of the Dimeric beta-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

    SciTech Connect

    Dellarole,M.; Sanchez, I.; Freire, E.; de Prat-Gay, G.

    2007-01-01

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric {beta}-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  11. Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast

    PubMed Central

    Xu, Ping; Robinson, Anne Skaja

    2009-01-01

    Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the unfolded protein response, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4−4−20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the unfolded protein response. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties. PMID:19415776

  12. Polyelectrolyte Properties in Mono and Multi-Valent Ionic Media: Brushes and Complex Coacervates

    NASA Astrophysics Data System (ADS)

    Farina, Robert M.

    Materials composed of polyelectrolytes have unique and interesting physical properties resulting primarily from their charged monomer segments. Polyelectrolytes, which exist in many different biological and industrial forms, have also been shown to be highly responsive to external environmental changes. Here, two specific polyelectrolyte systems, brushes and complex coacervates, are discussed in regards to how their properties can be tailored by adjusting the surrounding ionic environment with mono and multi-valent ions. End-tethered polyelectrolyte brushes, which constitute an interesting and substantial portion of polyelectrolyte applications, are well known for their ability to provide excellent lubrication and low friction when coated onto surfaces (e.g. articular cartilage and medical devices), as well as for their ability to stabilize colloidal particles in solution (e.g. paint and cosmetic materials). These properties have been extensively studied with brushes in pure mono-valent ionic media. However, polyelectrolyte brush interactions with multi-valent ions in solution are much less understood, although highly relevant considering mono and multi-valent counterions are present in most applications. Even at very low concentrations of multi-valent ions in solution, dramatic polyelectrolyte brush physical property changes can occur, resulting in collapsed chains which also adhere to one another via multi-valent bridging. Here, the strong polyelectrolyte poly(sodium styrene sulfonate) was studied using the Surface Forces Apparatus (SFA) and electrochemistry in order to investigate brush height and intermolecular interactions between two brushes as a function of multi-valent counterion population inside a brush. Complex coacervates are formed when polyanions and polycations are mixed together in proper conditions of an aqueous solution. This mixing results in a phase separation of a polymer-rich, coacervate phase composed of a chain network held together via

  13. [Construction of anti-B7-H4-scFv library and screening and identification of anti-B7-H4-scFv].

    PubMed

    Shao, Luanluan; Xu, Chaochao; Ji, Hongshuai; Mao, Weiping; Wang, Yingying; Liu, Xiaoqian; Zhu, Yanyan

    2016-09-01

    Objective To construct the ribosome display library of anti-B7-H4 extracellular domain, and select the antibody with high specificity. Methods The cDNA of B7-H4 extracellular domain was amplified from A549 cells by reverse transcription PCR (RT-PCR). To express ectodomains of B7-H4, the sequence of B7-H4 gene, which encodes the B7-H4 extracellular domains, was inserted into plasmid pET-28a(+). The purified recombinant protein of B7-H4 extracellular domain was used to immunize BALB/c mice. The total RNA was extracted from the spleen of BALB/c mice which had been immunized with B7-H4 recombinant protein. The genes of VH, Vκ and VH/Vκ were amplified separately by RT-PCR and splicing by overlap extension PCR (SOE-PCR). The gene of VH/ Vκ was ligated into pUM19-T vector and the ligated sample was transformed into competent E.coli DH5α. The resulting plasmid was isolated and then subjected to sequencing to verify the gene sequence. TNT(R)T7 Quick for PCR DNA kit was used to translate and screen the anti-B7-H4-scFv in vitro from the ribosome display library. Western blotting and an indirect ELISA were performed to detect the specificity of anti-B7-H4-scFv. Results The right sequences of VH, Vκ and VH/Vκ were acquired, which were 439, 680 and 1098 bp in length, respectively. The analysis of specificity demonstrated that the anti-B7-H4-scFv screened from the ribosome display library had a high specific combining ability with B7-H4. Conclusion The experiment has successively constructed the ribosome display library of anti-B7-H4 extracellular domain, and selected the anti-B7-H4-scFv which has a high specific binding ability with recombinant protein of B7-H4 extracelluar domain. PMID:27609584

  14. Universal Molecular Scaffold for Facile Construction of Multivalent and Multimodal Imaging Probes.

    PubMed

    Gai, Yongkang; Xiang, Guangya; Ma, Xiang; Hui, Wenqi; Ouyang, Qin; Sun, Lingyi; Ding, Jiule; Sheng, Jing; Zeng, Dexing

    2016-03-16

    Multivalent and multimodal imaging probes are rapidly emerging as powerful chemical tools for visualizing various biochemical processes. Herein, we described a bifunctional chelator (BFC)-based scaffold that can be used to construct such promising probes concisely. Compared to other reported similar scaffolds, this new BFC scaffold demonstrated two major advantages: (1) significantly simplified synthesis due to the use of this new BFC that can serve as chelator and linker simultaneously; (2) highly efficient synthesis rendered by using either click chemistry and/or total solid-phase synthesis. In addition, the versatile utility of this molecular scaffold has been demonstrated by constructing several multivalent/multimodal imaging probes labeled with various radioisotopes, and the resulting radiotracers demonstrated substantially improved in vivo performance compared to the two individual monomeric counterparts.

  15. Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip.

    PubMed

    Branderhorst, Hilbert M; Ruijtenbeek, Rob; Liskamp, Rob M J; Pieters, Roland J

    2008-07-21

    Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

  16. Design, synthesis and biological studies of efficient multivalent melanotropin ligands: tools towards melanoma diagnosis and treatment

    PubMed Central

    Brabez, Nabila; Lynch, Ronald M.; Xu, Liping; Gillies, Robert J.; Chassaing, Gerard; Lavielle, Solange; Hruby, Victor J.

    2011-01-01

    In order to achieve early detection and specific cancer treatment we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells, which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated leading to more insights into the effects of multivalent compounds on transduction mechanisms. PMID:21928837

  17. Controlling Orientational Order in 1-D Assemblies of Multivalent Triangular Prisms.

    PubMed

    Kohlstedt, Kevin L; Olvera de la Cruz, Monica; Schatz, George C

    2013-01-01

    Multivalent nanostructures are becoming an increasingly important player in the self-assembly of supramolecular lattices. Understanding the role that shape plays in the coordination of the assemblies is crucial for the functional response of the material. We develop a simple design rule for the assembly of multivalent Au triangular nanoprisms into 1-D ordered arrays based on both the length of the valent DNA and the aspect ratio of the prism. Using MD simulations, we describe an order parameter that captures the short-range order of the assembly controlled by the design parameters. The order parameter shows that even short chains (N = 4) of prisms have a high degree of orientational order that transitions to no orientational order when the DNA length is similar to the prism length. Unlike isotropic polyvalent assemblies, we find that the highly oriented chains of prisms lose orientational order in discrete steps during melting as the prisms in the arrays dissociate.

  18. Serum Multivalent Cationic Pattern: Speculation on the Efficient Approach for Detection of Alzheimer's Disease

    NASA Astrophysics Data System (ADS)

    Azhdarzadeh, Morteza; Noroozian, Maryam; Aghaverdi, Haniyeh; Akbari, Seyed Mostafa; Baum, Larry; Mahmoudi, Morteza

    2013-10-01

    Alzheimer's disease (AD) is increasingly becoming one of the greatest medical challenges. Due to the social and financial burden of AD, detection of AD in its early stages is a topic of major research interest. Thus, emergence of well-validated screening methods for fast detection of AD in the early stages would be of great importance. It is now recognized that the homeostasis and serum bioavailability of multivalent cations (e.g. zinc, copper, and iron) are disturbed in AD. Using a standard chemometric approach (hierarchical clustering analysis), we find that the serum concentrations of an array of such multivalent cations can be a fingerprint for identification of AD patients. This may pave the way for a reliable, efficient, and inexpensive method for early detection and treatment of AD.

