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Sample records for multivalent single-chain fv

  1. 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

  2. A single-chain Fv reactive with the Goodpasture antigen.

    PubMed

    Ross, C N; Turner, N; Savage, P; Cashman, S J; Spooner, R A; Pusey, C D

    1996-06-01

    Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

  3. Production and characterization of a single chain variable fragment (scFv) for the mycotoxin deoxynivalenol

    USDA-ARS?s Scientific Manuscript database

    Deoxynivalenol (DON)is a mycotoxin produced by certain fungi that infest cereal grains worldwide. A hybridoma cell line producing a monoclonal antibody (Mab) recognizing DON was used as the starting point in the development of a recombinant single chain variable fragment (scFv) antibody. The scFv wa...

  4. Avidity-mediated enhancement of in vivo tumor targeting by single-chain Fv dimers.

    PubMed

    Adams, Gregory P; Tai, Mei-Sheng; McCartney, John E; Marks, James D; Stafford, Walter F; Houston, L L; Huston, James S; Weiner, Louis M

    2006-03-01

    Radiolabeled single-chain Fv (sFv) molecules display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the absolute quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv molecules (Mr 27,000) and by dissociation of the monovalent sFv from tumor-associated antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFv')2] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFv'. However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention associated with the larger size of 54 kDa [(sFv')2] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent anti-c-erbB-2/HER2/neu 741F8-1 (sFv')2 homodimers with monovalent 741F8/26-10 (sFv')2 heterodimers (Mr 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over molecular weight in accounting for the superior tumor retention of 741F8-1 (sFv')2 homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 hours post i.v. injection was determined for 125I-741F8-1 (sFv')2 (3.57 %ID/g), 125I-741F8/26-10 (sFv')2 (1.13 %ID/g), and 125I-741F8-1 sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFv')2 homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers.

  5. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. 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. Published by Elsevier B.V.

  7. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    PubMed

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  8. Improved microtitre plate production of single chain Fv fragments in Escherichia coli.

    PubMed

    Hust, Michael; Steinwand, Miriam; Al-Halabi, Laila; Helmsing, Saskia; Schirrmann, Thomas; Dübel, Stefan

    2009-09-01

    The new era of functional genomics demands several antibodies as specific detection reagents for proteins, their complexes and post-translational modifications. Only in vitro antibody selection technologies are able to provide the required throughput to generate these large numbers. Phage display is the most widely used technology for in vitro selection of antibodies. The major bottleneck of a phage display selection pipeline is the production of monoclonal antibody fragments for screening and further analysis. In this study, we describe the development of improved protocols for the production of single chain Fv (scFv) antibody fragments in 96-well microtitre plates (MTPs) in Escherichia coli. Four scFvs were expressed using the antibody expression vector pOPE101-XP to analyse the influence of a set of different parameters on their production. Further, six scFvs were expressed using the phage display vector pHAL14 to investigate the effect on the production of functional scFvs using those parameters that improved production from pOPE101-XP. Yield in MTPs was influenced by a variety of conditions and was also strongly dependent on the individual scFv clone. Although it was not possible to deduce a single set of optimal parameters applicable to all the tested scFvs, a combined protocol was developed which improved the expression of scFv fragments over standard methods.

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

    USDA-ARS?s Scientific Manuscript database

    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. Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner.

    PubMed

    Tokunaga, Masao; Mizukami, Makoto; Yamasaki, Koji; Tokunaga, Hiroko; Onishi, Hiromasa; Hanagata, Hiroshi; Ishibashi, Matsujiro; Miyauchi, Akira; Tsumoto, Kouhei; Arakawa, Tsutomu

    2013-10-01

    Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.

  11. RECOMBINANT SINGLE CHAIN VARIABLE FRAGMENT ANTIBODIES (scFv) AGAINST Pro144-Leu155 FRAGMENT OF HUMAN PROTEIN C.

    PubMed

    Oliinyk, O S; Palyvoda, K O; Lugovskaya, N E; Kolibo, D V; Lugovskoy, E V; Komisarenko, S V

    2015-01-01

    The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.

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

    USDA-ARS?s Scientific Manuscript database

    ‘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. Isolation of BNYVV coat protein-specific single chain Fv from a mouse phage library antibody.

    PubMed

    Jahromi, Zahra Moghaddassi; Salmanian, Ali Hatef; Rastgoo, Nasrin; Arbabi, Mehdi

    2009-10-01

    Beet necrotic yellow vein virus (BNYVV) infects sugar beet plants worldwide and is responsible for the rhizomania disease and severe economic losses. Disease severity and lack of naturally occurring resistant plants make it very difficult to control the virus, both from epidemiological and economic standpoints. Therefore, early detection is vital to impose hygiene restrictions and prevent further spread of the virus in the field. Immunoassays are one of the most popular methodologies for the primary identification of plant pathogens including BNYVV since they are robust, sensitive, fast, and inexpensive. In this study, the major coat protein (CP21) of BNYVV was cloned and expressed in Escherichia coli. Thereafter, mice were immunized with purified CP21 and a phage antibody library was constructed from their PCR-amplified immunoglobulin repertoire. Following filamentous phage rescue of the library and four rounds of panning against recombinant CP21 antigen, several specific single chain Fv fragments were isolated and characterized. This approach may pave the way to develop novel immunoassays for a rapid detection of viral infection. Moreover, it will likely provide essential tools to establish antibody-mediated resistant transgenic technology in sugar beet plants.

  14. [Optimization of the preparation process for fusion protein Fv-LDP that composes lidamycin apoprotein and single-chain Fv antibody directed against type IV collagenase].

    PubMed

    Gao, Rui-Juan; Zhao, Chun-Yan; Li, Dian-Dong; Zhen, Yong-Su

    2013-10-01

    This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.

  15. Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y.

    PubMed

    Rouis, Souad; Lafaye, Pierre; Jaoua-Aydi, Leila; Sghaier, Zidani; Ayadi, Hammadi; Gargouri-Bouzid, Radhia

    2006-10-01

    Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

  16. Expression of single-chain Fv gene specific for gamma-seminoprotein by RTS and its biological activity identification.

    PubMed

    Han, Yuedong; Haun, Yi; Deng, Jinlan; Gao, Feng; Pan, Bifeng; Cui, Daxiang

    2006-01-01

    Fabricating a single-chain variable fragment specific for human seminoprotein is very important in antibody-directed enzyme prodrug therapy and NMR imaging for prostate cancer. Here a single-chain Fv specific for gamma-seminoprotein was expressed by RTS. Its activity and the efficiency of entry into prostate cancer cells are investigated by immunoprecipitation and Western blotting and immunofluorescent staining, as well as entry of conjugated magnetic beads into cells. Results showed that ScFv peptides specific for gamma-seminoprotein were successfully prepared, which can bind with the prostate cells specifically and can bring magnetic beads into prostate cancer cells within 15 min, the amount of magnetic beads inside prostate cancer cells increased as the culture time prolonged. ScFv-conjugated magnetic beads did not enter into control cells. In conclusion, the ScFv peptide against human gamma-seminoprotein with biological activity was successfully fabricated, which can take magnetic beads to prostate cancer cells specifically and not to the control cells. This ScFv peptide against human gamma-seminoprotein should be useful in improving the detection and therapy of prostate cancer at early stages and NMR imaging.

  17. Single chain Fv fragment specific for human GM-CSF: selection and expression using a bacterial expression library.

    PubMed

    Tapryal, Suman; Pal Khasa, Yogender; Mukherjee, K J

    2010-10-01

    Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti-human granulocyte macrophage colony-stimulating factor (hGM-CSF) scFv as an immunoassay bio-reagent, utilizing an easily scalable bacterial expression system. For this, the V(H) and V(L) gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre-sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross-reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross-reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM-CSF, measured by surface plasmon resonance, was found to be within the physiological range (K(D) =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM-CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein.

  18. Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Tadakuma, Tomomi; Hayashi, Hideki; Inoue, Kazuyuki; Itoh, Kunihiko

    2010-01-01

    We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  19. Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

    PubMed

    Khantasup, Kannika; Chantima, Warangkana; Sangma, Chak; Poomputsa, Kanokwan; Dharakul, Tararaj

    2015-12-01

    Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

  20. Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco.

    PubMed

    Almquist, Kurt C; McLean, Michael D; Niu, Yongqing; Byrne, Greg; Olea-Popelka, Fernando C; Murrant, Coral; Barclay, Jack; Hall, J Christopher

    2006-03-15

    Botulinum neurotoxins (BoNTs) are the most poisonous substances known and are thus classified as high-risk threats for use as bioterror agents. To examine the potential of transgenic plants as bioreactors for the production of BoNT antidotes, we transformed tobacco with an optimized, synthetic gene encoding a botulinum neurotoxin A (BoNT/A) neutralizing single-chain Fv (scFv) recombinant antibody fragment. In vitro mouse muscle twitch assays demonstrated the functional utility of this scFv extracted from tobacco for neutralizing the paralytic effects of BoNT/A at neuromuscular junctions. Based on the efficiency of the scFv capture process and the dose required to antidote a human being, 1-2 ha of this tobacco could yield up to 4 kg of scFv, which would be enough to contribute to the manufacture of 1,000,000 therapeutic doses of a monoclonal antibody (mAb) cocktail capable of neutralizing the effects of BoNT poisoning. Transgenic plants could provide an inexpensive production platform for expression of multiple mAbs toward the creation of polyclonal therapies (i.e. pooled mAbs) as the next improvement in recombinant antibody therapy.

  1. [Construction, expression and functional characterization of single chain variable fragments (scFv) against human CD33 antigen].

    PubMed

    Chen, Xiao-Jun; Wang, Yang; Qu, Hao; Ge, Xin-Shun; Zuo, Yu-Feng; Liao, Xiao-Long

    2007-12-01

    To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen, and characterize its bioactivity. The genes encoding the light and heavy chain variable regions were cloned by RT-PCR from a murine hybridoma cell line, which could produce monoclonal antibody(mAb) against human CD33 antigen. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid (Gly(4)Ser)(3) using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+) and expressed in E.coli Rosetta after induction by IPTG. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta, and the purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni(2+) metal affinity chromatography and protein refolding. Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen. Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.

  2. CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies.

    PubMed

    Vaday, Gayle G; Hua, Shao-Bing; Peehl, Donna M; Pauling, Michelle H; Lin, Yu-Huei; Zhu, Li; Lawrence, Diana M; Foda, Hussein D; Zucker, Stanley

    2004-08-15

    Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities. We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion. Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels. Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

  3. Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

    PubMed Central

    Kapelski, Stephanie; de Almeida, Melanie; Fischer, Rainer; Barth, Stefan

    2014-01-01

    We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B. PMID:25313223

  4. Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-single-chain Fv fusion protein.

    PubMed

    Kapelski, Stephanie; de Almeida, Melanie; Fischer, Rainer; Barth, Stefan; Fendel, Rolf

    2015-01-01

    We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B.

  5. 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.

  6. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum.

    PubMed

    Wajanarogana, Sumet; Prasomrothanakul, Teerawat; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2006-04-01

    Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

  7. Construction and expression of single-chain Fv antibody against human bladder carcinoma.

    PubMed

    Yu, L Z; Xiao, S; Huang, H L; Gu, Z; Gu, F L; Guo, Y L

    1996-01-01

    We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR). The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma. The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression. The recombinants were sequenced by Sanger's method. It was proved that the full lengths of the VH and VK genes were 366 and 324 bp, respectively. Compared with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from the rearrangement of VH, Dsp2.2 and JH4. The VK gene was VK subgroup IV and from VK and JK4. The VH and VK genes was inserted expression vector pWAI80. By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected. We suggest that ScFv antibody recognized the antigen specifically.

  8. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    PubMed

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.

  9. Production of single chain fragment variable (scFv) antibodies in Escherichia coli using the LEX™ bioreactor.

    PubMed

    Miethe, Sebastian; Meyer, Torsten; Wöhl-Bruhn, Stefanie; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Hust, Michael

    2013-01-20

    For proteome research, antibodies against a growing number of antigens must be generated and characterized. The high throughput generation of antibody fragments, using in vitro selection, requires bacterial expression of antibody fragments. This created a need to establish an expression method to improve the parallel production of many antibody fragments. In this study, we describe the development of a high throughput bacterial production method for single chain fragment variables (scFvs) using shaking flasks or the LEX™ bioreactor. We compared the influence of a set of production parameters on Escherichia coli production of four different scFv. The results led to an optimized protocol for the parallel production of multiple antibody fragments. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. The expression of a single-chain Fv antibody fragment in different plant hosts and tissues by using Potato virus X as a vector.

    PubMed

    Roggero, P; Ciuffo, M; Benvenuto, E; Franconi, R

    2001-06-01

    Some aspects of the expression of a single-chain Fv antibody fragment (scFv) driven by the plant viral vector Potato virus X (PVX) have been studied by quantitative ELISA. After inoculation of the infectious transcript, the vector was stable only for a few passages of sap transmission in the inoculated leaves of Nicotiana benthamiana and the reversal to wild type was more pronounced in the systemically invaded leaves. The amount of synthesized scFv varied when different solanaceous hosts were tested, being generally higher and less variable in inoculated than in systemically invaded leaves. In tomato and Datura stramonium the scFv was synthesized only in the inoculated leaves. The scFv was also synthesized in the PVX local hosts Chenopodium amaranticolor and C. quinoa. No correlation was found between PVX and scFv concentration in the inoculated and systemically invaded leaves of N. benthamiana and N. clevelandii. Copyright 2001 Academic Press.

  11. Design and synthesis of germline-based hemi-humanized single-chain Fv against the CD18 surface antigen.

    PubMed

    Caldas, C; Coelho, V P; Rigden, D J; Neschich, G; Moro, A M; Brígido, M M

    2000-05-01

    The 6.7 murine monoclonal antibody (mAb) recognizes the human CD18 antigen and is therefore of interest as an anti-inflammatory agent. The 6.7 heavy variable chain (VH) was humanized using the closest human germline sequence as the template on to which to graft the murine complementary determining regions (CDRs). Two versions were proposed, one in which the residue proline 45 of the murine form was maintained and another in which this framework residue was changed to the leucine found in the human sequence. These VH humanized versions were expressed in the yeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs), with the VL from the murine antibody. The scFv from the murine antibody was also expressed. The binding activities of the murine and both hemi-humanized scFvs were determined by flow cytometry analysis. All the constructions were able to recognize human lymphocytes harboring CD18, indicating successful humanization with transfer of the original binding capability. Some differences between the two hemi-humanized versions were observed. The method used was simple and straightforward, with no need for refined structural analyses and could be used for the humanization of other antibodies.

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

    PubMed

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

    2010-11-01

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

  13. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

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

    PubMed

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

    2014-06-01

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

  15. High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells.

    PubMed

    Gilmartin, Allissia A; Lamp, Benjamin; Rümenapf, Till; Persson, Mats A A; Rey, Félix A; Krey, Thomas

    2012-02-01

    Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (V(H)) and light (V(L)) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of V(H) and V(L) domains, further validating the here-reported expression system.

  16. Construction and analysis of three-dimensional graphic model of single-chain Fv derived from an anti-human placental acidic isoferritin monoclonal antibody by computer.

    PubMed

    Zhou, C; Shen, G; Zhu, H; Yang, J; Zhang, Y; Feng, J; Shen, B

    2000-01-01

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

  17. 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.

  18. Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants.

    PubMed

    McCormick, A A; Kumagai, M H; Hanley, K; Turpen, T H; Hakim, I; Grill, L K; Tusé, D; Levy, S; Levy, R

    1999-01-19

    Rapid production of protein-based tumor-specific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 micrograms/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.

  19. Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1

    PubMed Central

    Ray, K; Embleton, M J; Jailkhani, B L; Bhan, M K; Kumar, R

    2001-01-01

    We have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 109 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment. PMID:11472431

  20. 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

  1. 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.

  2. 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

  3. IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

    PubMed

    Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

    2017-06-26

    IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

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

    PubMed

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

    2014-09-01

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

  5. Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones.

    PubMed

    Sonoda, Hiroyuki; Kumada, Yoichi; Katsuda, Tomohisa; Yamaji, Hideki

    2010-04-01

    The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expression of functional scFvs is increased by co-expression of molecular chaperones. In the present study, we examined the effects of the combination of Trx fusion and molecular chaperone co-expression on the production of a functional scFv. A Trx-fused scFv was obtained in the oxidizing cytoplasm, and co-expression of GroELS and trigger factor had the greatest effect, resulting in a 2.8-fold increase in specific productivity. By contrast, the molecular chaperone DnaKJE had no effect. Moreover, co-expression of DnaKJE with GroELS negated the effects of GroELS. Trx-scFv was purified using a bovine ribonuclease A-coupled Sepharose column, and 2.7 mg/L of purified protein was obtained. Soluble Trx-scFv, expressed and purified as described above, exhibited pH-dependent binding similar to that of the parental full-length antibody. In addition, approximately 80% of the initial binding activity was retained after incubation at 37 degrees C for 2 weeks, indicating that the Trx-scFv fusion protein is quite stable. This strategy might be useful for the preparation of other recombinant scFvs. (c) 2009 Elsevier Inc. All rights reserved.