  19. Pillar[5]arene-Based Glycoclusters: Synthesis and Multivalent Binding to Pathogenic Bacterial Lectins.

    PubMed

    Buffet, Kevin; Nierengarten, Iwona; Galanos, Nicolas; Gillon, Emilie; Holler, Michel; Imberty, Anne; Matthews, Susan E; Vidal, Sébastien; Vincent, Stéphane P; Nierengarten, Jean-François

    2016-02-24

    The synthesis of pillar[5]arene-based glycoclusters has been readily achieved by CuAAC conjugations of azido- and alkyne-functionalized precursors. The lectin binding properties of the resulting glycosylated multivalent ligands have been studied by at least two complementary techniques to provide a good understanding. Three lectins were selected from bacterial pathogens based on their potential therapeutic applications as anti-adhesives, namely LecA and LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria. As a general trend, multivalency improved the binding to lectins and a higher affinity can be obtained by increasing to a certain limit the length of the spacer arm between the carbohydrate subunits and the central macrocyclic core.

  20. The effect of multivalent ions on the thermal transition of hydrated polyelectrolyte multilayers

    NASA Astrophysics Data System (ADS)

    Reid, Dariya; Lutkenhaus, Jodie

    2015-03-01

    Layer-by-layer (LbL) assembly is a commonly studied technique in the production of uniform thin films. Hydrate LbL assemblies made of model polyelectrolytes, poly(diallyldimethylammonium chloride) (PDAC) and poly(styrene sulfonate) (PSS), exhibit a thermal transition with features of a glass transition and a lower critical solution temperature transition when assembled in the presence of sodium chloride. The question remains as to how multivalent cations affect the nature of the transition. Here, we present results on the thermal transition of PDAC/PSS LbL assemblies exposed to various multivalent salts. Quartz crystal microbalance (QCM-D) and modulated differential scanning calorimetry (MDSC) is used to assess the transition.

  1. Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

    PubMed

    de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

    2016-06-10

    The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products. PMID:27085890

  2. Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

    PubMed

    de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

    2016-06-10

    The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products.

  3. Expression and Purification of the scFv from Hybridoma Cells Secreting a Monoclonal Antibody Against S Protein of PEDV

    PubMed Central

    Zhu, Qinghe; Guo, Donghua; Feng, Li

    2013-01-01

    The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. 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/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule. PMID:23600505

  4. Pentacyclic triterpenes grafted on CD cores to interfere with influenza virus entry: A dramatic multivalent effect.

    PubMed

    Xiao, Sulong; Si, Longlong; Tian, Zhenyu; Jiao, Pingxuan; Fan, Zibo; Meng, Kun; Zhou, Xiaoshu; Wang, Han; Xu, Renyang; Han, Xu; Fu, Ge; Zhang, Yongmin; Zhang, Lihe; Zhou, Demin

    2016-02-01

    Multivalent effect plays an important role in biological processes, particularly in the specific recognition of virus with its host cell during the first step of infection. Here we report the synthesis of multivalent pentacyclic triterpene grafted on cyclodextrin core and potency of against influenza entry activity. Nine star-shaped compounds containing six, seven and eight pentacyclic triterpene pharmacophore on cyclodextrin scaffold were prepared by way of copper-catalyzed azide-alkyl cycloaddition reaction under microwave activation. Some of the multimers exhibited much potent antiviral activity against H1N1 virus (A/WSN/33), even equivalent or superior to oseltamivir. The most active compound 31, a heptavalent oleanolic acid-β-cyclodextrin conjugate, shows an up to 125-fold potency enhancement by its IC50 value over the corresponding monovalent conjugate and oleanolic acid, disclosing a clear multivalent effect. Further studies show that three compounds 31-33 exhibited broad spectrum inhibitory activity against other two human influenza A/JX/312 (H3N2) and A/HN/1222 (H3N2) viruses with the IC50 values at 2.47-14.90 μM. Most importantly, we found that compound 31, one of the best representative conjugate, binds tightly to the viral envelope hemagglutinin with a dissociation constant of KD = 2.08 μM, disrupting the interaction of hemagglutinin with the sialic acid receptor and thus the attachment of viruses to host cells. Our study might establish a strategy for the design of new pharmaceutical agents based on multivalency so as to block influenza virus entry into host cells. PMID:26686050

  5. Partial protection against four species of chicken coccidia induced by multivalent subunit vaccine.

    PubMed

    Song, Xiaokai; Gao, Yunlu; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

    2015-09-15

    In this study, a multivalent subunit vaccine was designed to protect chickens against simultaneous infection by several Eimeria species. This vaccine contains recombinant proteins from four Eimeria species - E. tenella, E. necatrix, E. acervulina and E. maxima - and was evaluated for efficacy in animals. To produce this vaccine, candidate antigens from each Eimeria species were first screened in chickens via intramuscular inoculation and subsequent challenge. Antigens tested include recombinant proteins TA4 and SO7 from E. tenella, NA4 and NPmz19 from E. necatrix, LDH, 3-1E and MIF from E. acervulina, and Em6 and Em8 (two portions of EmTFP250) from E. maxima. A homologous challenge was then performed to identify which antigen from each species conferred the best protection. The antigens identified as most protective against its species were then challenged by heterologous species. Finally, the selected recombinant proteins from each of the four respective species were mixed with the final concentration of 400 μg/ml (100 μg of each protein/ml) to form the multivalent subunit vaccine, which was tested for efficacy in animals. The results indicated that TA4 from E. tenella, NA4 from E. necatrix, LDH from E. acervulina, and Em8 from E. maxima each induced the most effective protection from homologous challenge. Cross-protection results showed that TA4 provided partial cross-protection against E. necatrix, NA4 provided partial cross-protection against E. tenella and E. acervulina, LDH provided partial cross-protection against E. tenella and E. necatrix, and Em8 provided partial cross-protection against E. tenella and E. acervulina. The multivalent subunit vaccine provided partial protection against E. tenella, E. necatrix, E. acervulina and E. maxima challenge, and resulted in ACIs of more than 170. These results suggest that our candidate multivalent vaccine could protect chickens against simultaneous infection by several Eimeria species.

  6. Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

    PubMed

    Peck, Evan M; Battles, Paul M; Rice, Douglas R; Roland, Felicia M; Norquest, Kathryn A; Smith, Bradley D

    2016-05-18

    A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

  7. Influence of microorganisms on the oxidation state distribution of multivalent actinides under anoxic conditions

    SciTech Connect

    Reed, Donald Timothy; Borkowski, Marian; Lucchini, Jean - Francois; Ams, David; Richmann, M. K.; Khaing, H.; Swanson, J. S.

    2010-12-10

    The fate and potential mobility of multivalent actinides in the subsurface is receiving increased attention as the DOE looks to cleanup the many legacy nuclear waste sites and associated subsurface contamination. Plutonium, uranium and neptunium are the near-surface multivalent contaminants of concern and are also key contaminants for the deep geologic disposal of nuclear waste. Their mobility is highly dependent on their redox distribution at their contamination source as well as along their potential migration pathways. This redox distribution is often controlled, especially in the near-surface where organic/inorganic contaminants often coexist, by the direct and indirect effects of microbial activity. Under anoxic conditions, indirect and direct bioreduction mechanisms exist that promote the prevalence of lower-valent species for multivalent actinides. Oxidation-state-specific biosorption is also an important consideration for long-term migration and can influence oxidation state distribution. Results of ongoing studies to explore and establish the oxidation-state specific interactions of soil bacteria (metal reducers and sulfate reducers) as well as halo-tolerant bacteria and Archaea for uranium, neptunium and plutonium will be presented. Enzymatic reduction is a key process in the bioreduction of plutonium and uranium, but co-enzymatic processes predominate in neptunium systems. Strong sorptive interactions can occur for most actinide oxidation states but are likely a factor in the stabilization of lower-valent species when more than one oxidation state can persist under anaerobic microbiologically-active conditions. These results for microbiologically active systems are interpreted in the context of their overall importance in defining the potential migration of multivalent actinides in the subsurface.

  8. Spontaneous formation of bilayers and vesicles in mixtures of single-chain alkyl carboxylates: effect of pH and aging and cytotoxicity studies.

    PubMed

    Vlachy, N; Merle, C; Touraud, D; Schmidt, J; Talmon, Y; Heilmann, J; Kunz, W

    2008-09-16

    We report the observation of bilayer fragments, some of which close to form vesicles, over a large range of pH at room temperature from mixtures of single-chain biocompatible commercially available nontoxic alkyl carboxylic surfactants after neutralization with HCl. The pH at which the morphological transitions occur is varied only by changing the ratio between two surfactants: the alkyloligoethyleneoxide carboxylate and sodium laurate. The effect of aging of the mixed surfactant systems in the pH region desired for dermatologic application (4.5 < pH < 7) is also studied. Finally, we show results of cytotoxicity studies on the surfactant mixtures.

  9. Two Isostructural Coordination Polymers Showing Diverse Magnetic Behaviors: Weak Coupling (Ni(II)) and an Ordered Array of Single-Chain Magnets (Co(II)).

    PubMed

    Chen, Min; Zhao, Hui; Sañudo, E Carolina; Liu, Chun-Sen; Du, Miao

    2016-04-18

    Two isomorphic 3-D complexes with the formulas [M3(TPTA) (OH)2(H2O)4]n (M = Ni for 1 and Co for 2; H4TPTA = [1,1':4',1″-terphenyl]-2',3,3″,5'-tetracarboxylic acid) have been synthesized and magnetically characterized. Complexes 1 (Ni(II)) and 2 (Co(II)) have the same 1-D rod-shaped inorganic SBUs but exhibit significantly different magnetic properties. Complex 2(Co(II)) is a 3-D arrangement of a 1-D Co(II) single-chain magnet (SCM), while complex 1(Ni(II)) exhibits weak coupling. PMID:27022765

  10. Two Isostructural Coordination Polymers Showing Diverse Magnetic Behaviors: Weak Coupling (Ni(II)) and an Ordered Array of Single-Chain Magnets (Co(II)).

    PubMed

    Chen, Min; Zhao, Hui; Sañudo, E Carolina; Liu, Chun-Sen; Du, Miao

    2016-04-18

    Two isomorphic 3-D complexes with the formulas [M3(TPTA) (OH)2(H2O)4]n (M = Ni for 1 and Co for 2; H4TPTA = [1,1':4',1″-terphenyl]-2',3,3″,5'-tetracarboxylic acid) have been synthesized and magnetically characterized. Complexes 1 (Ni(II)) and 2 (Co(II)) have the same 1-D rod-shaped inorganic SBUs but exhibit significantly different magnetic properties. Complex 2(Co(II)) is a 3-D arrangement of a 1-D Co(II) single-chain magnet (SCM), while complex 1(Ni(II)) exhibits weak coupling.

  11. Novel Approach to Prepare {sup 99m}Tc-Based Multivalent RGD Peptides

    SciTech Connect

    Shuang Liu

    2012-10-24

    This project presents a novel approach to prepare the {sup 99m}Tc-bridged multivalent RGD (arginine-glycine-aspartate) peptides. This project will focus on fundamentals of {sup 99m}Tc radiochemistry. The main objective of this project is to demonstrate the proof-of-principle for the proposed radiotracers. Once a kit formulation is developed for preparation of the {sup 99m}Tc-bridged multivalent RGD peptides, various tumor-bearing animal models will be used to evaluate their potential for SPECT (single photon-emission computed tomography) imaging of cancer. We have demonstrated that (1) multimerization of cyclic RGD peptides enhances the integrin {alpha}{sub v}{beta}{sub 3} bonding affinity and radiotracer tumor uptake; (2) addition of G{sub 3} or PEG{sub 4} linkers makes it possible for two RGD motifs in 3P-RGD{sub 2} and 3G-RGD{sub 2} to achieve simultaneous integrin {alpha}{sub v}{beta}{sub 3} binding; and (3) multimers are actually bivalent (not multivalent), the presence of extra RGD motifs can enhance the tumor retention time of the radiotracer.

  12. Multivalent Polymers for Drug Delivery and Imaging: The Challenges of Conjugation

    PubMed Central

    2015-01-01

    Multivalent polymers offer a powerful opportunity to develop theranostic materials on the size scale of proteins that can provide targeting, imaging, and therapeutic functionality. Achieving this goal requires the presence of multiple targeting molecules, dyes, and/or drugs on the polymer scaffold. This critical review examines the synthetic, analytical, and functional challenges associated with the heterogeneity introduced by conjugation reactions as well as polymer scaffold design. First, approaches to making multivalent polymer conjugations are discussed followed by an analysis of materials that have shown particular promise biologically. Challenges in characterizing the mixed ligand distributions and the impact of these distributions on biological applications are then discussed. Where possible, molecular-level interpretations are provided for the structures that give rise to the functional ligand and molecular weight distributions present in the polymer scaffolds. Lastly, recent strategies employed for overcoming or minimizing the presence of ligand distributions are discussed. This review focuses on multivalent polymer scaffolds where average stoichiometry and/or the distribution of products have been characterized by at least one experimental technique. Key illustrative examples are provided for scaffolds that have been carried forward to in vitro and in vivo testing with significant biological results. PMID:25120091

  13. Synthetic Multivalent Ligands as Probes of Inter-Receptor Communication in Bacterial Chemotaxis

    NASA Astrophysics Data System (ADS)

    Gestwicki, Jason

    2004-03-01

    Bacteria can sense chemotactic signals, such as nutrients and toxins, with remarkable sensitivity. Escherichia coli, respond to changes in stimulant concentration of less than 10to maintain sensitivity, the relationship between ligand concentration and output response must be non-linear. For example, at low ligand levels, substantial amplification of the chemotactic signal is required to trigger locomotion. Signal amplification must be quickly suppressed, however, to restore proper sensitivity to small changes in ligand at higher concentrations. Because of the rapid flexibility of this system, it has been hypothesized that alterations in the organization of the chemotactic signaling proteins, rather than changes in their expression, provide this exquisite sensitivity. The interaction between chemoreceptors within lattices has been proposed to play a role in this process. Using a series of synthetic multivalent ligands directed at the chemoreceptors, we have demonstrated a requirement for dynamic changes in inter-receptor interactions for amplification and integration of sensory information. Multivalent ligands that interact through the galactose-sensing receptor Trg, enforce proximal interactions between chemoreceptors and enhance signal output. Further, upon treatment with multivalent ligands, the response to the attractant serine is amplified by at least 100-fold. These results, and those from genetic and structural studies by other laboratories, suggest that the entire array is involved in sensing. These results support general strategy by which biological responses may be regulated.

  14. Glycopolymer brushes for specific lectin binding by controlled multivalent presentation of N-acetyllactosamine glycan oligomers.