  6. High-Level Expression of Single-Chain Fv-Fc Fusion Protein in Serum and Egg White of Genetically Manipulated Chickens by Using a Retroviral Vector

    PubMed Central

    Kamihira, Masamichi; Ono, Ken-ichiro; Esaka, Kazuhisa; Nishijima, Ken-ichi; Kigaku, Ryoko; Komatsu, Hiroyuki; Yamashita, Takashi; Kyogoku, Kenji; Iijima, Shinji

    2005-01-01

    We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs. PMID:16103139

  7. 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.

  8. Construction of a single chain variable fragment antibody (scFv) against tetrodotoxin (TTX) and its interaction with TTX.

    PubMed

    Wang, Rongzhi; Huang, Ailing; Liu, Licai; Xiang, Shuangshuang; Li, Xiufeng; Ling, Sumei; Wang, Lei; Lu, Tun; Wang, Shihua

    2014-06-01

    Tetrodotoxin (TTX) is a small molecular weight neurotoxin that occludes voltage-gated sodium channels in nerve and muscle tissue, resulting in respiratory paralysis and death. A high affinity antibody that can neutralize the toxicity of TTX is still lacking, so it is very important to prepare an antibody for TTX therapy and detection. In the present study, a chemical method was used to prepare the tetrodotoxin complete antigen, and a small amount, repeatedly immunity way was carried to immunize 4 mice. The amplified genes encoding monoclonal antibodies against TTX were used to construct the phage display library. After six rounds of biopanning, an antibody named scFv-T53 was characterized from clones showing high affinity and specific to TTX, and its affinity constant was 1.1 × 10(6) L/mol. Three dimensional structure of the scFv-T53 was constructed by computer modeling, and TTX was docked to the scFv-T53 model to obtain the structure of the binding complex. Two predicted essential amino acids, K183 and I189, were mutated to verify the theoretical model. Both mutants lost binding activity significantly against TTX as predicted by the theoretical model. Hence, the above results will be useful for screening the high affinity anti-TTX scFv mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    PubMed

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

  10. Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro.

    PubMed

    Robinson, M K; Hodge, K M; Horak, E; Sundberg, A L; Russeva, M; Shaller, C C; von Mehren, M; Shchaveleva, I; Simmons, H H; Marks, J D; Adams, G P

    2008-11-04

    Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

  11. 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

  12. 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.

  13. 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.

  14. Construction of single-chain Fv with two possible CDR3H conformations but similar inter-molecular forces that neutralize bovine herpesvirus 1.

    PubMed

    Koti, Madhuri; Farrugia, William; Nagy, Eva; Ramsland, Paul A; Kaushik, Azad K

    2010-02-01

    Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V(lambda)-V(H) orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V(lambda) and V(H). Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V(lambda) and V(H). Despite such potential configurational diversity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle.

  15. Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation.

    PubMed

    Jung, Yuchul; Rhee, Yong; Auh, Chung-Kyoon; Shim, Hyekyung; Choi, Jung-Jin; Kwon, Suk-Tae; Yang, Joo-Sung; Kim, Donggiun; Kwon, Myung-Hee; Kim, Yong-Sung; Lee, Sukchan

    2009-10-01

    We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.

  16. Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture.

    PubMed

    Mousli, Mohamed; Turki, Imène; Kharmachi, Habib; Saadi, Mohamed; Dellagi, Koussay

    2007-12-01

    The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.

  17. 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.

  18. 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

  19. Crystal structure of anti-polysialic acid antibody single chain Fv fragment complexed with octasialic acid: insight into the binding preference for polysialic acid.

    PubMed

    Nagae, Masamichi; Ikeda, Akemi; Hane, Masaya; Hanashima, Shinya; Kitajima, Ken; Sato, Chihiro; Yamaguchi, Yoshiki

    2013-11-22

    Polysialic acid is a linear homopolymer of α2-8-linked sialic acids attached mainly onto glycoproteins. Cell surface polysialic acid plays roles in cell adhesion and differentiation events in a manner that is often dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies have DP-dependent antigenic specificity, and such antibodies are widely utilized in biological studies for detecting and distinguishing between different oligo/polysialic acids. A murine monoclonal antibody mAb735 has a unique preference for longer polymers of polysialic acid (DP >10), yet the mechanism of recognition at the atomic level remains unclear. Here, we report the crystal structure of mAb735 single chain variable fragment (scFv735) in complex with octasialic acid at 1.8 Å resolution. In the asymmetric unit, two scFv735 molecules associate with one octasialic acid. In both complexes of the unit, all the complementarity-determining regions except for L3 interact with three consecutive sialic acid residues out of the eight. A striking feature of the complex is that 11 ordered water molecules bridge the gap between antibody and ligand, whereas the direct antibody-ligand interaction is less extensive. The dihedral angles of the trisialic acid unit directly interacting with scFv735 are not uniform, indicating that mAb735 does not strictly favor the previously proposed helical conformation. Importantly, both reducing and nonreducing ends of the bound ligand are completely exposed to solvent. We suggest that mAb735 gains its apparent high affinity for a longer polysialic acid chain by recognizing every three sialic acid units in a paired manner.

  20. Intrathecal administration of single-chain immunotoxin, LMB-7 [B3(Fv)-PE38], produces cures of carcinomatous meningitis in a rat model.

    PubMed Central

    Pastan, I H; Archer, G E; McLendon, R E; Friedman, H S; Fuchs, H E; Wang, Q C; Pai, L H; Herndon, J; Bigner, D D

    1995-01-01

    LMB-7 [B3(Fv)-PE38] is a single-chain immunotoxin constructed from the murine monoclonal antibody B3 and a truncated from of Pseudomonas exotoxin PE38. Antibody B3 recognizes a carbohydrate epitope found on solid tumors that frequently invade the intrathecal space and cause neoplastic meningitis. We tested the therapeutic value of intrathecally administered LMB-7 by using a model of human neoplastic meningitis in athymic rats. This model is representative of a clinical situation in that antibody B3 cross-reacts with a number of normal tissues that can be used to monitor potential systemic toxicity. Treatment was begun 3 days after A431 tumor implantation. Without treatment, the animals median survival was 10 days. Intrathecal administration of 10 micrograms of LMB-7 in 40 microliters on days 3, 5, and 7 produced 4 of 10 and 8 of 10 long-term survivors (> 170 days) in two experiments. Of the long-term survivors, 2 of 4 and 7 of 8 survivors had no microscopic evidence of tumor and were considered histologic cures. Lack of significant toxicity in the effective dose range and specificity make LMB-7 an excellent candidate for intrathecal treatment of neoplastic meningitis in humans. PMID:7708720

  1. Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae.

    PubMed

    Jang, ShinA; Kim, Gyuhee; Oh, Jihye; Lee, Seungyeop; Kim, Dongho; Kim, Kook-Han; Kim, Yong Ho; Rhee, Dong-Kwon; Lee, Sangho

    2017-01-01

    Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.

  2. Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems.

    PubMed

    Della Cristina, Pietro; Castagna, Monica; Lombardi, Alessio; Barison, Erika; Tagliabue, Giovanni; Ceriotti, Aldo; Koutris, Ilias; Di Leandro, Luana; Giansanti, Francesco; Vago, Riccardo; Ippoliti, Rodolfo; Flavell, Sopsamorn U; Flavell, David J; Colombatti, Marco; Fabbrini, Maria Serena

    2015-02-13

    Antibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats. An anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range. We undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in

  3. Efficient production of anti-fluorescein and anti-lysozyme as single-chain anti-body fragments (scFv) by Brevibacillus expression system.

    PubMed

    Onishi, Hiromasa; Mizukami, Makoto; Hanagata, Hiroshi; Tokunaga, Masao; Arakawa, Tsutomu; Miyauchi, Akira

    2013-10-01

    Expression of scFv in Brevibacillus choshinensis was tested using combinations of three different promoters and four different secretion signals. Two model scFv constructs, i.e., His-scFvFLU and His-scFvHEL, were successfully expressed with some of the combinations. Ni Sepharose column and size exclusion chromatography resulted in fairly pure preparations of these two proteins. The purified His-scFvFLU inhibited fluorescence from fluorescein, while the purified His-scFvHEL inhibited lysozyme activity. Relatively high yield of His-scFvFLU (∼40%) and His-scFvHEL (∼30%) was achieved with the expression and purification system described here.

  4. Towards the improvement in stability of an anti-Aβ single-chain variable fragment, scFv-h3D6, as a way to enhance its therapeutic potential.

    PubMed

    Montoliu-Gaya, Laia; Murciano-Calles, Javier; Martinez, Jose C; Villegas, Sandra

    2017-09-01

    ScFv-h3D6 is a single-chain variable fragment derived from the monoclonal antibody bapineuzumab that prevents Aβ-induced cytotoxicity by capturing Aβ oligomers. The benefits of scFv-h3D6 treatment in Alzheimer's disease are known at the behavioural, cellular and molecular levels in the 3xTg-AD mouse model. Antibody-based therapeutics are only stable in a limited temperature range, so their benefit in vivo depends on their capability for maintaining the proper fold. Here, we have stabilized the scFv-h3D6 folding by introducing the mutation VH-K64R and combining it with the previously described elongation of the VL domain (C3). The stabilities of the different scFv-h3D6 constructs were calculated from urea and thermal denaturation followed by Trp-fluorescence, CD and DSC and resulted in the order C3 > K64R/C3 > VH-K64R ≥ scFv-h3D6; showing that the combination of both mutations was not additive, instead they partially cancelled each other. The three mutants assayed showed a decreased aggregation tendency but maintained their capability to aggregate in the form of worm-like fibrils, basis of the protective effect of scFv-h3D6. Cytotoxicity assays showed that all the mutants recovered cell viability of Aβ-treated neuroblastoma cell cultures in a dose-dependent manner and with efficiencies that correlated with stability, therefore improving the therapeutic ability of this antibody.

  5. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli.

    PubMed

    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing

    2014-12-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.

  6. The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

    PubMed

    Qian, Liren; Li, Dan; Ma, Lie; He, Ting; Qi, Feifei; Shen, Jianliang; Lu, Xin-An

    2016-01-01

    The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19.

  7. Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

    PubMed Central

    Rippmann, Jörg F.; Klein, Michaela; Hoischen, Christian; Brocks, Bodo; Rettig, Wolfgang J.; Gumpert, Johannes; Pfizenmaier, Klaus; Mattes, Ralf; Moosmayer, Dieter

    1998-01-01

    Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic

  8. In vivo distribution of single chain variable fragment (scFv) against atherothrombotic oxidized LDL/β2-glycoprotein I complexes into atherosclerotic plaques of WHHL rabbits: Implication for clinical PET imaging.

    PubMed

    Sasaki, Takanori; Kobayashi, Kazuko; Kita, Shoichi; Kojima, Kazuo; Hirano, Hiroyuki; Shen, Lianhua; Takenaka, Fumiaki; Kumon, Hiromi; Matsuura, Eiji

    2017-02-01

    Oxidized LDL (oxLDL) can exist as a complex with β2-glycoprotein I (β2GPI) in plasma/serum of patients with non-autoimmune atherosclerotic disease or antiphospholipid syndrome (APS). Nonetheless, direct in vivo evidence supporting the pathophysiological involvement of oxLDL/β2GPI complexes and specific autoantibody against the complexes in developing atherothrombosis has yet been established. In the present study, we demonstrated in vivo distribution of single chain variable fragment of IgG anti-oxLDL/β2GPI complexes (3H3-scFv) in Watanabe heritable hyperlipidemic (WHHL) rabbits by PET/CT imaging. An antibody-based PET probe, (64)Cu-3H3-scFv, was established, and WHHL rabbits were applied for a non-autoimmune atherosclerotic model to demonstrate in vivo distribution of the probe. 3H3-scFv has exhibits specificity towards β2GPI complexed with oxLDL but neither a free form of β2GPI nor oxLDL alone. Post-intravenous administration of (64)Cu-3H3-scFv into WHHL rabbits has demonstrated a non-invasive approach for in vivo visualization of atherosclerotic lesion. The imaging probe achieved ideal blood clearance and distribution for optimal imaging capacity in 24h, significantly shorter than that of an intact IgG-based imaging probe. (64)Cu-3H3-scFv targeted on atherosclerotic plaques in aortas of WHHL rabbits where extensive accumulation of lipid deposits was observed by lipid staining and autoradiography. The accumulation of (64)Cu-3H3-scFv in aortic segments of WHHL rabbits was 2.8-folds higher than that of controls (p=0.0045). The present in vivo evidence supports the pathophysiological involvement of oxLDL/β2GPI complexes in atherosclerotic complications of WHHL rabbits. (64)Cu-3H3-scFv represents a novel PET imaging probe for non-invasive pathophysiological assessment of oxLDL/β2GPI complexes accumulated in atherosclerotic plaques. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Seed-specific expression and analysis of recombinant anti-HER2 single-chain variable fragment (scFv-Fc) in Arabidopsis thaliana.

    PubMed

    Dong, Yuanyuan; Li, Jian; Yao, Na; Wang, Dezhong; Liu, Xiuming; Wang, Nan; Li, Xiaowei; Wang, Fawei; Li, Haiyan; Jiang, Chao

    2017-05-01

    Antibodies to human epidermal growth factor receptor 2 (HER2) are a key element of breast cancer therapy; however, they are expensive to produce and their availability is limited. A seed-specific expression system can be used to produce recombinant proteins. We report a seed-specific expression system for the manufacture of anti-HER2 ScFv-Fc in Arabidopsis thaliana, driven by the Phaseolus vulgaris β-phaseolin promoter. Recombinant anti-HER2 ScFv-Fc was successfully and specifically expressed in seeds, and identified by protein analysis. The highest protein accumulation level, with a maximum of 1.1% of total soluble protein, was observed in mature seeds. We also demonstrated the anti-tumor potency of the plant-derived antibody against SK-BR-3 cells. These results suggest that seed-expression systems could contribute to the manufacture of commercial antibodies such as anti-HER2 ScFv-Fc. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms

    PubMed Central

    Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada

    2017-01-01

    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future. PMID:28654662

  11. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

    PubMed

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

    2017-01-01

    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

  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. Development and Preclinical Testing of a High Affinity Single Chain Antibody against (+)-Methamphetamine

    PubMed Central

    Peterson, Eric C.; Laurenzana, Elizabeth M.; Atchley, William T.; Hendrickson, Howard; Owens, S. Michael

    2009-01-01

    Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κ light chain, KD = 11 nM) and found to have similar ligand affinity (KD = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified and formulated as a naturally occurring mixture of monomer (~75%) and dimer (~25%). To test the in vivo efficacy of the scFv6H4, male Sprague Dawley rats (n=5) were implanted with 3-day sc osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [3H]-scFv6H4 tracer. Serum pharmacokinetic (PCKN) analysis of METH and [3H]-scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0–480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t1/2λz of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t1/2λz (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum. PMID:18192498

  14. Method for preparation of single chain antibodies

    SciTech Connect

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

    2012-04-03

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

  15. 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

  16. Single-Chain Fragment Variable Antibody Piezoimmunosensors

    PubMed Central

    Shen, Zhihong; Stryker, Gabrielle A.; Mernaugh, Ray L.; Yu, Lei; Yan, Heping; Zeng, Xiangqun

    2008-01-01

    In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled mono-layer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW ≈ 27000) and the ease with which they can be modified using genetic engineering, scFv’s have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples. PMID:15679346

  17. Single chain Fab (scFab) fragment.

    PubMed

    Hust, Michael; Jostock, Thomas; Menzel, Christian; Voedisch, Bernd; Mohr, Anja; Brenneis, Mariam; Kirsch, Martina I; Meier, Doris; Dübel, Stefan

    2007-03-08

    The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabDeltaC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.

  18. Single chain Fab (scFab) fragment

    PubMed Central

    Hust, Michael; Jostock, Thomas; Menzel, Christian; Voedisch, Bernd; Mohr, Anja; Brenneis, Mariam; Kirsch, Martina I; Meier, Doris; Dübel, Stefan

    2007-01-01

    Background The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera

  19. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

    PubMed

    Turki, Imène; Hammami, Akil; Kharmachi, Habib; Mousli, Mohamed

    2014-02-01

    Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.