    PubMed

    Park, Hyunji; Rosencrantz, Ruben R; Elling, Lothar; Böker, Alexander

    2015-01-01

    A new multivalent glycopolymer platform for lectin recognition is introduced in this work by combining the controlled growth of glycopolymer brushes with highly specific glycosylation reactions. Glycopolymer brushes, synthetic polymers with pendant saccharides, are prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-O-(N-acetyl-β-d-glucosamine)ethyl methacrylate (GlcNAcEMA). Here, the fabrication of multivalent glycopolymers consisting of poly(GlcNAcEMA) is reported with additional biocatalytic elongation of the glycans directly on the silicon substrate by specific glycosylation using recombinant glycosyltransferases. The bioactivity of the surface-grafted glycans is investigated by fluorescence-linked lectin assay. Due to the multivalency of glycan ligands, the glycopolymer brushes show very selective, specific, and strong interactions with lectins. The multiarrays of the glycopolymer brushes have a large potential as a screening device to define optimal-binding environments of specific lectins or as new simplified diagnostic tools for the detection of cancer-related lectins in blood serum. PMID:25354386

  15. Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection.

    PubMed

    Muñoz, Antonio; Sigwalt, David; Illescas, Beatriz M; Luczkowiak, Joanna; Rodríguez-Pérez, Laura; Nierengarten, Iwona; Holler, Michel; Remy, Jean-Serge; Buffet, Kevin; Vincent, Stéphane P; Rojo, Javier; Delgado, Rafael; Nierengarten, Jean-François; Martín, Nazario

    2016-01-01

    The use of multivalent carbohydrate compounds to block cell-surface lectin receptors is a promising strategy to inhibit the entry of pathogens into cells and could lead to the discovery of novel antiviral agents. One of the main problems with this approach, however, is that it is difficult to make compounds of an adequate size and multivalency to mimic natural systems such as viruses. Hexakis adducts of [60]fullerene are useful building blocks in this regard because they maintain a globular shape at the same time as allowing control over the size and multivalency. Here we report water-soluble tridecafullerenes decorated with 120 peripheral carbohydrate subunits, so-called 'superballs', that can be synthesized efficiently from hexakis adducts of [60]fullerene in one step by using copper-catalysed azide–alkyne cycloaddition click chemistry. Infection assays show that these superballs are potent inhibitors of cell infection by an artificial Ebola virus with half-maximum inhibitory concentrations in the subnanomolar range.

  16. Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection

    NASA Astrophysics Data System (ADS)

    Muñoz, Antonio; Sigwalt, David; Illescas, Beatriz M.; Luczkowiak, Joanna; Rodríguez-Pérez, Laura; Nierengarten, Iwona; Holler, Michel; Remy, Jean-Serge; Buffet, Kevin; Vincent, Stéphane P.; Rojo, Javier; Delgado, Rafael; Nierengarten, Jean-François; Martín, Nazario

    2016-01-01

    The use of multivalent carbohydrate compounds to block cell-surface lectin receptors is a promising strategy to inhibit the entry of pathogens into cells and could lead to the discovery of novel antiviral agents. One of the main problems with this approach, however, is that it is difficult to make compounds of an adequate size and multivalency to mimic natural systems such as viruses. Hexakis adducts of [60]fullerene are useful building blocks in this regard because they maintain a globular shape at the same time as allowing control over the size and multivalency. Here we report water-soluble tridecafullerenes decorated with 120 peripheral carbohydrate subunits, so-called ‘superballs’, that can be synthesized efficiently from hexakis adducts of [60]fullerene in one step by using copper-catalysed azide-alkyne cycloaddition click chemistry. Infection assays show that these superballs are potent inhibitors of cell infection by an artificial Ebola virus with half-maximum inhibitory concentrations in the subnanomolar range.

  17. Use of a Genetically Engineered Protein for the Design of a Multivalent MRI Contrast Agent

    PubMed Central

    Karfeld, Lindsay S.; Bull, Steve R.; Davis, Nicolynn E.; Meade, Thomas J.; Barron, Annelise E.

    2008-01-01

    The majority of clinically used contrast agents (CAs) for magnetic resonance imaging have low relaxivities and thus require high concentrations for signal enhancement. Research has turned to multivalent, macromolecular CAs to increase CA efficiency. However, previously developed macromolecular CAs do not provide high relaxivities, have limited biocompatibility, and/or do not have a structure that is readily modifiable to tailor to particular applications. We report a new family of multivalent, biomacromolecular, genetically engineered protein polymer-based CAs; the protein backbone contains evenly spaced lysines that are derivatized with gadolinium (Gd(III)) chelators. The protein's length and repeating amino acid sequence are genetically specified. We reproducibly obtained conjugates with an average of 8 – 9 Gd(III) chelators per protein. These multivalent CAs reproducibly provide a high relaxivity of 7.3 mM-1s-1 per Gd(III) and 62.6 mM-1s-1 per molecule. Furthermore, they can be incorporated into biomaterial hydrogels via chemical crosslinking of remaining free lysines, and provide a dramatic contrast enhancement. Thus, these protein polymer CAs could be a useful tool for following the evolution of tissue engineering scaffolds. PMID:17927227

  18. Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

    PubMed Central

    Crespillo, Sara; Cámara-Artigas, Ana; Casares, Salvador; Morel, Bertrand; Cobos, Eva S.; Mateo, Pedro L.; Mouz, Nicolas; Martin, Christophe E.; Roger, Marie G.; El Habib, Raphaelle; Su, Bin; Moog, Christiane; Conejero-Lara, Francisco

    2014-01-01

    During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates. PMID:25489108

  19. The effect of secondary structures on the NLO properties of single chain oligopeptides: a comparison between β-strand and α-helix polyglycines.

    PubMed

    Alparone, Andrea

    2013-08-21

    The evolution of the electronic first-order longitudinal hyperpolarizability (βzzz) and the hyperpolarizability aligned along the direction of the dipole moment (βμ) of the α-helix and β-strand single chain H2N-(CH2-CO-NH)n-CH2-COOH (n = 1-9) oligoglycines, were investigated. For this purpose we have used Hartree-Fock, second-order Møller-Plesset perturbation theory and Coulomb-attenuating Density Functional Theory computations. For the longest chain, βμ(β-strand) is one order of magnitude greater than βμ(α-helix), due to the cooperative effect of the α-helices being unfavourable for the NLO properties. The βzzz and βμ values per unit cell of the β-strand conformation were determined, extrapolating the properties in the limit of the polymer. The calculated βzzz values were elucidated using the two-state model involving the characteristic π-π* NV1 electronic transition of peptides. Single chain β-strand polyglycines can be discriminated from the α-helices using second-order NLO effects.

  20. Negative Effects of Low Dose Atrazine Exposure on the Development of Effective Immunity to FV3 in Xenopus laevis

    PubMed Central

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B. Paige; Robert, Jacques

    2014-01-01

    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxiciology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses. PMID:24984115

  1. Negative effects of low dose atrazine exposure on the development of effective immunity to FV3 in Xenopus laevis.

    PubMed

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B Paige; Robert, Jacques

    2014-11-01

    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxicology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses.

  2. The Fusarium virguliforme toxin FvTox1 causes foliar sudden death syndrome-like symptoms in soybean.

    PubMed

    Brar, Hargeet K; Swaminathan, Sivakumar; Bhattacharyya, Madan K

    2011-10-01

    Fusarium virguliforme causes sudden death syndrome (SDS) in soybean. The pathogen has never been isolated from diseased foliar tissues; therefore, one or more toxins have been considered to cause foliar SDS development. Cell-free F. virguliforme culture filtrates containing a toxin causes foliar SDS in soybean. A low-molecular-weight protein of approximately 13.5 kDa (FvTox1), purified from F. virguliforme culture filtrates, produces foliar SDS-like symptoms in cut soybean seedlings. Anti-FvTox1 monoclonal antibodies raised against the purified FvTox1 were used in isolating the FvTox1 gene. In the presence of light, recombinant FvTox1 protein expressed in an insect cell line resulted in chlorosis and necrosis in soybean leaf disks that are typical foliar SDS symptoms. SDS-susceptible but not the SDS-resistant soybean lines were sensitive to the baculovirus-expressed toxin. The requirement of light for foliar SDS-like symptom development indicates that FvTox1 induces foliar SDS in soybean, most likely through production of free radicals by interrupting photosynthesis. PMID:21635141

  3. Role of Multivalent Cations in the Organization, Structure, and Assembly of the Cell Wall of Pseudomonas aeruginosa.

    PubMed

    Asbell, M A; Eagon, R G

    1966-08-01

    Asbell, Mary A. (University of Georgia, Athens), and R. G. Eagon. Role of multivalent cations in the organization, structure, and assembly of the cell wall of Pseudomonas aeruginosa. J. Bacteriol. 92:380-387. 1966. -Incubation of Pseudomonas aeruginosa with ethylenediaminetetraacetate induced the formation of osmotically fragile rods termed osmoplasts. These could be restored to osmotically stable forms by multivalent cations. Only those cells restored by divalent cations normally found in the cell wall were capable of multiplication. The respiration of restored cells, however, was unimpaired, irrespective of whether they were capable of multiplication. Moreover, the permeability characteristics of osmoplasts and restored cells were unimpaired. When multivalent cations were chelated from the cell wall and replaced by sodium, a weakened cell wall and an osmotically fragile cell resulted. This was apparently caused by the absence of cross-linkages in the cell wall via multivalent cations. Tris(hydroxymethyl)aminomethane buffer compounded the lethal effects of ethylenediaminetetraacetate. The lipopolysaccharide component was inferred to be the site of attack by ethylenediaminetetraacetate. A mechanism for the synthesis of the lipopolysaccharide sacculus was proposed whereby negatively charged subunits are "trapped" by forming ionic and coordinate bonds intermediated by multivalent cations.

  4. Phase changes in mixed lipid/polymer membranes by multivalent nanoparticle recognition.

    PubMed

    Olubummo, Adekunle; Schulz, Matthias; Schöps, Regina; Kressler, Jörg; Binder, Wolfgang H

    2014-01-14

    Selective addressing of membrane components in complex membrane mixtures is important for many biological processes. The present paper investigates the recognition between multivalent surface functionalized nanoparticles (NPs) and amphiphilic block copolymers (BCPs), which are successfully incorporated into lipid membranes. The concept involves the supramolecular recognition between hybrid membranes (composed of a mixture of a lipid (DPPC or DOPC), an amphiphilic triazine-functionalized block copolymer TRI-PEO13-b-PIB83 (BCP 2), and nonfunctionalized BCPs (PEO17-b-PIB87 BCP 1)) with multivalent (water-soluble) nanoparticles able to recognize the triazine end group of the BCP 2 at the membrane surface via supramolecular hydrogen bonds. CdSe-NPs bearing long PEO47-thymine (THY) polymer chains on their surface specifically interacted with the 2,4-diaminotriazine (TRI) moiety of BCP 2 embedded within hybrid lipid/BCP mono- or bilayers. Experiments with GUVs from a mixture of DPPC/BCP 2 confirm selective supramolecular recognition between the THY-functionalized NPs and the TRI-functionalized polymers, finally resulting in the selective removal of BCP 2 from the hybrid vesicle membrane as proven via facetation of the originally round and smooth vesicles. GUVs (composed of DOPC/BCP 2) show that a selective removal of the polymer component from the fluid hybrid membrane results in destruction of hybrid vesicles via membrane rupture. Adsorption experiments with mixed monolayers from lipids with either BCP 2 or BCP 1 (nonfunctionalized) reveal that the THY-functionalized NPs specifically recognize BCP 2 at the air/water interface by inducing significantly higher changes in the surface pressure when compared to monolayers from nonspecifically interacting lipid/BCP 1 mixtures. Thus, recognition of multivalent NPs with specific membrane components of hybrid lipid/BCP mono- and bilayers proves the selective removal of BCPs from mixed membranes, in turn inducing membrane rupture

  5. Display and selection of scFv antibodies on HEK-293T cells

    PubMed Central

    Ho, Mitchell; Pastan, Ira

    2009-01-01

    We describe a human cell display strategy to isolate high affinity single chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient protein expression. Flow cytometry enhances the screen's sensitivity thereby allowing us to isolate high affinity scFvs. Using human cell display one could isolate and engineer scFvs, single domains, Fabs or whole IgGs for increased affinity and other biological functions. PMID:19554290

  6. Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics.

    PubMed

    Jonas, Kristina; Van Der Vries, Erhard; Nilsson, Mikael T I; Widersten, Mikael

    2005-11-01

    New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

  7. Tunable crossover between one- and three-dimensional magnetic dynamics in C oII single-chain magnets organized by halogen bonding

    NASA Astrophysics Data System (ADS)

    Amjad, A.; Clemente-Juan, J. M.; Coronado, E.; Luis, F.; Evangelisti, M.; Espallargas, G. Mínguez; del Barco, E.

    2016-06-01

    Low-temperature magnetometry, ac susceptibility, and calorimetry have been employed to study Co-based single-chain magnets (SCMs) organized through halogen bonding. Magnetic hysteresis and maxima in the dc and ac susceptibilities, respectively, confirm the SCM behavior of the system. Several characteristic magnetic relaxation regimes are observed at different temperatures, which can be associated with both intra- and interchain exchange interactions. Remarkably, tweaking the rate at which an external magnetic field is swept along the axis of the chains enables a controlled transition between the one- and three-dimensional dynamics. Experiments on an isostructural Co-based SCM system crystallized with different halogens do not show three-dimensional dynamics, illustrating the importance of halogen bonding on the control of interchain interactions.

  8. Fe2+-Ti4+ vs. Fe2+-Fe3+ charge-transfer and short-range order in single chains of face-sharing octahedra: ellenbergerite and dumortierite

    NASA Astrophysics Data System (ADS)

    Chopin, C.; Langer, K.; Khomenko, V.

    2009-04-01

    In zoned pyrope megacrysts from the Dora-Maira UHP terrane, new, dark-violet colour varieties of the hexagonal, high-pressure silicate ellenbergerite extend the range of known Fe contents for this mineral from 0-0.1 to 0-0.4 atom pfu, for Ti contents commonly in the range 0.2-0.4 pfu. The new varieties show an extremely intense pleochroism, colourless for E perpendicular to c to deep Prussian blue for E//c, as compared to colourless to lilac or reddish purple for classical Fe-poor ellenbergerite. These features were the incentive for an electronic absorption spectroscopic study and a reappraisal of the interpretation of the charge transfers (CT), colour and ordering schemes in this group and the structurally related borosilicate dumortierite. Both structures are characterized by the presence of infinite single chains of face-sharing, partly vacant octahedra along the 6-fold screw axis and pseudo-hexad axis, respectively, in which the Fe and Ti atoms are partitioned. In the spectra of Fe-poor ellenbergerite, the presence of a single Fe2+-Ti4+ CT band near 19000 cm˘1 was taken as evidence for complete short-range ordering of Mg(Fe), Ti and vacancies in the octahedral single chain [1]. The E//c spectra of Fe-rich ellenbergerite show the same absorption band near 19000 cm˘1 but consistently flanked by another CT band near 14000 cm˘1 , the intensity of which increases with total Fe content. The latter is similar to the 12400 cm˘1 CT band observed as the single feature in E//c spectra of the isotructural (Ti-free and Fe-bearing) phosphoellenbergerite, and clearly assigned to Fe2+-Fe3+ CT in the octahedral single chain [1]. The same colour pattern occurs in the dumortierite group, with red Fe-poor, Ti-rich crystals showing a single CT band near 20000 cm˘1, blue Ti-poor crystals showing a single CT band near 16500 cm˘1, and violet Fe- and Ti-rich crystals showing a combination of the two bands [2]. In the light of the new data, we reinterpret the dumortierite colour