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

    PubMed

    Louis, John M; Aniana, Annie; Lohith, Katheryn; Sayer, Jane M; Roche, Julien; Bewley, Carole A; Clore, G Marius

    2014-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    SciTech Connect

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

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

  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. 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

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

    PubMed Central

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

    2009-01-01

    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 the present 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 α-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. PMID:19114030

  6. Purification, characterization, and biotinylation of single-chain antibodies.

    PubMed

    Kipriyanov, S M

    1998-01-01

    The variable region (Fv) portion of an antibody is comprised of the antibody V(H) and V(L) domains and is the smallest antibody fragment containing a complete antigen-binding site. To stabilize the association of the recombinant V(H) and V(L) domains, they have been linked in single-chain Fv constructs with a short peptide that bridges the approx 3.5 nm between the carboxy terminus of one domain and the ammo terminus of the other (1-3). An NMR comparison of the unlinked Fv fragment of the antibody McPC603 with the corresponding scFv containing a V(H)-(Gly(4)Ser)(3)-V(L) linker has shown no perturbation of the folding of the variable domains by the linker (4,5). In comparison to the much larger Fab', F(ab')(2), and IgG forms of monoclonal antibody (MAb) from which they are derived, scFvs have lower retention times in nontarget tissues, more rapid blood clearance, and better tumor penetration (6-8). ScFvs, therefore, represent potentially very useful molecules for the targeted delivery of drugs, toxins, or radionuclides to a tumor site.

  7. A label-free immunosensor array using single-chain antibody fragments.

    PubMed

    Backmann, Natalija; Zahnd, Christian; Huber, Francois; Bietsch, Alexander; Plückthun, Andreas; Lang, Hans-Peter; Güntherodt, Hans-Joachim; Hegner, Martin; Gerber, Christoph

    2005-10-11

    We report a microcantilever-based immunosensor operated in static deflection mode with a performance comparable with surface plasmon resonance, using single-chain Fv (scFv) antibody fragments as receptor molecules. As a model system scFv fragments with specificity to two different antigens were applied. We introduced a cysteine residue at the C terminus of each scFv construct to allow covalent attachment to gold-coated sensor interfaces in directed orientation. Application of an array enabled simultaneous deflection measurements of sensing and reference cantilevers. The differential deflection signal revealed specific antigen binding and was proportional to the antigen concentration in solution. Using small, oriented scFv fragments as receptor molecules we increased the sensitivity of microcantilevers to approximately 1 nM.

  8. A label-free immunosensor array using single-chain antibody fragments

    PubMed Central

    Backmann, Natalija; Zahnd, Christian; Huber, Francois; Bietsch, Alexander; Plückthun, Andreas; Lang, Hans-Peter; Güntherodt, Hans-Joachim; Hegner, Martin; Gerber, Christoph

    2005-01-01

    We report a microcantilever-based immunosensor operated in static deflection mode with a performance comparable with surface plasmon resonance, using single-chain Fv (scFv) antibody fragments as receptor molecules. As a model system scFv fragments with specificity to two different antigens were applied. We introduced a cysteine residue at the C terminus of each scFv construct to allow covalent attachment to gold-coated sensor interfaces in directed orientation. Application of an array enabled simultaneous deflection measurements of sensing and reference cantilevers. The differential deflection signal revealed specific antigen binding and was proportional to the antigen concentration in solution. Using small, oriented scFv fragments as receptor molecules we increased the sensitivity of microcantilevers to ≈1 nM. PMID:16192357

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

    PubMed

    Pleckaityte, Milda; Mistiniene, Edita; Lasickiene, Rita; Zvirblis, Gintautas; Zvirbliene, Aurelija

    2011-11-03

    Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G₄S)₄ were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused

  10. Construction and expression of a bispecific single-chain antibody that penetrates mutant p53 colon cancer cells and binds p53.

    PubMed

    Weisbart, Richard H; Wakelin, Rika; Chan, Grace; Miller, Carl W; Koeffler, Phillip H

    2004-10-01

    A bispecific, single-chain antibody Fv fragment (Bs-scFv) was constructed from a single-chain Fv fragment of mAb 3E10 that penetrates living cells and localizes in the nucleus, and a single-chain Fv fragment of a non-penetrating antibody, mAb PAb421 that binds the C-terminal of p53. PAb421 binding restores wild-type functions of some p53 mutants, including those of SW480 human colon cancer cells. The Bs-scFv penetrated SW480 cells and was cytotoxic, suggesting an ability to restore activity to mutant p53. COS-7 cells (monkey kidney cells with wild-type p53) served as a control since they are unresponsive to PAb421 due to the presence of SV40 large T antigen that inhibits binding of PAb421 to p53. Bs-scFv penetrated COS-7 cells but was not cytotoxic, thereby eliminating non-specific toxicity of Bs-scFv unrelated to binding p53. A single mutation in CDR1 of PAb421 VH eliminated binding of the Bs-scFv to p53 and abrogated cytotoxicity for SW480 cells without altering cellular penetration, further supporting the requirement of PAb421 binding to p53 for cytotoxicity. Our study demonstrates the use of an antibody that penetrates living cells in the design of a bispecific single chain antibody to target and restore the function of an intracellular protein.

  11. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    PubMed

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  12. Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

    PubMed Central

    Monedero, Vicente; Rodríguez-Díaz, Jesús; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar

    2004-01-01

    Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies. PMID:15528568

  13. Enzymatic Assembly for scFv Library Construction.

    PubMed

    Kato, Mieko; Hanyu, Yoshiro

    2017-01-01

    Recombinant monoclonal antibodies can be established by displaying single-chain variable fragment (scFv) antibody libraries on phages and then biopanning against the target. For constructing superior scFv libraries, antibody light-chain variable region (VL) and heavy-chain variable region (VH) fragments must be assembled into scFvs without loss of diversity. A high-quality scFv library is a prerequisite for obtaining strong binders from the scFv library. However, the technical challenges associated with the construction of a diverse library have been the bottleneck in the establishment of recombinant antibodies through biopanning. Here, we describe a simple and efficient method for assembling VL and VH fragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.

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

    PubMed Central

    2010-01-01

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

  15. 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.

  16. 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

  17. Chimeric Antigen Receptor T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate Virus Reactivated from Latency in CD4+ T Lymphocytes Isolated from HIV-1-Infected Individuals Receiving Suppressive Combined Antiretroviral Therapy.

    PubMed

    Liu, Bingfeng; Zou, Fan; Lu, Lijuan; Chen, Cancan; He, Dalian; Zhang, Xu; Tang, Xiaoping; Liu, Chao; Li, Linghua; Zhang, Hui

    2016-11-01

    Despite the advent of combined antiretroviral therapy (cART), the persistence of viral reservoirs remains a major barrier to curing human immunodeficiency virus type 1 (HIV-1) infection. Recently, the shock and kill strategy, by which such reservoirs are eradicated following reactivation of latent HIV-1 by latency-reversing agents (LRAs), has been extensively practiced. It is important to reestablish virus-specific and reliable immune surveillance to eradicate the reactivated virus-harboring cells. In this report, we attempted to reach this goal by using newly developed chimeric antigen receptor (CAR)-T cell technology. To generate anti-HIV-1 CAR-T cells, we connected the single-chain variable fragment of the broadly neutralizing HIV-1-specific antibody VRC01 to a third-generation CAR moiety as the extracellular and intracellular domains and subsequently transduced this into primary CD8(+) T lymphocytes. We demonstrated that the resulting VC-CAR-T cells induced T cell-mediated cytolysis of cells expressing HIV-1 Env proteins and significantly inhibited HIV-1 rebound after removal of antiviral inhibitors in a viral infectivity model in cell culture that mimics the termination of the cART in the clinic. Importantly, the VC-CAR-T cells also effectively induced the cytolysis of LRA-reactivated HIV-1-infected CD4(+) T lymphocytes isolated from infected individuals receiving suppressive cART. Our data demonstrate that the special features of genetically engineered CAR-T cells make them a particularly suitable candidate for therapeutic application in efforts to reach a functional HIV cure. The presence of latently infected cells remains a key obstacle to the development of a functional HIV-1 cure. Reactivation of dormant viruses is possible with latency-reversing agents, but the effectiveness of these compounds and the subsequent immune response require optimization if the eradication of HIV-1-infected cells is to be achieved. Here, we describe the use of a chimeric

  18. Single-Chain Fragment Variable Passive Immunotherapies for Neurodegenerative Diseases

    PubMed Central

    Huang, Liang; Su, Xiaomin; Federoff, Howard J.

    2013-01-01

    Accumulation of misfolded proteins has been implicated in a variety of neurodegenerative diseases including prion diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). In the past decade, single-chain fragment variable (scFv) -based immunotherapies have been developed to target abnormal proteins or various forms of protein aggregates including Aβ, SNCA, Htt, and PrP proteins. The scFvs are produced by fusing the variable regions of the antibody heavy and light chains, creating a much smaller protein with unaltered specificity. Because of its small size and relative ease of production, scFvs are promising diagnostic and therapeutic reagents for protein misfolded diseases. Studies have demonstrated the efficacy and safety of scFvs in preventing amyloid protein aggregation in preclinical models. Herein, we discuss recent developments of these immunotherapeutics. We review efforts of our group and others using scFv in neurodegenerative disease models. We illustrate the advantages of scFvs, including engineering to enhance misfolded conformer specificity and subcellular targeting to optimize therapeutic action. PMID:24048248

  19. Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs.

    PubMed

    Pavlinkova, G; Colcher, D; Booth, B J; Goel, A; Wittel, U A; Batra, S K

    2001-12-01

    One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28' CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.

  20. Rapid refolding and polishing of single-chain antibodies from Escherichia coli inclusion bodies.

    PubMed

    Sinacola, Jessica R; Robinson, Anne S

    2002-11-01

    An inexpensive and fast-folding strategy for single-chain antibody (scFv) recovered from Escherichia coli inclusion bodies has been developed. Two anti-fluorescein single-chain antibodies, 4-4-20 and 4M5.3, were expressed as inclusion bodies in E. coli for use in a comparative refolding study. Active protein yields as well as degree of aggregation were evaluated for scFv produced by stepwise dialysis, redox dialysis, and a newly developed controlled dilution and filtration strategy. Although all three methods produced active protein for both 4-4-20 and 4M5.3, the extent of aggregation differed greatly among the methods. For 4-4-20, the controlled dilution and filtration strategy reduced aggregation by half, allowed batch processing times of 8h (an 18-fold improvement), and significantly reduced denaturant usage while increasing active yields by 150%. A hydroxyapatite resin polishing step was used to remove completely the aggregate species and inactive monomeric scFv from active scFv.

  1. Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells.

    PubMed

    Zuhaida, A A; Ali, A M; Tamilselvan, S; Alitheen, N B; Hamid, M; Noor, A M; Yeap, S K

    2013-11-18

    A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

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

    PubMed

    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.

  3. Specific Targeting of Hepatitis C Virus Core Protein by an Intracellular Single-Chain Antibody of Human Origin

    PubMed Central

    Karthe, Juliane; Tessmann, Kathi; Li, Jisu; Machida, Raiki; Daleman, Maaike; Häussinger, Dieter; Heintges, Tobias

    2009-01-01

    The hepatitis C virus (HCV) core protein is essential for viral genome encapsidation and plays an important role in steatosis, immune evasion, and hepatocellular carcinoma. It may thus represent a promising therapeutic target to interfere with the HCV life-cycle and related pathogenesis. In this study, we used phage display to generate single-chain variable domain antibody fragments (scFv) to the core protein from bone marrow plasma cells of patients with chronic hepatitis C. An antibody with high-affinity binding (scFv42C) was thus identified, and the binding site was mapped to the PLXG motif (residues 84–87) of the core protein conserved among different genotypes. Whereas scFv42C displayed diffuse cytoplasmic fluorescence when expressed alone in the Huh7 human hepatoma cell line, cotransfection with the core gene shifted its subcellular distribution into that of core protein. The intracellular association of scFv42C with its target core protein was independently demonstrated by the fluorescence resonance energy transfer technique. Interestingly, expression of the single-chain antibody reduced core protein levels intracellularly, particularly in the context of full HCV replication. Moreover, cell proliferation as induced by the core protein could be reversed by scFv4C coexpression. Therefore, scFv42C may represent a novel anti-HCV agent, which acts by sequestering core protein and attenuating core protein–mediated pathogenesis. PMID:18697213

  4. Oligomeric forms of single chain immunoglobulin (scIgG)

    PubMed Central

    Menzel, Christian; Hust, Michael; Prilop, Jessica; Jostock, Thomas; Dübel, Stefan

    2010-01-01

    Assembly of immunoglobulin G (igG) molecules from two heavy and two light chains can be facilitated by connecting the light chain to the heavy chain by an oligopeptide linker. production of the anti-lysozyme D1.3-single chain (sc) igG1 in HeK293t cells yielded up to 8 mg/L functional scigG polypeptide. Size exclusion chromatography of material purified by protein-A affinity chromatography revealed that the majority of the D1.3-scigG1 molecules were 150 kDa monomers, with a KD of 1.8 × 10−10 M measured by surface plasmon resonance; however, significant fractions of scigG dimers and oligomers with molecular masses of 300 kDa and >600 kDa, respectively, were identified. the oligomerization resulted in an increased avidity. observed oligomerization capability may allow new approaches for the generation of bispecific/multivalent antibodies. PMID:20081378

  5. 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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

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

    2016-05-01

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

  7. Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

    PubMed

    Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2017-04-28

    The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

  8. Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

    PubMed

    Li, Benqiang; Ye, Jiaxin; Lin, Yuan; Wang, Man; Jia, Rui; Zhu, Jianguo

    2014-09-01

    Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

  9. Amelioration of amyloid load by anti-Aβ single-chain antibody in Alzheimer mouse model

    PubMed Central

    Fukuchi, Ken-ichiro; Accavitti-Loper, Mary Ann; Kim, Hong-Duck; Tahara, Kazuki; Cao, Yunpeng; Lewis, Terry L.; Caughey, Robert C.; Kim, Helen; Lalonde, Robert

    2008-01-01

    Parenteral immunization of transgenic mouse models of Alzheimer disease (AD) with synthetic amyloid β-peptide (Aβ) prevented or reduced Aβ deposits and attenuated their memory and learning deficits. A clinical trial of immunization with synthetic Aβ, however, was halted due to brain inflammation, presumably induced by a toxic Aβ, T-cell- and/or Fc-mediated immune response. Another issue relating to such immunizations is that some AD patients may not be able to raise an adequate immune response to Aβ vaccination due to immunological tolerance or age-associated decline. Because peripheral administration of antibodies against Aβ also induced clearance of amyloid plaques in the model mice, injection of humanized Aβ antibodies has been proposed as a possible therapy for AD. By screening a human single-chain antibody (scFv) library for Aβ immunoreactivity, we have isolated a scFv that specifically reacts with oligomeric Aβ as well as amyloid plaques in the brain. The scFv inhibited Aβ amyloid fibril formation and Aβ-mediated cytotoxicity in vitro. We have tested the efficacy of the human scFv in a mouse model of AD (Tg2576 mice). Relative to control mice, injections of the scFv into the brain of Tg2576 mice reduced Aβ deposits. Because scFvs lack the Fc portion of the immunoglobulin molecule, human scFvs against Aβ may be useful to treat AD patients without eliciting brain inflammation. PMID:16630540

  10. 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.

  11. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1.

  12. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    PubMed

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  13. Development of a Novel Human Single Chain Antibody Against EGFRVIII Antigen by Phage Display Technology

    PubMed Central

    Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Akbari, Bahman; Ahdi khosroshahi, Shiva

    2016-01-01

    Purpose: EGFRvIII as the most common mutant variant of the epidermal growth factor receptor is resulting from deletion of exons 2–7 in the coding sequence and junction of exons 1 and 8 through a novel glycine residue. EGFRvIII is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. The aim of the present study was identification of a novel single chain antibody against EGFRvIII as a promising target for cancer therapy. Methods: In this study, a synthetic peptide corresponding to EGFRvIII protein was used for screening a naive human scFv phage library. A novel five-round selection strategy was used for enrichment of rare specific clones. Results: After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, only three clones had expected size in PCR reaction. The specific interaction of two of the scFv clones with EGFRvIII was confirmed by indirect ELISA. One phage clone with higher affinity in scFv ELISA was purified for further analysis. The purity of the produced scFv antibody was confirmed using SDS-PAGE and Western blotting analyses. Conclusion: In the present study, a human anti- EGFRvIII scFv with high affinity was first identified from a scFv phage library. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers. PMID:28101463

  14. Engineering Peptide Linkers for scFv Immunosensors

    PubMed Central

    Shen, Zhihong; Yan, Heping; Zhang, Ying; Mernaugh, Raymond L.; Zeng, Xiangqun

    2008-01-01

    Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors. PMID:18290668

  15. 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.

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

    PubMed

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-12-01

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

  17. Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

    PubMed

    Sato, Ryuta; Obonai, Toshifumi; Tsumura, Ryo; Tsumoto, Kouhei; Koga, Yoshikatsu; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2014-12-01

    Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

  18. Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell.

    PubMed

    Sakamoto, Seiichi; Taura, Futoshi; Tsuchihashi, Ryota; Putalun, Waraporn; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-12-01

    Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

  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.