  9. Structural and Functional Characterization of a Single-chain Peptide-MHC Molecule that Modulates both Naive and Activated CD8plus T Cells

    SciTech Connect

    D Samanta; G Mukherjee; U Ramagopal; R Chaparro; S Nathenson; T DiLorenzo; S Almo

    2011-12-31

    Peptide-MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8{sup +} T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8{sup +} T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scK{sup d}.IGRP) by using the class I MHC molecule H-2K{sup d} and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP{sub 206-214}), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scK{sup d}.IGRP tetramers bound specifically to cognate CD8{sup +} T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-{gamma} response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8{sup +} T cells makes them a potential intervention strategy in early and late stages of disease.

  10. Facile Synthesis of Chevrel Phase Nanocubes and their Applications for Multivalent Energy Storage

    SciTech Connect

    Cheng, Yingwen; Parent, Lucas R.; Shao, Yuyan; Wang, Chong M.; Sprenkle, Vincent L.; Li, Guosheng; Liu, Jun

    2014-08-14

    The Chevrel phases (CPs, MxMo6T8, M=metal, T=S or Se) are capable of rapid and reversible intercalation of multivalent ions and are the most practical cathode materials for rechargeable magnesium batteries. For the first time, we report a facile method for synthesizing Mo6S8 nanoparticles and demonstrate that these nanoparticles have significantly better Mg2+ intercalation kinetics compared with microparticles. The results described in this work could inspire the synthesis of nanoscale CPs, which could substantially impact their application.

  11. Self-assembly of heteroleptic dinuclear metallosupramolecular kites from multivalent ligands via social self-sorting

    PubMed Central

    Benkhäuser, Christian

    2015-01-01

    Summary A Tröger's base-derived racemic bis(1,10-phenanthroline) ligand (rac)-1 and a bis(2,2'-bipyridine) ligand with a central 1,3-diethynylbenzene unit 2 were synthesized. Each of these ligands acts as a multivalent entity for the binding of two copper(I) ions. Upon coordination to the metal ions these two ligands undergo selective self-assembly into heteroleptic dinuclear metallosupramolecular kites in a high-fidelity social self-sorting manner as evidenced by NMR spectroscopy and mass spectrometry. PMID:26124873

  12. Design, synthesis, and testing of multivalent compounds targeted to melanocortin receptors

    NASA Astrophysics Data System (ADS)

    Dehigaspitiya, Dilani Chathurika

    Our focus is on developing non-invasive molecular imaging reagents, which target human cancers that presently are difficult to detect, such as melanoma. We wish to apply the multivalency concept to differentiate between healthy cells and melanoma cells. Melanoma cells are known to over-express alpha melanocyte stimulating hormone receptors. A successful multivalent construct should show greater avidity towards melanoma cells than healthy cells due to the synergistic effects arising from multivalency. Both oligomeric and shorter linear constructs bearing the minimum active sequence of melanocyte stimulating hormone, His-DPhe-Arg-Trp-NH2(MSH4), which binds with low micromolar affinity to alpha melanocyte stimulating hormone receptors, were synthesized. Binding affinities of these constructs were evaluated in a competitive binding assay by competing with labeled ligands, Eu-DTPA-PEGO-MSH7 and/or Eu-DTPA-PEGO-NDP-alpha-MSH on the engineered cell line HEK293 CCK2R/hMC4R, which is genetically modified to over-express both the cholecystokinin 2 receptor (CCK2R) and human melanocortin 4 receptor (hMC4R). The oligomers were rapidly assembled using microwave-assisted copper catalyzed azide-alkyne cycloaddition between a dialkyne derivative of MSH4 and a diazide derivative of (Pro-Gly)3 as co-monomers. Three oligomer mixtures were further analyzed based on their degree of oligomerization and the route by which the MSH4 monomers were oligomerized, protected vs deprotected. Completive binding assay against Eu-DTPA-PEGO-MSH7 showed only a statistical enhancement of binding when calculated based on the total MSH4 concentration. However, when the calculation of avidity is based on an estimation of the particles numbers, there was a seven times enhancement of binding compared to a monovalent MSH4 control. The shorter linear multivalent MSH4 constructs were synthesized using ethylene glycol, glycerol, and mannitol as core scaffolds with maximum inter-ligand distances ranging from 27

  13. Spinel compounds as multivalent battery cathodes: A systematic evaluation based on ab initio calculations

    SciTech Connect

    Liu, Miao; Rong, Ziqin; Malik, Rahul; Canepa, Pieremanuele; Jain, Anubhav; Ceder, Gerbrand; Persson, Kristin A.

    2014-12-16

    In this study, batteries that shuttle multivalent ions such as Mg2+ and Ca2+ ions are promising candidates for achieving higher energy density than available with current Li-ion technology. Finding electrode materials that reversibly store and release these multivalent cations is considered a major challenge for enabling such multivalent battery technology. In this paper, we use recent advances in high-throughput first-principles calculations to systematically evaluate the performance of compounds with the spinel structure as multivalent intercalation cathode materials, spanning a matrix of five different intercalating ions and seven transition metal redox active cations. We estimate the insertion voltage, capacity, thermodynamic stability of charged and discharged states, as well as the intercalating ion mobility and use these properties to evaluate promising directions. Our calculations indicate that the Mn2O4 spinel phase based on Mg and Ca are feasible cathode materials. In general, we find that multivalent cathodes exhibit lower voltages compared to Li cathodes; the voltages of Ca spinels are ~0.2 V higher than those of Mg compounds (versus their corresponding metals), and the voltages of Mg compounds are ~1.4 V higher than Zn compounds; consequently, Ca and Mg spinels exhibit the highest energy densities amongst all the multivalent cation species. The activation barrier for the Al³⁺ ion migration in the Mn₂O₄ spinel is very high (~1400 meV for Al3+ in the dilute limit); thus, the use of an Al based Mn spinel intercalation cathode is unlikely. Amongst the choice of transition metals, Mn-based spinel structures rank highest when balancing all the considered properties.

  14. Programmable Nanoscaffolds That Control Ligand Display to a G-Protein-Coupled Receptor in Membranes To Allow Dissection of Multivalent Effects

    PubMed Central

    2015-01-01

    A programmable ligand display system can be used to dissect the multivalent effects of ligand binding to a membrane receptor. An antagonist of the A2A adenosine receptor, a G-protein-coupled receptor that is a drug target for neurodegenerative conditions, was displayed in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics was developed to interpret the binding data, suggesting the importance of receptor dimers. Using this model, extended multivalent arrangements of ligands were constructed with progressive improvements in binding to A2A. The results highlight the ability to use a highly controllable multivalent approach to determine optimal ligand valency and spacing that can be subsequently optimized for binding to a membrane receptor. Models explaining the multivalent binding data are also presented. PMID:25116377

  15. Programmable nanoscaffolds that control ligand display to a G-protein-coupled receptor in membranes to allow dissection of multivalent effects.

    PubMed

    Dix, Andrew V; Moss, Steven M; Phan, Khai; Hoppe, Travis; Paoletta, Silvia; Kozma, Eszter; Gao, Zhan-Guo; Durell, Stewart R; Jacobson, Kenneth A; Appella, Daniel H

    2014-09-01

    A programmable ligand display system can be used to dissect the multivalent effects of ligand binding to a membrane receptor. An antagonist of the A2A adenosine receptor, a G-protein-coupled receptor that is a drug target for neurodegenerative conditions, was displayed in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics was developed to interpret the binding data, suggesting the importance of receptor dimers. Using this model, extended multivalent arrangements of ligands were constructed with progressive improvements in binding to A2A. The results highlight the ability to use a highly controllable multivalent approach to determine optimal ligand valency and spacing that can be subsequently optimized for binding to a membrane receptor. Models explaining the multivalent binding data are also presented.

  16. Block Liposome and Nanotube Formation is a General Phenomenon of Two-Component Membranes Containing Multivalent Lipids.

    PubMed

    Zidovska, Alexandra; Ewert, Kai K; Quispe, Joel; Carragher, Bridget; Potter, Clinton S; Safinya, Cyrus R

    2011-01-01

    We report a study on the formation of block liposomes (BLs) and nanotubes from membranes comprised of mixtures of membrane curvature-stabilizing multivalent cationic lipids MVL3(3+) and MVL5(5+) with neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In conjunction with prior work on MVLBG2(16+), our experiments suggest that BL and nanotube formation is a general phenomenon in membranes containing multivalent lipids, thus enhancing the relevance of BLs for applications such as gene/drug storage and delivery or templating.

  17. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    PubMed

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

  18. Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations.

    PubMed

    Hellwig, S; Emde, F; Raven, N P; Henke, M; van Der Logt, P; Fischer, R

    2001-08-20

    In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.

  19. Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW

    PubMed Central

    Bertazzon, Miriam; Marczynke, Michaela; Seitz, Oliver; Volkmer, Rudolf; Haag, Rainer

    2015-01-01

    Summary The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein–protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 μM and 150 µM to the individual WW domains and with a K D of 150 μM to the tandem-WW1–WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome. PMID:26124874

  20. Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors.

    PubMed

    Junquera, Elena; Aicart, Emilio

    2016-07-01

    Due to the potential use as transfecting agents of nucleic acids (DNA or RNA), multivalent cationic non-viral vectors have received special attention in the last decade. Much effort has been addressed to synthesize more efficient and biocompatible gene vectors able to transport nucleic acids into the cells without provoking an immune response. Among them, the mostly explored to compact and transfect nucleic acids are: (a) gemini and multivalent cationic lipids, mixed with a helper lipid, by forming lipoplexes; and (b) cationic polymers, polycations, and polyrotaxanes, by forming polyplexes. This review is focused on the progress and recent advances experimented in this area, mainly during the present decade, devoting special attention to the lipoplexes and polyplexes, as follows: (a) to its biophysical characterization (mainly electrostatics, structure, size and morphology) using a wide variety of experimental methods; and (b) to its biological activity (transfection efficacy and cytotoxicity) addressed to confirm the optimum formulations and viability of these complexes as very promising gene vectors of nucleic acids in nanomedicine.

  1. Multivalent group A streptococcal vaccine elicits bactericidal antibodies against variant M subtypes.

    PubMed

    Dale, James B; Penfound, Thomas; Chiang, Edna Y; Long, Valerie; Shulman, Stanford T; Beall, Bernard

    2005-07-01

    Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.

  2. Multivalent IDP assemblies: Unique properties of LC8-associated, IDP duplex scaffolds.

    PubMed

    Clark, Sarah A; Jespersen, Nathan; Woodward, Clare; Barbar, Elisar

    2015-09-14

    A wide variety of subcellular complexes are composed of one or more intrinsically disordered proteins (IDPs) that are multivalent, flexible, and characterized by dynamic binding of diverse partner proteins. These multivalent IDP assemblies, of broad functional diversity, are classified here into five categories distinguished by the number of IDP chains and the arrangement of partner proteins in the functional complex. Examples of each category are summarized in the context of the exceptional molecular and biological properties of IDPs. One type - IDP duplex scaffolds - is considered in detail. Its unique features include parallel alignment of two IDP chains, formation of new self-associated domains, enhanced affinity for additional bivalent ligands, and ubiquitous binding of the hub protein LC8. For two IDP duplex scaffolds, dynein intermediate chain IC and nucleoporin Nup159, these duplex features, together with the inherent flexibility of IDPs, are central to their assembly and function. A new type of IDP-LC8 interaction, distributed binding of LC8 among multiple IDP recognition sites, is described for Nup159 assembly. PMID:26226419

  3. Multivalency of Sn in Cu2ZnSnS4

    NASA Astrophysics Data System (ADS)

    Biswas, Koushik; Lany, Stephan; Zunger, Alex

    2010-03-01

    The highly efficient ternary chalcopyrites such as 2(CuInSe2) = Cu2In2Se4 can be replaced as absorber materials in photovoltaic cells by Cu2ZnSnS4 (band gap ˜ 1.5 eV) in order to avoid the costly element In. Yet, the question remains whether a Fermi-level pinning defect can form spontaneously in these quaternary materials just as the InCu intrinsic DX centers in CuInSe2 [1]. Here we study theoretically the deep gap levels introduced by the SnCu and SnZn defects in Cu2ZnSnS4. We find that these originate from the multi-valency of Sn, which can change into a +II oxidation state instead of the normal +IV state. Such a transition can even occur for Sn on its native site. Thus, we compare to the respective defect behavior in the kesterite Cu2ZnGeSe4, which has a similar band gap, but a less pronounced multi-valency of the respective IV-valent element Ge. [4pt] [1] S. Lany and A. Zunger, Phys. Rev. Lett. 100, 016401 (2008).

  4. Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

    SciTech Connect

    Ratto, T V; Rudd, R E; Langry, K C; Balhorn, R L; McElfresh, M W

    2005-07-15

    We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

  5. Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans

    PubMed Central

    Broecker, Felix; Hanske, Jonas; Martin, Christopher E.; Baek, Ju Yuel; Wahlbrink, Annette; Wojcik, Felix; Hartmann, Laura; Rademacher, Christoph; Anish, Chakkumkal; Seeberger, Peter H.

    2016-01-01

    Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan–antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort. PMID:27091615

  6. Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

    PubMed Central

    Banjade, Sudeep; Wu, Qiong; Mittal, Anuradha; Peeples, William B.; Pappu, Rohit V.; Rosen, Michael K.

    2015-01-01

    The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins. PMID:26553976

  7. Phase transitions of multivalent proteins can promote clustering of membrane receptors

    PubMed Central

    Banjade, Sudeep; Rosen, Michael K

    2014-01-01

    Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones. DOI: http://dx.doi.org/10.7554/eLife.04123.001 PMID:25321392

  8. Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines from China.

    PubMed

    Cook, Matthew C; Gibeault, Sabrina; Filippenko, Vasilisa; Ye, Qiang; Wang, Junzhi; Kunkel, Jeremy P

    2013-07-01

    The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine - which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.

  9. Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy.