  20. Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development

    PubMed Central

    Samuel, A.S.; Naz, R.K.

    2008-01-01

    BACKGROUND Contraceptive vaccines can provide valuable alternatives to current methods of contraception. We describe here the development of sperm-reactive human single chain variable fragment (scFv) antibodies of defined sperm specificity for immunocontraception. METHODS Peripheral blood leukocytes (PBL) from antisperm antibody-positive immunoinfertile and vasectomized men were activated with human sperm antigens in vitro, and the complementary DNA prepared and PCR-amplified using primers based on all the variable regions of heavy and light chains of immunoglobulins. The scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. RESULTS Panning of the library against specific sperm antigens yielded several clones, and the four strongest reactive were selected for further analysis. These clones had novel sequences with unique complementarity-determining regions. ScFv antibodies were expressed, purified and analyzed for human sperm reactivity and effect on human sperm function. AFA-1 and FAB-7 scFv antibodies both reacted with fertilization antigen-1 antigen, but against different epitopes. YLP20 antibody reacted with the expected human sperm protein of 48 ± 5 kDa. The fourth antibody, AS16, reacted with an 18 kDa sperm protein and seems to be a human homologue of the mouse monoclonal recombinant antisperm antibody that causes sperm agglutination. All these antibodies inhibited human sperm function. CONCLUSIONS This is the first study to report the use of phage display technology to obtain antisperm scFv antibodies of defined antigen specificity. These antibodies will find clinical applications in the development of novel immunocontraceptives, and specific diagnostics for immunoinfertility. PMID:18372255

  1. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    PubMed

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  2. Production of single chain Fab (scFab) fragments in Bacillus megaterium

    PubMed Central

    Jordan, Eva; Al-Halabi, Laila; Schirrmann, Thomas; Hust, Michael; Dübel, Stefan

    2007-01-01

    Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli. PMID:18042285

  3. Novel single chain antibodies to the prion protein identified by phage display.

    PubMed

    Adamson, Catherine S; Yao, Yongxiu; Vasiljevic, Snezana; Sy, Man-Sun; Ren, Junyuan; Jones, Ian M

    2007-02-05

    A well defined structure is available for the carboxyl half of the cellular prion protein (PrP(c)), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrP(c) recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrP(c) as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised 1 predominant and 3 additional scFv-Ps that contained different V(H) and V(L) sequences and that bound specifically to the PrP(c) target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrP(c) residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrP(c) that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the amino-central region of PrP(c). In addition, the binding of MAbs to known linear epitopes within PrP(c) depends strongly on the endpoints of the target PrP(c) fragment used.

  4. Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity.

    PubMed

    Tarasuk, Mayuri; Poungpair, Ornnuthchar; Ungsupravate, Duangporn; Bangphoomi, Kunan; Chaicumpa, Wanpen; Yenchitsomanus, Pa-Thai

    2014-03-01

    Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.

  5. Duplex Microfluidic SERS Detection of Pathogen Antigens with Nanoyeast Single-Chain Variable Fragments

    PubMed Central

    2015-01-01

    Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications. PMID:25192256

  6. Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

    PubMed

    Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

    2014-10-07

    Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

  7. [Preparation and identification of a single chain antibody against the shedding site of N-cadherin cleaved by ADAM10].

    PubMed

    Li, Xiaoou; Huang, Wei; Li, Li; Zhou, Lirong

    2013-09-01

    To construct and express a specific single chain Fv antibody (scFv) against the shedding site of N-cadherin cleaved by ADAM10 and identify its biological activity. The VH; and VL; genes were amplified by RT-PCR from a hybridoma cell line 2B3 which produced the monoclonal antibody (mAb) against the shedding site of N-cadherin cleaved by ADAM10. SOE-PCR was used to splice the VH; and VL; genes to construct ScFv, which was subcloned into the prokaryotic expression vector pET-28a. The positive clones were transformed into E.coli BL21 (DE3) and induced with IPTG. The target protein was purified and refolded via Ni-NTA column, and was analyzed by SDS-PAGE, ELISA and Western blotting. The VH; and VL; genes of mAb were cloned successfully and the prokaryotic expression vector of scFv was constructed. The analysis of DNA sequencing showed that the full-length of the constructed scFv gene was 744 bp, and coded 248 amino acids. ScFv was expressed in E.coli BL21 (DE3) as inclusion body under the induction of IPTG, and showed relative molecular mass (Mr;) of 29 000 as analyzed by SDS-PAGE and Western blotting. About 90% purity of scFv was obtained following denaturing, purifying and renaturing via Ni-NTA, and ELISA and Western blotting revealed that the soluble scFv exhibited the binding activity to the shedding site of N-cadherin cleaved by ADAM10. The scFv against the shedding site of N-cadherin cleaved by ADAM10 has been successfully constructed, which lays the foundation for its diagnostic and therapeutic application.

  8. Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis.

    PubMed

    Kazuma, Soraya M; Cavalcante, Marcela F; Telles, Andréia E R; Maranhão, Andrea Queiroz; Abdalla, Dulcineia S P

    2013-01-01

    The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr(-/-) mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr(-/-) mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).

  9. 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

  10. 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.

  11. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor

    PubMed Central

    Stylianou, DC; Auf der Maur, A; Kodack, DP; Henke, RT; Hohn, S; Toretsky, JA; Riegel, AT; Wellstein, A

    2013-01-01

    The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically. PMID:19633684

  12. Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein.

    PubMed

    Krishnaswamy, Senthilkumar; Kabir, M Enamul; Rahman, M Mamunur; Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

    2011-03-07

    Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.

  13. 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).

  14. In silico experiments of single-chain antibody fragment against drugs of abuse.

    PubMed

    Hu, Guodong; Chen, L Y

    2010-12-01

    Three sets of in silico experiments have been conducted to elucidate the binding mechanics of two drugs, (+)-methamphetamine (METH) and amphetamine (AMP) to the single-chain variable fragment (scFv) recently engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κlight chain, K(d)=11nM). The first set of in silico experiments are long time equilibration runs of scFv:drug complexes and of drug-free scFv both in the solution. They demonstrate how the solution structures of scFv deviate from its crystallographic form with or without drug molecules bound to it. They lead to the prediction that the Arrhenius activation barrier is nearly zero for transitions from the dissociated state to the bound state. The second set of in silico experiments are nonequilibrium dynamics of pulling the drug molecules out of the binding pocket of scFv and the equilibration runs for drugs to fall back into the binding pocket. They demonstrate that extra water molecules (in addition to the two crystallographic waters) exist inside the binding pocket, underneath the drug molecules. These extra waters must have been evaporated from the binding pockets during the crystallization process of the in vitro experiments of structural determination. The third set of in silico experiments are nonequilibrium steered molecular dynamics simulations to determine the absolute binding free energies of METH and AMP to scFv. The center of mass of a drug molecule (METH or AMP) is steered (pulled) towards (forward) and away from (reverse) the binding site, sampling forward and reverse pulling paths. Mechanic work is measured along the pulling paths. The work measurements are averaged through the Brownian dynamics fluctuation dissipation theorem to produce the free-energy profiles of the scFv:drug complexes as a function of the drug-scFv separation. These experiments lead to the theoretical prediction of absolute binding energies of METH and AMP that are in agreement with the in vitro experimental results.

  15. 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.

  16. Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment.

    PubMed

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

    2011-02-25

    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 LD(50) 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.

  17. Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

    PubMed Central

    Fisher, Adam; DeLisa, Matthew P.

    2008-01-01

    One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway (Fisher et al., 2006 Protein Sci). This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We also show that ISELATE enables focusing of an scFv library in soluble sequence space prior to functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. In these ways, we show that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli. PMID:18992254

  18. 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

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

    PubMed

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

    2016-10-01

    In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B.

    PubMed

    Makvandi-Nejad, Shokouh; McLean, Michael D; Hirama, Tomoko; Almquist, Kurt C; Mackenzie, C Roger; Hall, J Christopher

    2005-10-01

    Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T(0)) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 mug of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T(1) plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T(0) plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T(1) generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.

  1. Expression and characterization of a single-chain variable fragment against human LOX-1 in Escherichia coli and Brevibacillus choshinensis.

    PubMed

    Hu, Wei; Xiang, Jun-Yan; Kong, Ping; Liu, Ling; Xie, Qiuhong; Xiang, Hongyu

    2017-03-09

    The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and the response surface methodology (RSM), under which the yield reached up to 1.5 g/L in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature and particle diameter size is 1.01E-07 M, 55.2 ± 0.3°C and 9.388 nm for the scFv expressed by B. choshinensis and 4.53E-07 M, 52.5 ± 0.3°C and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

  2. 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.

  3. Elementary excitations in single-chain magnets

    NASA Astrophysics Data System (ADS)

    Lutz, Philipp; Aguilà, David; Mondal, Abhishake; Pinkowicz, Dawid; Marx, Raphael; Neugebauer, Petr; Fâk, Björn; Ollivier, Jacques; Clérac, Rodolphe; van Slageren, Joris

    2017-09-01

    Single-chain magnets (SCMs) are one-dimensional coordination polymers or spin chains that display slow relaxation of the magnetization. Typically their static magnetic properties are described by the Heisenberg model, while the description of their dynamic magnetic properties is based on an Ising-like model. The types of excitations predicted by these models (collective vs localized) are quite different. Therefore we probed the nature of the elementary excitations for two SCMs abbreviated Mn2Ni and Mn2Fe , as well as a mononuclear derivative of the Mn2Fe chain, by means of high-frequency electron paramagnetic resonance spectroscopy (HFEPR) and inelastic neutron scattering (INS). We find that the HFEPR spectra of the chains are clearly distinct from those of the monomer. The momentum transfer dependence of the INS intensity did not reveal significant dispersion, indicating an essentially localized nature of the excitations. At the lowest temperatures these are modified by the occurrence of short-range correlations.

  4. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    PubMed

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

  5. Enhanced transport of plant-produced rabies single chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device.

    PubMed

    Phoolcharoen, Waranyoo; Prehaud, Christophe; van Dolleweerd, Craig J; Both, Leonard; da Costa, Anaelle; Lafon, Monique; Ma, Julian K-C

    2017-03-08

    The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In the present study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system (CNS). This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralising single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognises the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralisation activity against RABV in cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVG(P) fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVG(P) fusion protein was able to cross the in cellulo BBB and neutralise RABV. This article is protected by copyright. All rights reserved.

  6. 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

  7. 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.

  8. One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector.

    PubMed

    Zhao, Qi; Chan, Yin-Wah; Lee, Susanna Sau-Tuen; Cheung, Wing-Tai

    2009-12-01

    Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.

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

    PubMed

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

    2016-06-15

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

  10. Insights into scFv:drug binding using the molecular dynamics simulation and free energy calculation.

    PubMed

    Hu, Guodong; Zhang, Qinggang; Chen, L Y

    2011-08-01

    Molecular dynamics simulations and free energy calculation have been performed to study how the single-chain variable fragment (scFv) binds methamphetamine (METH) and amphetamine (AMP). The structures of the scFv:METH and the scFv:AMP complexes are analyzed by examining the time-dependence of their RMSDs, by analyzing the distance between some key atoms of the selected residues, and by comparing the averaged structures with their corresponding crystallographic structures. It is observed that binding an AMP to the scFv does not cause significant changes to the binding pocket of the scFv:ligand complex. The binding free energy of scFv:AMP without introducing an extra water into the binding pocket is much stronger than scFv:METH. This is against the first of the two scenarios postulated in the experimental work of Celikel et al. (Protein Science 18, 2336 (2009)). However, adding a water to the AMP (at the position of the methyl group of METH), the binding free energy of the scFv:AMP-H2O complex, is found to be significantly weaker than scFv:METH. This is consistent with the second of the two scenarios given by Celikel et al. Decomposition of the binding energy into ligand-residue pair interactions shows that two residues (Tyr175 and Tyr177) have nearly-zero interactions with AMP in the scFv:AMP-H2O complex, whereas their interactions with METH in the scFv:METH complex are as large as -0.8 and -0.74 kcal mol(-1). The insights gained from this study may be helpful in designing more potent antibodies in treating METH abuse.

  11. [Construction and panning of scFv phage display library against recombinant interleukin 4 receptor].

    PubMed

    Yang, Guangyong; Guo, Haitao; Liu, Ximing; He, Guangzhi; Tian, Weiyi; Cai, Kun; Wang, Ping; Wang, Wenjia

    2016-06-01

    Objective To construct the recombinant human interleukin 4 receptor (rhIL-4R) single-chain Fv (scFv) antibody library by phage display technique to obtain the anti-IL-4R scFv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rhIL-4R. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv by splice overlap extension PCR (SOE-PCR). Both scFv and the pCANTAB5E vector were respectively double-digested with restriction endonuclease Sfi I and Not I, connected with T4 ligase, and then transformed into the competent cells E.coli TG1; it was cultured in medium to obtain the phage scFv antibody library; after three rounds of enrichment and panning, the specific antigen scFv with high affinity was selected for the sequencing. Results After three rounds of panning, we obtained a diversity of approximately 2×10(8) anti-rhIL-4R scFv antibody library. Sequencing analysis of one positive clone showed that the anti-rhIL-4R scFv was 741 bp and coded 247 amino acids. The analysis of VBASE2 database indicated that VH and VL gene sequences of anti-rhIL-4R protein all had three complementarity determining regions and four backbone areas.Conclusion The anti-rhIL-4R scFv was obtained from the scFv antibody library.

  12. 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.

  13. scFv antibodies against infectious bursal disease virus isolated from a combinatorial antibody library by flow cytometry.

    PubMed

    Xu, Li-Ming; Li, Tian-He; Zhou, Bing; Guo, Mo; Liu, Miao; Zhao, Jing-Zhuang; Cao, Hong-Wei; Li, De-Shan

    2014-05-01

    Infectious bursal disease is an economically important disease that affects chickens worldwide. Here, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry against VP2 protein through a bacteria display technology, resulting in the enrichment of scFv. Three scFv clones with different fluorescence intensity were obtained by random colony pick up. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis. The potential use of the selected IBDV-specific scFv antibodies was demonstrated by the successful application of the isolated antibodies in western blotting assay and ELISA.

  14. 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.

  15. Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

    PubMed

    Kumano-Kuramochi, Miyuki; Fujimura, Takashi; Komba, Shiro; Maeda-Yamamoto, Mari; Machida, Sachiko

    2016-09-01

    Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.

  16. Expression, characterization, and evaluation of a RANK-binding single chain fraction variable: an osteoclast targeting drug delivery strategy.

    PubMed

    Newa, Madhuri; Lam, Michael; Bhandari, Krishna Hari; Xu, Biwen; Doschak, Michael R

    2014-01-06

    A single chain Fraction variable (scFv) employs antibody-like target recognition specificity. Osteoclasts, responsible for bone resorption, express Receptor Activator of Nuclear factor Kappa B (RANK) receptors. This study aimed to express, characterize, and evaluate scFv against RANK receptors that may serve as a platform to target osteoclasts. Using phage display technology, scFv against RANK receptor was expressed and characterized by DNA sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption-ionization time-of-flight (MALDI TOF), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry. The potential for cytotoxicity was evaluated using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, and its cross reactivity was evaluated using ELISA. Osteoclast-like cells were generated from RAW 264.7 cells, and the osteoclast targeting ability of scFv was evaluated using immunocytochemistry. ScFv's antiresorptive efficacy was studied using a tartrate-resistant acid phosphatase (TRAP) assay and resorption assay. Anti-RANK scFv was successfully expressed and characterized. No cross reactivity with other tumor necrosis factor receptor (TNFR) members and no cytotoxic effect on a non-RANK bearing cell line were observed. It showed specificity toward a RANK receptor and an inhibitory effect on osteoclast activity. With the increase in development trends for biologics as therapeutics and growing knowledge on the importance of osteoclast targeted therapy, this study may provide a drug delivery strategy to target osteoclasts, thereby leading to a promising therapy for resorptive bone diseases.

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

    PubMed

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

    2011-05-01

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

  18. 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

  19. Selection of single-chain variable fragments specific for Mycobacterium tuberculosis ESAT-6 antigen using ribosome display

    PubMed Central

    Ahangarzadeh, Shahrzad; Bandehpour, Mojgan; Kazemi, Bahram

    2017-01-01

    Objective(s): Tuberculosis (TB) is still one of the problematic infectious diseases in developing countries, especially in Iran. In the present study, we applied ribosome display technique to select single chain variable fragments (scFvs) specific for the 6-kDa early secretory antigenic target (ESAT-6) antigen of Mycobacterium tuberculosis from a mouse scFv library. Materials and Methods: The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed in Escherichia coli BL21 (DE3). The purified recombinant ESAT-6 protein was injected into female BALB/c mice for immunization, and then m-RNA was extracted from the spleen of immunized mice. The anti-ESAT-6 VH/k chain library was assembled by joining of VH and k into the VH/k chain with a 72-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-6 using a single round of ribosome display via a rabbit reticulocyte lysate system. Results: ELISA assay showed that one of the selected scFvs had higher affinity against the recombinant ESAT-6 protein. The affinity of the candidate scFv was ~ 3.74×108 M-1. Conclusion: It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB. PMID:28392906

  20. Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.

    PubMed

    Fujiki, Yuya; Kumada, Yoichi; Kishimoto, Michimasa

    2015-08-01

    The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture. Furthermore, specific proteins, such as alcohol oxidase, which is strongly related to scFv expression, and proteinase A, which could biodegrade scFv in the latter phases of production, were identified via the PMF method. The proteomics technique provided valuable information about the effect of the methanol concentration on scFv production.

  1. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

    PubMed

    Zhang, Kathy; Geddie, Melissa L; Kohli, Neeraj; Kornaga, Tad; Kirpotin, Dmitri B; Jiao, Yang; Rennard, Rachel; Drummond, Daryl C; Nielsen, Ulrik B; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

  2. 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

  3. Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development.

    PubMed

    Isaacs, Alison T; Jasinskiene, Nijole; Tretiakov, Mikhail; Thiery, Isabelle; Zettor, Agnès; Bourgouin, Catherine; James, Anthony A

    2012-07-10

    Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.

  4. Selection of single chain variable fragments specific for the human-inducible costimulator using ribosome display.

    PubMed

    Pan, Yangbin; Mao, Weiping; Liu, Xuanxuan; Xu, Chong; He, Zhijuan; Wang, Wenqian; Yan, Hao

    2012-11-01

    We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody-ribosome-mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.

  5. 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.

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

    PubMed

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

    2010-07-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which 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 medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) 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 nonselective medium. 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. (c)2010 AACR.

  7. Preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII.

    PubMed

    Zollner, Sabine B; Raquet, Elmar; Müller-Cohrs, Jochen; Metzner, Hubert J; Weimer, Thomas; Pragst, Ingo; Dickneite, Gerhard; Schulte, Stefan

    2013-08-01

    The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1-150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF(®)), or full-length rFVIII products (Advate(®), Helixate(®)). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY). Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. [Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b].

    PubMed

    Hil'chuk, P V; Okuniev, O V; Pavlova, M V; Irodov, D M; Horbatiuk, O B

    2006-01-01

    The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.

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

    PubMed Central

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

    2011-01-01

    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 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, 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 antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support. PMID:21538339

  10. 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.

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

    PubMed Central

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

    2006-01-01

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

  12. Single-chain antibody-delivered Livin siRNA inhibits human malignant melanoma growth in vitro and in vivo.

    PubMed

    Wang, Hao; Yang, Yifei; Wang, Wei; Guan, Bing; Xun, Meng; Zhang, Hai; Wang, Ziling; Zhao, Yong

    2017-05-01

    Although gene therapy has brought new insights into the treatment of malignant melanoma, targeting delivery of nucleic acid which targets critical oncogene/anti-oncogene in vivo is still a bottleneck in the therapeutic application. Our previous in vitro studies have found that the oncogene Livin could serve as a potential molecular target by small interfering RNA for gene therapy of malignant melanoma. However, how to transport Livin small interfering RNA into malignant melanoma cells specifically and efficiently in vivo needs further investigation. Cumulative evidence has suggested that single-chain antibody-mediated small interfering RNA targeted delivery is an effective way to silence specific genes in human cancer cells. Indeed, this study designed a protamine-single-chain antibody fusion protein, anti-MM scFv-tP, to deliver Livin small interfering RNA into LiBr cells. Further experiments confirmed the induction of cell apoptosis and suppression of cell proliferation by anti-MM scFv-tP in LiBr cells, along with efficient silence of Livin gene both in vitro and in vivo. Altogether, our findings provide a feasible approach to transport Livin small interfering RNA to malignant melanoma cells which would be a new therapeutic strategy for combating malignant melanoma.

  13. A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting.

    PubMed

    Schubert, Ingo; Kellner, Christian; Stein, Christoph; Kügler, Markus; Schwenkert, Michael; Saul, Domenica; Mentz, Kristin; Singer, Heiko; Stockmeyer, Bernhard; Hillen, Wolfgang; Mackensen, Andreas; Fey, Georg H

    2011-01-01

    A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.

  14. High affinity scFv-hapten pair as a tool for quantum dot labeling and tracking of single proteins in live cells.

    PubMed

    Iyer, Gopal; Michalet, Xavier; Chang, Yun-Pei; Pinaud, Fabien F; Matyas, Stephanie E; Payne, Gregory; Weiss, Shimon

    2008-12-01

    We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.

  15. 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.

  16. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

    PubMed Central

    Wang, Rongzhi; Xiang, Shuangshuang; Feng, Youjun; Srinivas, Swaminath; Zhang, Yonghui; Lin, Mingshen; Wang, Shihua

    2013-01-01

    Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody. PMID:24224158

  18. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique.

    PubMed

    Schlebusch, H; Reinartz, S; Kaiser, R; Grünn, U; Wagner, U

    1997-02-01

    The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.

  20. 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. Copyright © 2015. Published by Elsevier Ltd.

  1. Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy.

    PubMed

    Yuasa, Noriyuki; Koyama, Tsubasa; Subedi, Ganesh P; Yamaguchi, Yoshiki; Matsushita, Misao; Fujita-Yamaguchi, Yoko

    2013-12-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.

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

    USDA-ARS?s Scientific Manuscript database

    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. Xylella fastidiosa subsp pauca causes citrus variegat...

  3. 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.

  4. Selection of single chain variable fragments (scFv) against Xylella fastidiosa subsp. pauca by phage display

    USDA-ARS?s Scientific Manuscript database

    Xylella fastidiosa is a gram-negative member of the gamma proteobacteria. Xylella fastidiosa subsp pauca causes citrus variegated chlorosis in Brazil and enjoys ‘select agent’ status in the United States. Antibody based detection assays are commercially available for Xylella fastidiosa, and are ef...

  5. A Top-Down Approach to Mechanistic Biological Modeling: Application to the Single-Chain Antibody Folding Pathway

    PubMed Central

    Hildebrandt, Scott; Raden, David; Petzold, Linda; Robinson, Anne Skaja; Doyle, Francis J.

    2008-01-01

    A top-down approach to mechanistic modeling of biological systems is presented and exemplified with the development of a hypothesis-driven mathematical model for single-chain antibody fragment (scFv) folding in Saccharomyces cerevisiae by mediators BiP and PDI. In this approach, model development starts with construction of the most basic mathematical model—typically consisting of predetermined or newly-elucidated biological behavior motifs—capable of reproducing desired biological behaviors. From this point, mechanistic detail is added incrementally and systematically, and the effects of each addition are evaluated. This approach follows the typical progression of experimental data availability in that higher-order, lumped measurements are often more prevalent initially than specific, mechanistic ones. It also necessarily provides the modeler with insight into the structural requirements and performance capabilities of the resulting detailed mechanistic model, which facilitates further analysis. The top-down approach to mechanistic modeling identified three such requirements and a branched dependency-degradation competition motif critical for the scFv folding model to reproduce experimentally observed scFv folding dependencies on BiP and PDI and increased production when both species are overexpressed and promoted straightforward prediction of parameter dependencies. It also prescribed modification of the guiding hypothesis to capture BiP and PDI synergy. PMID:18641066

  6. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

    PubMed

    Thatikonda, Naresh; Delfani, Payam; Jansson, Ronnie; Petersson, Linn; Lindberg, Diana; Wingren, Christer; Hedhammar, My

    2016-03-01

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.

  7. Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

    PubMed

    Du, Xin-jun; Zhou, Xiao-nan; Li, Ping; Sheng, Wei; Ducancel, Frédéric; Wang, Shuo

    2016-04-13

    Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (V(H )and V(L)) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the V(H) and V(L) chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection.

  8. Molecular Recognition-Mediated Transformation of Single-Chain Polymer Nanoparticles into Crosslinked Polymer Films.

    PubMed

    Mahon, Clare S; McGurk, Christopher J; Watson, Scott M D; Fascione, Martin A; Sakonsinsiri, Chadamas; Turnbull, W Bruce; Fulton, David A

    2017-08-14

    We describe single-chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition-mediated crosslinking process. The SCNPs utilise molecular recognition with surface-immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra- to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to "wrap" surfaces displaying molecular recognition motifs-which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs-within a layer of polymer film. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  9. Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX ▿

    PubMed Central

    Poulin, Kathy L.; Lanthier, Robert M.; Smith, Adam C.; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L.; O'Meara, Ryan W.; Kothary, Rashmi; Lorimer, Ian A.; Parks, Robin J.

    2010-01-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions. PMID:20631131

  10. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    PubMed

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

  11. ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

    PubMed

    Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

    2014-10-01

    Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

  12. 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.

  13. Enhanced production of functional extracellular single chain variable fragment against HIV-1 matrix protein from Escherichia coli by sequential simplex optimization.

    PubMed

    Intachai, Kannaporn; Singboottra, Panthong; Leksawasdi, Noppol; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Butr-Indr, Bordin

    2015-01-01

    The optimal culture condition for extracellular recombinant single chain variable fragment anti HIV-1 p17 protein (scFv anti-p17) production in Escherichia coli HB2151 was investigated by the sequential simplex optimization (SS) method. Five variable parameters were submitted in the fermentation process. The most favorable condition obtained from 19 independent experiments was as followed: 58 µM of IPTG induction to 1.7 OD600 nm at 25.5°C for 16 h with 202 rpm agitation rate. The amount of secreted scFv anti-p17 at the optimal condition was 38% higher than under the control condition. The binding activity of soluble extracellular scFv anti-p17 protein increased 95.5% and 73.2% in comparison with the control condition and non-optimized condition respectively. The soluble scFv anti-p17 from crude HB2151 lysated was subsequently purified by immobilized metal ion affinity chromatography (IMAC) with His-tag. The purified scFv anti-p17 was intact and retained its antigen-binding affinity against HIV-1 p17. We demonstrated that the sequential simplex optimization method was a key for exertion of high yield with fewer experimental requirements for acquiring of large scale secretory protein production.

  14. Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses.

    PubMed

    Hurwitz, Amy M; Huang, Wanzhi; Kou, Baijun; Estes, Mary K; Atmar, Robert L; Palzkill, Timothy

    2017-01-01

    Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)-named NJT-R3-A2 and NJT-R3-A3-that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.

  15. 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

  16. Production of recombinant scFv against p24 of human immunodeficiency virus type 1 by phage display technology.

    PubMed

    Mohammadzadeh, Sara; Rajabibazl, Masoumeh; Fourozandeh, Mehdi; Rasaee, Mohammad Javad; Rahbarizadeh, Fatemeh; Mohammadi, Mohammad

    2014-02-01

    Phage display has a fundamental role in protein isolation and engineering. Isolated proteins produced with this method can be modified for specific binding and affinity. P24 is the most produced protein during human immune deficiency virus (HIV) replication; especially in the early steps of HIV-1 infection, its evaluation may have diagnostic values. To test the HIV-1 infection, p24 antigen assay appears to be a very promising alternative to RNA assays. In this study, we have generated a recombinant mouse single chain antibody fragment against p24 of the HIV-1 with the use of phage display technology. After isolation of antibody variable-region (V) gene of B cells extracted from the spleen of an immunized mouse, a library of single chain Fv fragments (scFv) was constructed. The library was used in a series of bio-panning processes against recombinant p24 protein expressed from Escherichia coli. The isolated scFv antibody specifically recognizes the HIV-1 capsid protein p24. The affinity constant of the isolated scFv antibody (MF85) was found to be 2×10(-9) M. Our studies showed that the MF85 scFV antibody has similar properties as that of monoclonal antibodies produced by the hybridoma technology.

  17. 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

  18. A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

    PubMed Central

    Lee, Gunsup; Yu, Jaelim; Cho, Seungchan; Byun, Sung-June; Kim, Dae Hyun; Lee, Taek-Kyun; Kwon, Myung-Hee; Lee, Sukchan

    2014-01-01

    Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system. PMID:24968358

  19. A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

    PubMed Central

    Jansson, Bo; Stuhr-Hansen, Nicolai; Kovács, András; Welinder, Charlotte

    2016-01-01

    We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation. PMID:28002485

  20. Generation and expression in plants of a single-chain variable fragment antibody against the immunodominant membrane protein of Candidatus phytoplasma aurantifolia.

    PubMed

    Shahryari, F; Safarnejad, M R; Shams-Bakhsh, M; Schillberg, S; Nölke, G

    2013-08-01

    Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

  1. A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

    PubMed

    Frigerio, B; Fracasso, G; Luison, E; Cingarlini, S; Mortarino, M; Coliva, A; Seregni, E; Bombardieri, E; Zuccolotto, G; Rosato, A; Colombatti, M; Canevari, S; Figini, M

    2013-06-01

    Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers. Published by Elsevier Ltd.

  2. Development of a hyperimmune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer.

    PubMed

    Winthrop, M D; DeNardo, S J; DeNardo, G L

    1999-10-01

    Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy. Optimizing the approach has proven difficult thus far. Antibody phage display libraries provide unique molecules that could improve RIT. A phage display library of single chain antibody fragments (scFv) against the MUC-1 mucin molecule, which is expressed on 90% of human breast cancers, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice. Increased serum IgG levels, 15 times baseline, were detected following the third immunization. RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions were amplified using PCR technology. The variable heavy and variable light chain gene regions were combined with a flexible linker, ligated into the pCANTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells. A library of 10(7) initial colonies was compiled. Forty-six of 288 colonies screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 breast cancer cell membrane fragments. Anti-MUC-1 library diversity evaluated by BstNI digest demonstrated that 52% of the anti-MUC-1 scFv binding MCF-7 possessed individual banding patterns representative of approximately 5 x 10(5) colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers. In summary, the anti-MUC-1 scFv antibody phage library contains diverse scFv molecules, which should provide unique characteristics and epitope recognition. These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer.

  3. Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

    PubMed

    Hexham, J M; Dudas, D; Hugo, R; Thompson, J; King, V; Dowling, C; Neville, D M; Digan, M E; Lake, P

    2001-09-01

    The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly

  4. 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.

  5. Phage display of ScFv peptides recognizing the thymidine(6–4)thymidine photoproduct

    PubMed Central

    Zavala, Anamaria G.; Lancaster, Thaddeus; Groopman, John D.; Strickland, Paul T.; Chandrasegaran, Srinivasan

    2000-01-01

    Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, αUVssDNA-1 (mAb C3B6), recognizing the thymidine(6–4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure–function studies and application to human investigations. PMID:10710441

  6. 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

  7. Small molecular peptide-ScFv αvβ3 conjugates specifically inhibit lung cancer cell growth in vitro and in vivo

    PubMed Central

    Qiu, Qianqian; Wang, Qiongyao; Deng, Changxu; Sun, Yanqin; Chen, Taoliang; Guo, Linlang; Zhang, Fan

    2016-01-01

    Integrin αvβ3 (ITG) is highly expressed in various cancers and is considered a major target for anti-angiogensis cancer therapy. The single chain fragment variable of which (ScFv αvβ3) has been reported to inhibit tumor growth both in vitro and in vivo. Here, we conjugated cdGIGPQc which can exclusively bind to NSCLC cells according to our previous study synthesized by SPPS with ScFv αvβ3 expressed in E. coli BL21 (DE3) to develop a novel lung cancer specific targeted drug. Specific cell targeting of cdGIGPQc-ScFv was assessed in parallel with the single ScFv and a control nonspecific peptide-ScFv through immunofluorescence and flow cytometry while the αvβ3-binding property was examined by Western blot. Our results showed that cdGIGPQc-ScFv retained both the lung cancer-binding activity of cdGIGPQc and the antigen-recognizing ability of ScFv αvβ3 in vitro. CCK8 assays and in animal experiments suggested that cdGIGPQc-ScFv possessed a superior antitumor effect than ScFv and nonspecific peptide-ScFv both in vitro and vivo. Further immunohistochemical staining revealed that cdGIGPQc-ScFv retarded lung cancer growth through inhibiting tumor angiogensis and proliferation. Therefore, cdGIGPQc delivery of ScFv αvβ3 to lung cancer may be a hopeful new strategy for enhancing specific antitumor efficacy and cdGIGPQc-ScFv could be a potential drug for lung cancer targeted treatment. PMID:28042504

  8. Study of the Interactions of Fusarium virguliforme Toxin FvTox1 with Synthetic Peptides by Molecular Simulations and a Label-Free Biosensor.