    PubMed

    Jiang, Kuo; Li, Jia; Yin, Jipeng; Ma, Qiong; Yan, Bo; Zhang, Xiang; Wang, Lei; Wang, Lifeng; Liu, Tao; Zhang, Yinglong; Fan, Qingyu; Yang, Angang; Qiu, Xiuchun; Ma, Baoan

    2015-08-01

    Therapeutics based on short interfering RNAs (siRNAs) have great potential to treat human diseases. However, the clinical application of siRNAs has been limited by their poor intracellular uptake, low serum stability, and inability to target specific cells. In this study, we addressed this lack of specificity by synthesizing a molecularly targeted CXCR4-siRNA (CXCR4si) for the treatment of HER2(+) breast cancers using a HER2-scFv-arginine nonamer peptide fusion protein (e23sFv-9R) as an siRNA carrier. The e23sFv-9R binding siRNA is able to specifically deliver the siRNA to HER2(+) breast cancer cells and concentrate and persist in orthotopic HER2(+) breast cancer xenografts for at least 36 h. CXCR4si delivered by e23sFv-9R inhibited CXCR4 gene expression, reduced proliferation and metastasis and induced apoptosis in the HER2(+) breast cancer BT-474 cell line in vitro. Moreover, the systemic delivery of CXCR4si by e23sFv-9R is able to suppress tumor growth, reduce metastasis and prolong survival in mice bearing HER2(+) xenografts. This approach causes no systemic toxicity and does not activate the innate immune response, suggesting that a fusion protein carrying CXCR4si shows promise in the treatment of HER2-overexpressing breast cancer. PMID:25956853

  10. FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing.

    PubMed

    Wang, Z; Dove, P; Wang, X; Shamas-Din, A; Li, Z; Nachman, A; Oh, Y J; Hurren, R; Ruschak, A; Climie, S; Press, B; Griffin, C; Undzys, E; Aman, A; Al-awar, R; Kay, L E; O'Neill, D; Trudel, S; Slassi, M; Schimmer, A D

    2015-01-01

    Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.

  11. Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

    PubMed Central

    Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E.; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D.; Heywood, Sam; Humphreys, David P.

    2016-01-01

    ABSTRACT An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG. PMID:27532598

  12. The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides.

    PubMed

    Zhang, Yueping; Choi, Yoon-E; Zou, Xuexiao; Xu, Jin-Rong

    2011-02-01

    Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity. PMID:20887797

  13. The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides.

    PubMed

    Zhang, Yueping; Choi, Yoon-E; Zou, Xuexiao; Xu, Jin-Rong

    2011-02-01

    Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity.

  14. Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

    PubMed

    Onda, M; Nagata, S; Tsutsumi, Y; Vincent, J J; Wang, Q; Kreitman, R J; Lee, B; Pastan, I

    2001-07-01

    Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity. PMID:11431343

  15. Prediction of FV520B Steel Flow Stresses at High Temperature and Strain Rates

    NASA Astrophysics Data System (ADS)

    Han, Xiaolan; Zhao, Shengdun; Zhang, Chenyang; Fan, Shuqin; Xu, Fan

    2015-10-01

    In order to develop reliable constitutive equations for the simulation, the hot deformation behavior of FV520B steel was investigated through isothermal compression tests in a wide range of temperatures from 900 °C to 1100 °C at an interval of 50 °C and strain rate from 0.01 to 10 s-1 on Gleeble-1500D simulator. The effects of temperature and strain rate on deformation behavior were represented by Zener-Holloman parameter in an exponent-type equation of Arrhenius constitutive. The influence of strain was incorporated in the constitutive analysis by material constants expressed as a polynomial function of strain. The constitutive equation (considering the compensation of strain) could precisely predict the flow stress only at strain rate 0.01 s-1 except at the temperatures of 900 °C and 1000 °C, whereas the flow stress predicted by a modified equation (incorporating both the strain and strain rate) demonstrated a well agreement with the experimental data throughout the entire range of temperatures and strain rates. Correlation coefficient (R) of 0.988 and average absolute relative error (AARE) of 5.7% verified the validity of developed equation from statistical analysis, which further confirmed that the modified constitutive equation could accurately predict the flow stress of FV520B steel.

  16. Membrane Fusion Mediated Targeted Cytosolic Drug Delivery Through scFv Engineered Sendai Viral Envelopes.

    PubMed

    Kumar, M; Mani, P; Pratheesh, P; Chandra, S; Jeyakkodi, M; Chattopadhyay, P; Sarkar, D P; Sinha, S

    2015-01-01

    Antibody targeted cytoplasmic delivery of drugs is difficult to achieve as antigen-antibody interaction results in the payload being directed to the endosomal compartment. However, Sendai viral envelopes can bring about cytoplasmic delivery due to F-protein mediated membrane fusion. In this study we have generated and fused a recombinant scFv directed to the onco-fetal antigen, the Placental isozyme of Alkaline Phosphatase (PAP) with the trans-membrane and part of the cytoplasmic domain of the Sendai F protein (F(TMC)). Reconstituted virosomes, having both the fusion protein as well as the native F-protein were able to specifically bind and deliver drugs to PAP expressing cells. About 75% of the delivery was cytoplasmic in nature. Hence, this immuno-virosome, which is devoid of the comparatively more toxic HN protein, has the novel ability to combine specific antibody mediated targeting with cytoplasmic delivery. The scFv ensured specific binding to PAP expressing cells, without cross reacting with the other isozymes of alkaline phosphatase. The advantages of cytoplasmic delivery would include reduced degradation and lowered immunogenicity of the payload and carrier. The ubiquitous expression of PAP on a variety of cancers like seminoma, choriocarcinoma, cervical and breast cancers also suggests its potential usefulness in a number of malignancies.

  17. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity.

    PubMed

    Bai, Xuelian; Kim, Jihye; Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo

    2015-01-01

    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications.

  18. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity

    PubMed Central

    Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo

    2015-01-01

    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications. PMID:26484868

  19. Pyrene-based heparin sensors in competitive aqueous media - the role of self-assembled multivalency (SAMul).

    PubMed

    Chan, Ching W; Smith, David K

    2016-03-01

    Amine-functionalised pyrene derivatives are reported and their ability to detect heparin via a fluorescent response determined - different responses are observed dependent on whether self-assembled multivalent binding between sensor and analyte takes place, and ratiometric heparin sensors which can detect this surgically-relevant polyanion in competitive media are reported.

  20. Optimizing the Multivalent Binding of the Bacterial Lectin LecA by Glycopeptide Dendrimers for Therapeutic Purposes.

    PubMed

    Bouvier, Benjamin

    2016-06-27

    Bacterial lectins are nonenzymatic sugar-binding proteins involved in the formation of biofilms and the onset of virulence. The weakness of individual sugar-lectin interactions is compensated by the potentially large number of simultaneous copies of such contacts, resulting in high overall sugar-lectin affinities and marked specificities. Therapeutic compounds functionalized with sugar residues can compete with the host glycans for binding to lectins only if they are able to take advantage of this multivalent binding mechanism. Glycopeptide dendrimers, featuring treelike topologies with sugar moieties at their leaves, have already shown great promise in this regard. However, optimizing the dendrimers' amino acid sequence is necessary to match the dynamics of the lectin active sites with that of the multivalent ligands. This work combines long-time-scale coarse-grained simulations of dendrimers and lectins with a reasoned exploration of the dendrimer sequence space in an attempt to suggest sequences that could maximize multivalent binding to the galactose-specific bacterial lectin LecA. These candidates are validated by simulations of mixed dendrimer/lectin solutions, and the effects of the dendrimers on lectin dynamics are discussed. This approach is an attractive first step in the conception of therapeutic compounds based on the dendrimer scaffold and contributes to the understanding of the various classes of multivalency that underpin the ubiquitous "sugar code". PMID:27223679