    PubMed

    Zhang, Bailin; Wang, Bing; Morales, Andres W; Scudder, Jonathan; Bhattacharyya, Madan K; Ye, Jing Yong

    2016-03-15

    Fusarium virguliforme is a soil borne pathogen that causes sudden death syndrome (SDS) in soybean plants. This pathogenic disease may result in severe soybean yield suppression and can cause serious economic harm. It has been shown that the FvTox1 toxin produced by the pathogen may be the root cause of foliar SDS. Anti-FvTox1 single-chain variable fragment antibody expressed in transgenic soybean plants was shown to neutralize the FvTox1 toxin involved in foliar SDS development. Here, we have investigated the binding affinities of FvTox1 with four FvTox1-interacting peptides of 7 to 12 amino acids identified from phage display libraries using both bioinformatics-based molecular simulations and label-free bioassays with a unique photonic crystal biosensor. Results from the molecular simulations have predicted the interaction energies and 3-dimensional (3D) structures of FvTox1 and FvTox1-interacting peptide complexes. Our label-free binding assays have further provided the interaction strength of FvTox1 with four different FvTox1-interacting peptides and experimentally confirmed the simulation results obtained from bioinformatics-based molecular calculations.

  9. [Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

    PubMed

    Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

    2012-08-01

    To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

  10. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    PubMed Central

    Pavoni, Emiliano; Flego, Michela; Dupuis, Maria Luisa; Barca, Stefano; Petronzelli, Fiorella; Anastasi, Anna Maria; D'Alessio, Valeria; Pelliccia, Angela; Vaccaro, Paola; Monteriù, Giorgia; Ascione, Alessandro; De Santis, Rita; Felici, Franco; Cianfriglia, Maurizio; Minenkova, Olga

    2006-01-01

    Background CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. Methods The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. Results The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. Conclusion The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance. PMID:16504122

  11. CNS delivery of vectored prion-specific single-chain antibodies delays disease onset.

    PubMed

    Wuertzer, Charles A; Sullivan, Mark A; Qiu, Xing; Federoff, Howard J

    2008-03-01

    A unifying characteristic of prion diseases is the conversion of a normal cellular protein (PrP(c)) to an abnormal pathogenic conformation, designated PrP(sc). Antibodies directed against PrP(c), when added to scrapie-infected cell cultures or passively administered in vivo, can result in elimination of PrP(sc) or prevent its replication, respectively. In our efforts to develop an approach with potential prophylactic utility we employed a recombinant adeno-associated vector type 2 (rAAV2) viral vector platform to express PrP(c)-specific single-chain fragment variable (scFv) antibodies within the central nervous system (CNS) of susceptible mice that were subsequently inoculated peripherally with infectious prions. Vector expressed scFvs delayed onset of prion pathogenesis as evidenced by improvements in clinical signs and rotarod performance, in extended incubation periods, and in decreased PrP(sc) burden in the CNS. This novel antibody delivery platform enables the in vivo translation of prion prophylactics to other species afflicted by transmissible spongiform encephalopathies (TSEs) and which also has relevance to the development of therapeutics for other protein-misfolding diseases such as Alzheimer's or Parkinson's disease.

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

    PubMed

    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

    2014-09-01

    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 noncomplementarity 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.

  14. 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.

  15. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    PubMed

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  16. 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

    2007-09-05

    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.

  17. Chiral imprinting of diblock copolymer single-chain particles.

    PubMed

    Njikang, Gabriel; Liu, Guojun; Hong, Liangzhi

    2011-06-07

    This Article reports the molecular imprinting of polymer single-chain particles that have a radius ∼3.7 nm. For this, the template L-phenylalanine anilide or L-ΦAA and a diblock copolymer PtBA-b-P(CEMA-r-CA) were used. Here, PtBA denotes poly(tert-butyl acrylate), and P(CEMA-r-CA) denotes a random block consisting of cinnamoyloxyethyl methacrylate (CEMA) and carboxyl-bearing (CA) units. In CHCl(3)/cyclohexane (CHX) with 64 vol % of CHX or at f(CHX) = 64%, a block-selective solvent for PtBA, PtBA-b-P(CEMA-r-CA) formed spherical micelles. The core consisted of the insoluble P(CEMA-r-CA) block and L-ΦAA, which complexed with the CA groups. Pumping slowly this micellar solution into stirred CHCl(3)/(CHX) at f(CHX) = 64% triggered micelle dissociation into single-chain micelles, which comprised presumably a solubilized PtBA tail and a collapsed P(CEMA-r-CA)/L-ΦAA head. Because the solvent reservoir was under constant UV irradiation, the photo-cross-linkable units in the P(CEMA-r-CA) head cross-linked, and the single-chain micelles were converted into cross-linked single-chain micelles or tadpoles. Synchronizing the micelle addition and photoreaction rates allowed the preparation, from this protocol, of essentially pure tadpoles at high final polymer concentrations. Imprinted tadpoles were procured after L-ΦAA was extracted from the tadpole heads. Under optimized conditions, the produced imprinted tadpoles had exceptionally high binding capacity and high selectivity for L-ΦAA. In addition, the rates of L-ΦAA release from and rebinding by the particles were high.

  18. Enzymatic glycosylation of multivalent scaffolds.

    PubMed

    Bojarová, Pavla; Rosencrantz, Ruben R; Elling, Lothar; Křen, Vladimír

    2013-06-07

    The design of glycoclusters, glycodendrimers, glycopolymers and other complex glycostructures that mimic the multivalent carbohydrate display on the cell surface is of immense interest for diagnosis and therapy. This review presents a detailed insight into the exciting possibilities of multiple glycosylation using enzymes, particularly glycosyltransferases (EC 2.4). A representative choice of available scaffolds for the enzyme action is practically infinite and comprises synthetic polymers, carbosilane dendrimers, multiantennary glycans or hyperbranched conjugates. The introduced glyco-patterns range from common sialyl Lewis(x) and sialyl lacto-chains to chemically functionalized carbohydrate units for detection purposes. The possibilities of in vitro enzymatic production of N- and O-glycans and other natural polymers are also discussed. In harmony with their natural tasks, glycosyltransferases may in vitro complete the imperfect glycosylation pattern of proteins, recombinantly produced in pro- and eukaryotic hosts. What is more, the required enzymatic battery may be directly co-expressed with the protein, in order to elegantly accomplish the production of eukaryotic glycans. Ingenious metabolic labeling enables facile imaging of glycostructures. The boom of glycoarray technology opens vast possibilities in high-throughput screening for novel enzymes and substrate specificities as well as in the synthesis. Though there is still a long way until the Nature's ideal of multivalent glycans is achievable in the laboratory, the sketched pathways to multivalent glycostructures open tremendous possibilities for the future glycobiological research.

  19. Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro.

    PubMed

    Wang, Hai-rong; Xiao, Zhen-yu; Chen, Miao; Wang, Fei-long; Liu, Jia; Zhong, Hua; Zhong, Ji-hua; Ou-Yang, Ren-rong; Shen, Yan-lin; Pan, Shu-ming

    2012-06-01

    Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus. The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

  20. 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

  1. Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

    PubMed Central

    Rivera-Hernández, Geovanny; Marín-Argany, Marta; Blasco-Moreno, Bernat; Bonet, Jaume; Oliva, Baldomero; Villegas, Sandra

    2013-01-01

    Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ1–42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest. PMID:23924802

  2. Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency.

    PubMed

    Rivera-Hernández, Geovanny; Marin-Argany, Marta; Blasco-Moreno, Bernat; Bonet, Jaume; Oliva, Baldo; Villegas, Sandra

    2013-01-01

    Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ 1-42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest.

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

    PubMed

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

    2014-07-01

    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.

  4. 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

  5. PROTECTIVE EFFECT OF ScFv-DAF FUSION PROTEIN ON THE COMPLEMENT ATTACK TO ACETYLCHOLINE RECEPTOR: A POSSIBLE OPTION FOR TREATMENT OF MYASTHENIA GRAVIS

    PubMed Central

    XU, JIANG; WU, XINGAN; ZHANG, FANGLIN; LIN, HONG; LI, ZHUYI; KAMINSKI, HENRY J.

    2015-01-01

    Introduction Autoantibody-induced complement activation, which causes disruption of the postsynaptic membrane, is recognized as a key pathogenic factor in myasthenia gravis (MG). Therefore, specific targeting of complement inhibitors to the site of complement activation is a potential therapeutic strategy for treatment of MG. Methods We assessed expression of single-chain antibody fragment–decay accelerating factor (scFv-DAF), comprising a single-chain fragment scFv1956 based on the rat complement inhibitor DAF in prokaryotic systems, and studied its inhibitory effect on complement deposition in vitro. Results The recombinant conjugate scFv-DAF completely retained the wild-type binding activity of scFv1956 to AChR and inhibited complement activation of DAF in vitro. Conclusions We found that scFv-DAF could bind specifically to TE671 cells, and it is significantly more potent at inhibiting complement deposition than the untargeted parent molecule DAF. scFv-DAF may be a candidate for in vivo protection of the AChR in MG. PMID:22499093

  6. 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.

  7. 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.

  8. Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability.

    PubMed

    Quintero-Hernández, Veronica; Del Pozo-Yauner, Luis; Pedraza-Escalona, Martha; Juárez-González, Victor R; Alcántara-Recillas, Israel; Possani, Lourival D; Becerril, Baltazar

    2012-04-30

    The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

  9. Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones.

    PubMed

    Wen, Kai; Nölke, Greta; Schillberg, Stefan; Wang, Zhanhui; Zhang, Suxia; Wu, Congming; Jiang, Haiyang; Meng, Hui; Shen, Jianzhong

    2012-07-01

    Fluoroquinolones (FQs) are a group of synthetic, broad-spectrum antibacterial agents. Due to its extensive use in animal industry and aquaculture, residues of these antibiotics and the emergence of bacteria resistant to FQs have become a major public health issue. To prepare a generic antibody capable of recognizing nearly all FQs, a single-chain variable fragment (scFv) was generated from the murine hybridoma cells C49H1 producing a FQ-specific monoclonal antibody. This scFv was characterized by indirect competitive enzyme-linked immunosorbent assay (ciELISA), and it showed identical binding properties to parental monoclonal antibody: it was capable of recognizing 17 of 20 targeted FQs below maximum residue limits, except for sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO) which are highly concerned members in the FQs family. In order to broaden the specificity of this scFv to SAR and its analogues (DIF and TRO), protein homology modeling and antibody-ligands docking analysis were employed to identify the potential key amino acid residues involved in hapten antibody. A mutagenesis phage display library was generated by site directed mutagenesis randomizing five aminoacid residues in the third heavy-chain complementarity determining region. After one round of panning against biotinylated norfloxacin (NOR) and four rounds of panning against biotinylated SAR, scFv variants we screened showed up to 10-fold improved IC(50) against SAR, DIF, and TRO in ciELISA while the specificity against other FQs was fully retained.

  10. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    PubMed

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    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 a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (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 amounts 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 × 10(13) 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 nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  11. Efficient expression of single chain variable fragment antibody against paclitaxel using the Bombyx mori nucleopolyhedrovirus bacmid DNA system and its characterizations.

    PubMed

    Yusakul, Gorawit; Sakamoto, Seiichi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    A single chain variable fragment (scFv), the smallest unit of functional recombinant antibody, is an attractive format of recombinant antibodies for various applications due to its small fragment and possibility of genetic engineering. Hybridoma clone 3A3 secreting anti-paclitaxel monoclonal antibody was used to construct genes encoding its variable domains of heavy (VH) and light (VL) chains. The VH and VL domains were linked to be the PT-scFv3A3 using flexible peptide linker in a format of VH-(GGGGS)5-VL. The PT-scFv3A3 was primarily expressed using the pET28a(+) vector in the Escherichia coli system, and was then further expressed by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Interestingly, the reactivity of PT-scFv3A3 expressed in the hemolymph of B. mori using the BmNPV bacmid DNA system was much higher than that expressed in the E. coli system. Using indirect competitive enzyme-linked immunosorbent assay (icELISA), the PT-scFv3A3 (B. mori) reacted not only with immobilized paclitaxel, but also with free paclitaxel in a concentration-dependent manner, with the linear range of free paclitaxel between 0.156 and 5.00 µg/ml. The PT-scFv3A3 (B. mori) exhibited less cross-reactivity (%) than its parental MAb clone 3A3 against paclitaxel-related compounds, including docetaxel (31.1 %), 7-xylosyltaxol (22.1 %), baccatin III (<0.68 %), 10-deacetylbaccatin III (<0.68 %), 1-hydroxybaccatin I (<0.68 %), and 1-acetoxy-5-deacetylbaccatin I (<0.68 %). With the exception of cephalomannine, the cross-reactivity was slightly increased to 8.50 %. The BmNPV bacmid DNA system was a highly efficient expression system of active PT-scFv3A3, which is applicable for PT-scFv3A3-based immunoassay of paclitaxel. In addition, the PT-scFv3A3 can be applied to evaluate its neutralizing property of paclitaxel or docetaxel toxicity.

  12. 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.

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

    PubMed Central

    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

  14. The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease.

    PubMed

    Hust, Michael; Maiss, Edgar; Jacobsen, Hans-Jörg; Reinard, Thomas

    2002-12-01

    A single chain variable fragment antibody (scFv; anti-NIa scFv102) was selected from a synthetic human antibody library by using a NIa protease of Plum pox virus (PPV) as an antigen, which was expressed in bacteria. The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. The NIa protein was detected with anti-NIa scFv102 after expression in Escherichia coli cells as well as from PPV-infected Nicotiana benthamiana plants. Furthermore, the scFv102 has the ability to identify not only PPV from infected plants but also can detect other infections with members of the potyviruses. Nineteen different potyviruses were recognized by the scFv102 in various infected plants tested through dot blot assays. Therefore, the antibody scFv102 has the potential of becoming a general tool to detect potyvirus infections in different plant species. Copyright 2002 Elsevier Science B.V.

  15. Schistosoma japonicum: screening of cercariae cDNA library by specific single-chain antibody against SIEA26-28 ku and immunization experiment of the recombinant plasmids containing the selected genes.

    PubMed

    Gao, Dong-mei; Wang, Shi-ping; He, Zhuo; Fung, Ming-chiu; Liu, Ming-she; Yu, Lu-xin; Chen, Xiu-chun

    2010-06-01

    To obtain the gene encoding SIEA26-28 ku, which has been proven to be a potential anti-schistosomiasis vaccine candidate, screening Schistosoma japonicum (Sj) cercariae cDNA library with soluble specific single-chain antibody (SIEA26-28 ku-scFv) was performed. A large amount of specific single-chain antibody was harvested through construction of recombinant expression vector pET32a/scFv. The protein was purified and characterized. By using this protein (PET32a-scFv) as a probe, S. japonicum cercariae cDNA library was screened. Two strong positive clones were selected, and their eukaryotic recombinant plasmids were constructed. These genes were named as S. japonicum ribosomal protein S4 (SjRPS4) and S. japonicum ribosomal protein L7 (SjRPL7), respectively. Experiments of mice showed that both SjRPS4 and SjRPL7 DNA vaccines could induce significant immunoprotection. Result of these experiments further proved that the specific single-chain antibody is a very valuable tool in screening of cDNA library to get the corresponding molecules.

  16. Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library.

    PubMed

    Bagheri, Vahid; Nejatollahi, Foroogh; Esmaeili, Seyed Alireza; Momtazi, Amir Abbas; Motamedifar, Mohamad; Sahebkar, Amirhossein

    2017-01-15

    The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31-43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB1 and scFv-gB2, with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB1 and gB2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB1 and scFv-gB2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

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

    PubMed

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

    2011-04-01

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

  18. A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

    PubMed Central

    Kellner, Christian; Stein, Christoph; Kügler, Markus; Schwenkert, Michael; Saul, Domenica; Mentz, Kristin; Singer, Heiko; Stockmeyer, Bernhard; Hillen, Wolfgang; Mackensen, Andreas; Fey, Georg H

    2011-01-01

    A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all three antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All three fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis. PMID:21081841

  19. Delivery of anti-platelet-endothelial cell adhesion molecule single-chain variable fragment-urokinase fusion protein to the cerebral vasculature lyses arterial clots and attenuates postischemic brain edema.

    PubMed

    Danielyan, Kristina; Ding, Bi-Sen; Gottstein, Claudia; Cines, Douglas B; Muzykantov, Vladimir R

    2007-06-01

    Efficacy and safety of current means to prevent cerebrovascular thrombosis in patients at high risk of stroke are suboptimal. In theory, anchoring fibrinolytic plasminogen activators to the luminal surface of the cerebral endothelium might arrest formation of occlusive clots in this setting. We tested this approach using the recombinant construct antiplatelet-endothelial cell adhesion molecule (PECAM) single-chain variable fragment (scFv)-urokinase-type plasminogen activator (uPA), fusing low-molecular-weight single-chain urokinase-type plasminogen activator with a scFv of an antibody directed to the stably expressed endothelial surface determinant PECAM-1, implicated in inflammation and thrombosis. Studies in mice showed that scFv-uPA, but not unconjugated uPA 1) accumulates in the brain after intravascular injection, 2) lyses clots lodged in the cerebral arterial vasculature without hemorrhagic complications, 3) provides rapid and stable cerebral reperfusion, and 4) alleviates post-thrombotic brain edema. Effective and safe thromboprophylaxis in the cerebral arterial circulation by anti-PECAM scFv-uPA represents a prototype of a new paradigm to prevent recurrent cerebrovascular thrombosis.

  20. Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

    PubMed Central

    Hurwitz, Amy M.; Huang, Wanzhi; Kou, Baijun; Estes, Mary K.; Atmar, Robert L.; Palzkill, Timothy

    2017-01-01

    Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics. PMID:28095447

  1. 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

  2. Suppression of pancreatic tumor growth by targeted arsenic delivery with anti-CD44v6 single chain antibody conjugated nanoparticles.

    PubMed

    Qian, Chenchen; Wang, Yong; Chen, Yinting; Zeng, Linjuan; Zhang, Qiubo; Shuai, Xintao; Huang, Kaihong

    2013-08-01

    Arsenic trioxide (As2O3) is a promising anticancer agent for solid tumors. However, the high toxicity to normal tissues resulting from the lack of tumor specificity remains a huge challenge in its systemic application. Targeted vectors enabling drug delivery to specific cancer cells bring about great potential for better therapeutic efficacy whereas low side effects in cancer treatments. Our previous work has demonstrated that the anti-CD44v6 single chain variable fragment (scFv(CD44v6)) screened out from the human phage-displayed scFv library possesses high specificity and affinity to membrane antigen CD44v6 over-expressing in a subset of epithelium-derived cancers, such as pancreatic, hepatocellular, colorectal and gastric cancers. Herein, a maleimide-functionalized amphiphilic diblock copolymer of poly (ethylene glycol) and poly (D, L-lactide) (mal-PEG-PDLLA) was synthesized and assembled to vesicles with arsenite ion (As) encapsulated in their cores (As-NPs). Conjugation of scFv(CD44v6) with mal-PEG-PDLLA (scFv-As-NPs) enabled more efficient delivery of As and exhibited higher cytotoxic activity than non-targeted ones (As-NPs) in human pancreatic cancer cells PANC-1. Furthermore, the targeted delivery of As induced more significant gene suppression in terms of the expression of anti-apoptotic Bcl-2 protein. Consequently, the expression level of cleaved caspase-3 which is a molecular indicator of cell apoptosis was remarkably elevated. In animal tests, scFv-As-NPs were found to greatly increase accumulation of drug in tumor site and potentiate the efficacy of As in inhibiting tumor growth owing to the enhanced cell apoptosis. These results imply that our tumor specific nanocarriers provide a highly efficient and safe platform for pancreatic cancer therapy.

  3. Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide.

    PubMed

    Kabir, M Enamul; Karim, Nurul; Krishnaswamy, Senthilkumar; Selvakumar, Dakshnamurthy; Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

    2011-12-01

    Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.

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

    PubMed

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

    2013-02-18

    Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv-AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10(-2) μg mL(-1), superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10(-3) mg g(-1) of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Ledezma-Candanoza, Luis M; Contreras-Ferrat, Luis Gabriel; Olamendi-Portugal, Timoteo; Possani, Lourival D; Becerril, Baltazar; Riaño-Umbarila, Lidia

    2012-10-26

    Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

  7. An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

    PubMed

    Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

    2015-03-31

    Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples.

  8. Single chain stochastic polymer modeling at high strain rates.

    SciTech Connect

    Harstad, E. N.; Harlow, Francis Harvey,; Schreyer, H. L.

    2001-01-01

    Our goal is to develop constitutive relations for the behavior of a solid polymer during high-strain-rate deformations. In contrast to the classic thermodynamic techniques for deriving stress-strain response in static (equilibrium) circumstances, we employ a statistical-mechanics approach, in which we evolve a probability distribution function (PDF) for the velocity fluctuations of the repeating units of the chain. We use a Langevin description for the dynamics of a single repeating unit and a Lioville equation to describe the variations of the PDF. Moments of the PDF give the conservation equations for a single polymer chain embedded in other similar chains. To extract single-chain analytical constitutive relations these equations have been solved for representative loading paths. By this process we discover that a measure of nonuniform chain link displacement serves this purpose very well. We then derive an evolution equation for the descriptor function, with the result being a history-dependent constitutive relation.

  9. The dynamics of single chains within a model polymer melt

    NASA Astrophysics Data System (ADS)

    McCormick, Julie A.; Hall, Carol K.; Khan, Saad A.

    2005-03-01

    Discontinuous molecular dynamics simulations are performed on a system containing 32 hard chains of length 192 at a volume fraction of ϕ =0.45 to explore the idea that localized entanglements have a significant effect on the dynamics of the individual chains within an entangled polymer melt. Anomalous behavior can still be observed when studying the dynamics of the individual chains, although increased time averaging causes the anomalous relaxation-memory-release behavior that was observed previously in the system to smooth out. First, the individual chain mean squared displacements and apparent diffusion coefficients are calculated, and a wide distribution of diffusive behavior is found. Although the apparent diffusion coefficient curve averaged over all chains displays the predicted long-time diffusive behavior, the curves for the individual chains differ both qualitatively and quantitatively. They display superdiffusive, diffusive, and subdiffusive behavior, with the largest percentage of chains exhibiting superdiffusive behavior and the smallest percentage exhibiting the predicted diffusive behavior. Next, the individual chain end-to-end vector autocorrelation functions and relaxation times are determined, and a wide distribution of stress relaxation behavior is found. The times when the end-to-end vector autocorrelation functions relax completely span almost an order of magnitude in reduced time. For some chains, the end-to-end vector autocorrelation function relaxes smoothly toward zero similar to the system average; however, for other chains the relaxation is slowed greatly, indicating the presence of additional entanglements. Almost half of the chains exhibit the anomalous behavior in the end-to-end vector autocorrelation function. Finally, the dynamic properties are displayed for a single chain exhibiting anomalous relaxation-memory-release behavior, supporting the idea that the relaxation-memory-release behavior is a single-chain property.

  10. Design of an Active Ultrastable Single-chain Insulin Analog

    PubMed Central

    Hua, Qing-xin; Nakagawa, Satoe H.; Jia, Wenhua; Huang, Kun; Phillips, Nelson B.; Hu, Shi-quan; Weiss, Michael A.

    2008-01-01

    Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 ± 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (ΔΔGu = 0.7 ± 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 ± 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts ValA3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 α-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world. PMID:18332129

  11. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    PubMed

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  12. Use of antibody fragments (Fv) in immunocytochemistry.

    PubMed

    Kleymann, G; Ostermeier, C; Heitmann, K; Haase, W; Michel, H

    1995-06-01

    We developed a novel antibody fragment (Fv) technique for localization and determination of the surface topology of membrane protein complexes by immunogold electron microscopy. Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) raised against bacterial membrane proteins were established. The cDNAs coding for the variable domains of the MAbs were cloned and expressed in Escherichia coli. The engineered Fv fragments served as trifunctional adapter molecules. The Fv fragment binds to the epitope of the membrane protein. The Strep tag fused to the VH chain was used for one-step affinity purification of the Fv fragments. Immunological detection of the membrane protein-bound Fv fragments in electron microscopy was accomplished either via the Strep tag with colloidal gold-labeled streptavidin or via the c-myc tag, which was fused to the VL chain, in combination with the c-myc tag-specific antibody 9E10 and a colloidal gold-labeled secondary antibody. We examined four Fv fragments directed against the cytochrome c oxidase or the ubiquinol-cytochrome c oxidoreductase of Paracoccus denitrificans and bacteriorhodopsin of Halobacterium halobium to show that this method is generally applicable. In all cases the Fv fragments showed the same results as their corresponding parent antibodies in electron microscopic immunostaining and other applications.

  13. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

    PubMed

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen

    2002-01-01

    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

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

    PubMed

    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

    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. 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. 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.

  15. 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

  16. In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner.

    PubMed

    Grage, Katrin; Rehm, Bernd H A

    2008-01-01

    Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively

  17. 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.

  18. Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

    PubMed

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

    2011-06-10

    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 Å 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 β-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.

  19. Characterization of a Single-Chain Variable Fragment Recognizing a Linear Epitope of Aβ: A Biotechnical Tool for Studies on Alzheimer’s Disease?

    PubMed Central

    Dornieden, Silke; Müller-Schiffmann, Andreas; Sticht, Heinrich; Jiang, Nan; Cinar, Yeliz; Wördehoff, Michael; Korth, Carsten; Funke, Susanne Aileen; Willbold, Dieter

    2013-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aβ encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs), Aβ binding single-chain variable fragments (scFvs) were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aβ binding IgG, designated IC16, which binds the N-terminal region of Aβ (Aβ(1-8)). scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aβ species and its influence on Aβ fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aβ and could be applied as an Aβ detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD. PMID:23555792

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

    PubMed Central

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

    2011-01-01

    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 Å 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 β-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. PMID:21489992

  1. 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

  2. CD40-directed scFv-TRAIL fusion proteins induce CD40-restricted tumor cell death and activate dendritic cells

    PubMed Central

    El-Mesery, M; Trebing, J; Schäfer, V; Weisenberger, D; Siegmund, D; Wajant, H

    2013-01-01

    Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction. PMID:24232092

  3. 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.

  4. 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

  5. [Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

    PubMed

    Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

    2017-03-01

    Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A2+(A1-A2)/(1+(x/x0)(p)) (A1=1.28; A2=-0.066; x0=12560.75; p=0.74) with a correlation coefficient (R(2)) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

  6. PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody.

    PubMed

    Gargouri-Bouzid, Radhia; Jaoua, Leïla; Rouis, Souad; Saïdi, Mohamed Najib; Bouaziz, Donia; Ellouz, Radhouane

    2006-06-01

    A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commercial potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVYO strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.

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

    PubMed

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

    2016-01-01

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

  8. 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

  9. 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

  10. A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

    PubMed

    Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

    2016-08-01

    Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

  11. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.

    PubMed

    Dong, Jie-Xian; Li, Zhen-Feng; Lei, Hong-Tao; Sun, Yuan-Ming; Ducancel, Frédéric; Xu, Zhen-Lin; Boulain, Jean-Claude; Yang, Jin-Yi; Shen, Yu-Dong; Wang, Hong

    2012-07-29

    A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25±0.03 and 0.02±0.004 ng mL(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-1). The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between the data of dc-CLEIA and HPLC-MS (R(2)>0.99), indicating that the assay was an efficient analytical method for monitoring food safety. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Dendritic cells transfected with scFv from Mab 7.B12 mimicking original antigen gp43 induces protection against experimental Paracoccidioidomycosis.

    PubMed

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

    2011-01-07

    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.

  13. 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

  14. The design of target specific antibodies (scFv) by applying de novo workflow: Case study on BmR1 antigen from Brugia malayi.

    PubMed

    Khor, Bee Yin; Lim, Theam Soon; Noordin, Rahmah; Choong, Yee Siew

    2017-09-01

    De novo approach was applied to design single chain fragment variable (scFv) for BmR1, a recombinant antigen from Bm17DIII gene which is the primary antigen used for the detection of anti-BmR1 IgG4 antibodies in the diagnostic of lymphatic filariasis. Three epitopes of the BmR1 was previously predicted form an ab initio derived three-dimensional structure. A collection of energetically favourable conformations was generated via hot-spot-centric approach. This resulted in a set of three different scFv scaffolds used to compute the high shape complementary conformations via dock-and-design approach with the predicted epitopes of BmR1. A total of 4227 scFv designs were generated where 200 scFv designs produced binding energies of less than -20 R.E.U with shape complementarity higher than 0.5. We further selected the design with at least one hydrogen bond and one salt bridge with the epitope, thus resulted in a total of 10, 1 and 19 sFv designs for epitope 1, 2 and 3, respectively. The results thus showed that de novo design can be an alternative approach to yield high affinity in silico scFv designs as a starting point for antibody or specific binder discovery processes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  16. Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display.

    PubMed

    Eteshola, Edward

    2010-06-30

    Iterative affinity selection procedures were used to isolate a number of single chain Fv (scFv) antibody fragment clones from naïve Tomlinson I+J phage display libraries that specifically recognize and bind a chemokine, monokine induced by interferon-gamma (MIG/CXCL9). MIG is an important transplant rejection/biology chemokine protein. ELISA-based affinity characterization results indicate that selectants preferentially bind to MIG in the presence of key biopanning component materials and closely related chemokine proteins. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific MIG binding capability with potential applications in transplant rejection monitoring, and other biomedical applications where detection of MIG level is important. Published by Elsevier B.V.

  17. 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.

  18. Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor.

    PubMed

    Sotiriadis, A; Keshavarz, T; Keshavarz-Moore, E

    2001-01-01

    A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.

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

    PubMed Central

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

    2011-01-01

    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 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å 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. PMID:21217145

  20. 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.

  1. Functional characterization of an scFv-Fc antibody that immunotherapeutically targets the common cancer cell surface proteoglycan CSPG4.

    PubMed

    Wang, Xinhui; Katayama, Akihiro; Wang, Yangyang; Yu, Ling; Favoino, Elvira; Sakakura, Koichi; Favole, Alessandra; Tsuchikawa, Takahiro; Silver, Susan; Watkins, Simon C; Kageshita, Toshiro; Ferrone, Soldano

    2011-12-15

    Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

  2. 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

  3. Construction of scFv derived from a tumor-associated monoclonal antibody having tumoricidal activity on human hepatocellular carcinoma.

    PubMed

    Tungpradabkul, Sumalee; Sandee, Duanpen; Puthong, Songchan; Laohathai, Kingkarn

    2005-04-01

    A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4 mg of the Mab over 1-month period. A single-chain variable fragment (scFv) molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavy- and light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma.

  4. 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.

  5. Generation and characterization of chicken-sourced single-chain variable fragments (scFvs) against porcine interferon-gamma (pIFN-γ).

    PubMed

    Chen, Hong-Xiu; He, Fan; Sun, Yuan; Luo, Yuzi; Qiu, Hua-Ji; Zhang, Xiao-Ying; Sutton, Brian J

    2015-01-01

    Development of chicken-sourced antibodies offers an alternative strategy for the development of highly specific antibodies against mammalian proteins with conserved epitopes due to the phylogenetic distance between avian and mammalian species. In this study, the single-chain variable fragments (scFvs) against porcine interferon-gamma was screened and characterized from a hyperimmunized chicken phage display library. The expressed soluble scFvs exhibited highly specific recognition of porcine interferon-gamma in ELISA, Western blot, and immunofluorescence staining assays. Results of the current study indicate that it is possible to develop scFv IgY antibodies to a mammalian interferon by using Biopanning technology. Furthermore, it also confirms that monoclonal avian IgY antibody technique could be applied as a promising tool to produce immunoglobulin molecules with high specificity and affinity towards conserved mammalian epitopes or antigens.

  6. Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen.

    PubMed

    Frigerio, Barbara; Benigni, Fabio; Luison, Elena; Seregni, Ettore; Pascali, Claudio; Fracasso, Giulio; Morlino, Sara; Valdagni, Riccardo; Mezzanzanica, Delia; Canevari, Silvana; Figini, Mariangela

    2015-11-01

    Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution.

  7. [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.

  8. Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo

    PubMed Central

    2010-01-01

    Background Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. Methods DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of γH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells. Results Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of γH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy. Conclusion The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential

  9. Bridging lectin binding sites by multivalent carbohydrates.

    PubMed

    Wittmann, Valentin; Pieters, Roland J

    2013-05-21

    Carbohydrate-protein interactions are involved in a multitude of biological recognition processes. Since individual protein-carbohydrate interactions are usually weak, multivalency is often required to achieve biologically relevant binding affinities and selectivities. Among the possible mechanisms responsible for binding enhancement by multivalency, the simultaneous attachment of a multivalent ligand to several binding sites of a multivalent receptor (i.e. chelation) has been proven to have a strong impact. This article summarizes recent examples of chelating lectin ligands of different size. Covered lectins include the Shiga-like toxin, where the shortest distance between binding sites is ca. 9 Å, wheat germ agglutinin (WGA) (shortest distance between binding sites 13-14 Å), LecA from Pseudomonas aeruginosa (shortest distance 26 Å), cholera toxin and heat-labile enterotoxin (shortest distance 31 Å), anti-HIV antibody 2G12 (shortest distance 31 Å), concanavalin A (ConA) (shortest distance 72 Å), RCA120 (shortest distance 100 Å), and Erythrina cristagalli (ECL) (shortest distance 100 Å). While chelating binding of the discussed ligands is likely, experimental proof, for example by X-ray crystallography, is limited to only a few cases.

  10. Multivalent Inhibitors of Channel-Forming Bacterial Toxins.

    PubMed

    Yamini, Goli; Nestorovich, Ekaterina M

    2016-07-29

    Rational design of multivalent molecules represents a remarkable modern tool to transform weak non-covalent interactions into strong binding by creating multiple finely-tuned points of contact between multivalent ligands and their supposed multivalent targets. Here, we describe several prominent examples where the multivalent blockers were investigated for their ability to directly obstruct oligomeric channel-forming bacterial exotoxins, such as the pore-forming bacterial toxins and B component of the binary bacterial toxins. We address problems related to the blocker/target symmetry match and nature of the functional groups, as well as chemistry and length of the linkers connecting the functional groups to their multivalent scaffolds. Using the anthrax toxin and AB5 toxin case studies, we briefly review how the oligomeric toxin components can be successfully disabled by the multivalent non-channel-blocking inhibitors, which are based on a variety of multivalent scaffolds.

  11. Expression and characterization of recombinant interleukin-21 receptor and its targeting single-chain variable fragment antibodies selected from a human phage display library.

    PubMed

    Wu, Qinhang; Zhang, Juan; Luo, Chen; Zhang, Tao; Wang, Tong; Wang, Min

    2012-10-01

    Interleukin-21 receptor (IL-21R) is widely expressed in lymphocytes, and plays an important role in immunological cell proliferation and cytokine production. The present study aims to express a recombinant extracellular domain of human IL-21R (rhIL-21R-ECD) with high yield, and to screen the anti-IL-21R single-chain variable fragments (scFvs) from a synthetic human phage display library. The rhIL-21R-ECD, being expressed mainly as insoluble inclusion bodies in Escherichia coli BL21 (DE3), was purified and refolded. ELISA analysis showed that the refolded rhIL-21R-ECD bound to its ligand IL-21 in a concentration-dependent manner. Using a phage display technique, anti-IL-21R scFvs were screened from a naïve human phage display library by biopanning. After four rounds of panning, positive clones were isolated, sequenced, and characterized. The clone with highest activity was designated as C2. Flow cytometry analysis showed that the scFv C2 could recognize IL-21R on Jurkat cells. Furthermore, proliferation assay revealed a concentration-dependent inhibitory effect of C2 on the Jurkat cell, with fifty percent inhibitory concentration (IC(50)) of 78 nM. A human scFv antibody C2 with a high binding specificity to IL-21R was isolated and characterized. The antibody showed a concentration-dependent inhibitory effect on Jurkat cell proliferation.

  12. Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

    PubMed

    Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

    2009-11-01

    Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, K(D)= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name "ecstasy." The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C--H...O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 A, and 2.4 A, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

  13. Insect cell-based expression and characterization of a single-chain variable antibody fragment directed against blood coagulation factor VIII.

    PubMed

    Kurasawa, James H; Shestopal, Svetlana A; Jha, Naveen K; Ovanesov, Mikhail V; Lee, Timothy K; Sarafanov, Andrey G

    2013-04-01

    A recombinant single-chain variable antibody fragment (scFv) KM33 was previously described as a ligand that can inhibit the function of blood coagulation factor VIII (FVIII). This scFv was previously derived from an individual with anti-FVIII antibodies manifested in FVIII functional deficiency (Hemophilia A) and expressed in bacteria. In the present work, we describe an alternative approach for fast and easy production of KM33 in insect cells (Spodoptera frugiperda). The KM33 gene was codon-optimized and expressed in secreted form using a baculovirus system. The protein was isolated using metal-affinity and size-exclusion chromatography to purity of about 96% and yield of 0.4-1.2 mg per 120 mL of culture, based on several independent expression experiments. In a binding assay using surface plasmon resonance, the insect cell-derived KM33 (iKM33) was qualified as a high-affinity ligand for FVIII. Epitope specificity of iKM33 on FVIII (C1 domain) was confirmed by testing the binding with a relevant mutant of FVIII. In several FVIII functional tests (factor Xa generation, APTT clotting, thrombin generation and video microscopy clot growth assays), iKM33 strongly inhibited FVIII activity in accordance with the clinical effect of the parental antibody. Therefore, the expressed protein was concluded to be fully functional and applicable in various assays with FVIII. Published by Elsevier Inc.

  14. Bicistronic expression plasmid encoding allergen and anti-IgE single chain variable fragment antibody as a novel DNA vaccine for allergy therapy and prevention.

    PubMed

    Bandbon Balenga, Nariman Aghaei; Thalhamer, Josef; Weiss, Richard

    2006-01-01

    Several approaches have been applied in order to alleviate the difficulties allergic patients are suffering from. Among them DNA vaccination and anti-IgE antibody have shown promising results. Herewith, a combination of both strategies is proposed to minimize IgE production while inducing high levels of blocking IgG and strong Th1 immune responses. A bicistronic expression plasmid including an internal ribosomal entry site (IRES) can express both, allergen and a single chain variable fragment (scFv) antibody against human IgE within antigen presenting cells (APCs) including B cells. Presentation of allergen derived peptides via MHC I and MHC II stimulates specific Th1 responses resulting in high levels of IFN-gamma and IgG. Anti-IgE scFv antibody binds to newly synthesized IgE molecules within B cell cytoplasm and also to free serum IgE, thereby inhibiting attachment of IgE to its receptors on basophils and mast cells. Also, IgE-anti-IgE complex functions as blocking antibody and neutralizes allergens entering the body. Additionally, anti-IgE scFv antibody binds to membrane bound IgE (mIgE) on B cells and interferes with IgE expression. Using assays, such as enzyme linked immunosorbent assay (ELISA), IgG and IgE production in response to this expression system can be evaluated. Also, rat basophil leukemia cell assay (using RBL-2H3 cells) can show the amount of functional IgE in sera as basophil mediator release is regarded as an indicator of the allergic hypersensitive reactions. The proposed approach may result in high levels of blocking IgG and low levels of IgE secretion from B cells. Additionally, it can inhibit activity of IgE in degranulation of basophils and mast cells.

  15. Single-chain antibody-based immunotoxins targeting Her2/neu: design optimization and impact of affinity on antitumor efficacy and off-target toxicity.

    PubMed

    Cao, Yu; Marks, James D; Huang, Qian; Rudnick, Stephen I; Xiong, Chiyi; Hittelman, Walter N; Wen, Xiaoxia; Marks, John W; Cheung, Lawrence H; Boland, Kim; Li, Chun; Adams, Gregory P; Rosenblum, Michael G

    2012-01-01

    Recombinant immunotoxins, consisting of single-chain variable fragments (scFv) genetically fused to polypeptide toxins, represent potentially effective candidates for cancer therapeutics. We evaluated the affinity of various anti-Her2/neu scFv fused to recombinant gelonin (rGel) and its effect on antitumor efficacy and off-target toxicity. A series of rGel-based immunotoxins were created from the human anti-Her2/neu scFv C6.5 and various affinity mutants (designated ML3-9, MH3-B1, and B1D3) with affinities ranging from 10(-8) to 10(-11) mol/L. Against Her2/neu-overexpressing tumor cells, immunotoxins with increasing affinity displayed improved internalization and enhanced autophagic cytotoxicity. Targeting indices were highest for the highest affinity B1D3/rGel construct. However, the addition of free Her2/neu extracellular domain (ECD) significantly reduced the cytotoxicity of B1D3/rGel because of immune complex formation. In contrast, ECD addition had little impact on the lower affinity constructs in vitro. In vivo studies against established BT474 M1 xenografts showed growth suppression by all immunotoxins. Surprisingly, therapy with the B1D3-rGel induced significant liver toxicity because of immune complex formation with shed Her2/neu antigen in circulation. The MH3-B1/rGel construct with intermediate affinity showed effective tumor growth inhibition without inducing hepatotoxicity or complex formation. These findings show that while high-affinity constructs can be potent antitumor agents, they may also be associated with mistargeting through the facile formation of complexes with soluble antigen leading to significant off-target toxicity. Constructs composed of intermediate-affinity antibodies are also potent agents that are more resistant to immune complex formation. Therefore, affinity is an exceptionally important consideration when evaluating the design and efficacy of targeted therapeutics.

  16. 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

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

    PubMed Central

    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

    2016-01-01

    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

  18. 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

  19. 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.

  20. Selection of scFv phages specific for chloramphenicol acetyl transferase (CAT), as alternatives for antibodies in CAT detection assays.

    PubMed

    Van Dorst, Bieke; Mehta, Jaytry; Rouah-Martin, Elsa; Backeljau, Jelke; De Coen, Wim; Eeckhout, Dominique; De Jaeger, Geert; Blust, Ronny; Robbens, Johan

    2012-10-01

    Reporter gene assays are commonly used in applied toxicology to measure the transcription of genes involved in toxic responses. In these reporter gene assays, transgenic cells are used, which contain a promoter-operator region of a gene of interest fused to a reporter gene. The transcription of the gene of interest can be measured by the detection of the reporter protein. Chloramphenicol acetyl transferase (CAT) is frequently used as a reporter protein in mammalian reporter gene assays. Although CAT can be measured by different detection systems, like enzymatic and immune assays, most of these tests are expensive, time-consuming and labor-intensive. The excellent characteristics of phages, like their high affinity and specificity, their fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and their stability, make them appropriate as alternatives for antibodies in detection assays. Therefore, in this study single-chain variable fragment (scFv) phages were selected with affinity for CAT. Several scFv phages were selected that showed affinity towards CAT in a screening ELISA. Surface plasmon resonance analyses showed that the tested scFv phages have an affinity for CAT with a dissociation constant (K(d)) around 1 µM. The selected scFv phages in this study could be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng ml⁻¹ or 4 pM. This low detection limit demonstrates the potential of the scFv phages as an alternative for capturing antibodies in a highly sensitive detection test to measure CAT concentrations in reporter gene assays.

  1. Phage display library selection of a hypoxia-binding scFv antibody for liver cancer metabolic marker discovery

    PubMed Central

    Chen, Hang; Gao, Zhihui; Li, Yao; Sun, Zhongyuan; Xiang, Rong; Zhang, Sihe

    2016-01-01

    Hypoxia, which is frequently observed in liver cancer and metastasis, influences tumor progression and resistance to therapy. Although hypoxia-associated biomarkers are of use in other cancers, none is recognized as a surrogate for hypoxia in liver cancer. In this study, we generated seven unique human single-chain Fv (scFv) antibodies (Abs) specific to hypoxic liver cancer cells, using normoxia-depleted vs hypoxia-selected phage library panning technology. By developing the scFv immunoprecipitation-based mass spectrometry method, the antigen that bound with one of the Abs (H103) was identified as the M2 splice isoform of pyruvate kinase (PKM2), an enzyme that is a key regulator of aerobic glycolysis in cancer cells. Increased expression of PKM2 was induced by hypoxia in liver cancer cell lines. Immunohistochemical (IHC) staining showed that PKM2 was highly expressed in moderately and well differentiated hepatocellular carcinoma (HCC) tissues with a hypovascular staining pattern. High expression of PKM2 was also localized in the perinecrotic area of intrahepatic cholangiocarcinoma (ICC) tissues. The percentage of the HCC or ICC tumor expressing PKM2 was significantly higher with more tumor necrosis, low microvessel density, and advanced stage. Moreover, the H103 scFv Ab was efficiently internalized into hypoxic liver cancer cells and could have potential for targeted drug delivery. Conclusion: our study, for the first time, developed hypoxia-specific scFv Ab H103 to liver cancer cells, and revealed that PKM2 is a promising biomarker for hypoxia in HCC and ICC tissues. These allow further exploration of this valuable Ab and PKM2 antigen for hypoxia targeting in liver cancer. PMID:27203546

  2. Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro.

    PubMed

    Chen, Yinting; Huang, Kaihong; Li, Xuexian; Lin, Xiangan; Zhu, Zhaohua; Wu, Ying

    2010-06-01

    CD44v6 is a cancer-associated antigen that mainly expresses in a subset of adenocarcinomas. Therefore, in this study, anti-human CD44v6 single-chain variable fragment (scFv) has been selected and characterized because it is the first step of primary importance towards the construction of a novel cancer-targeted agent for cancer diagnosis and therapy. In our study, anti-human CD44v6 scFv was selected from a human phage-displayed scFv library based on its ability to bind in vitro to CD44v6 antigen. Subsequently, immunofluorescent staining and Western blot analyses were performed to measure the binding characteristics of this scFv. In addition, flow cytometric analysis was done to verify its cancer-targeting ability in vitro. And a flow cytometry-based assay was used to determine its equilibrium dissociation constant (K (D)). Finally, one functional anti-CD44v6 scFv was selected and characterized. Nucleotide sequencing verified that it was an incomplete scFv gene but had a variable heavy chain (V(H)) alone. However, anti-CD44v6 scFv demonstrated cell-binding and antigen-binding activities by immunofluorescent staining and Western blot analyses. Furthermore, flow cytometric analysis proved that this scFv specifically targeted CD44v6-expressing cancer cells other than CD44v6 non-expressing normal cells or tumor cells in vitro. The K (D) of this scFv was calculated to be 7.85 +/- 0.93 x 10(-8) M. In summary, the selected human scFv against CD44v6 has specific binding activity and favorable binding affinity despite lacking a variable light chain (V(L)). Moreover, it can effectively and specifically target CD44v6-expressing cancer cells. All these characteristics make anti-CD44v6 scFv a promising agent for cancer detection and anti-cancer therapy.

  3. Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium.

    PubMed

    Meyer, Torsten; Stratmann-Selke, Janin; Meens, Jochen; Schirrmann, Thomas; Gerlach, Gerald F; Frank, Ronald; Dübel, Stefan; Strutzberg-Minder, Katrin; Hust, Michael

    2011-01-10

    Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-Salmonella antibodies in serum. Recombinant antibody fragments (scFvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scFv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-OmpD IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Production and characterization of recombinant scFv against digoxin by phage display technology.

    PubMed

    Alirezapour, Behruz; Rajabibazl, Masoumeh; Rasaee, Mohhamad Javad; Omidfar, Kobra

    2013-06-01

    The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of treated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).

  5. 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

  6. ScFv-decorated PEG-PLA-based nanoparticles for enhanced siRNA delivery to Her2⁺ breast cancer.

    PubMed

    Dou, Shuang; Yang, Xian-Zhu; Xiong, Meng-Hua; Sun, Chun-Yang; Yao, Yan-Dan; Zhu, Yan-Hua; Wang, Jun

    2014-11-01

    Patients with Her2-overexpressing (Her2(+)) breast cancers generally have a poorer prognosis due to the high aggressiveness and chemoresistance of the disease. Small interfering RNA (siRNA) targeting the gene encoding polo-like kinase 1 (Plk1; siPlk1) has emerged as an efficient therapeutic agent for Her2(+) breast cancers. Poly(ethylene glycol)-block-poly(D,L-lactide) (PEG-PLA)-based nanoparticles for siRNA delivery were previously developed and optimized. In this study, for targeted delivery of siPlk1 to Her2(+) breast cancer, anti-Her2 single-chain variable fragment antibody (ScFv(Her2))-decorated PEG-PLA-based nanoparticles with si Plk1 encapsulation (ScFv(Her2)-NP(si) Plk1) are developed. With the rationally designed conjugation site, ScFv(Her2)-NP(siRNA) can specifically bind to the Her2 antigen overexpressed on the surface of Her2(+) breast cancer cells. Therefore, ScFv(Her2)-NP(si) Plk1 exhibits improved cellular uptake, promoted Plk1 silencing efficiency, and induced enhanced tumor cell apoptosis in Her2(+) breast cancer cells, when compared with nontargeted NP(si) Plk1. More importantly, ScFv(Her2)-NP(siRNA) markedly enhances the accumulation of siRNA in Her2(+) breast tumor tissue, and remarkably improves the efficacy of tumor suppression. Dose-dependent anti-tumor efficacy further demonstrates that ScFvHer2 -decorated PEG-PLA-based nanoparticles with siPlk1 encapsulation can significantly enhance the inhibition of Her2(+) breast tumor growth and reduce the dose of injected siRNA. These results suggest that ScFvHer2 -decorated PEG-PLA-based nanoparticles show great potential for targeted RNA interference therapy of Her2(+) breast tumor. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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

  8. 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.

  9. Therapeutic treatment with scFv-PLGA nanoparticles decreases pulmonary fungal load in a murine model of paracoccidioidomycosis.

    PubMed

    Jannuzzi, Grasielle Pereira; Souza, Nicole de Araújo; Françoso, Kátia Sanches; Pereira, Roney Henrique; Santos, Raquel Possemozer; Kaihami, Gilberto Hideo; Almeida, José Roberto Fogaça de; Batista, Wagner Luiz; Amaral, André Corrêa; Maranhão, Andrea Queiroz; Almeida, Sandro Rogério de; Ferreira, Karen Spadari

    2017-09-23

    Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  10. Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus.

    PubMed

    Wang, Man; Zhang, Yan; Li, Benqiang; Zhu, Jianguo

    2015-06-01

    Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. CD70-restricted specific activation of TRAILR1 or TRAILR2 using scFv-targeted TRAIL mutants

    PubMed Central

    Trebing, J; El-Mesery, M; Schäfer, V; Weisenberger, D; Siegmund, D; Silence, K; Wajant, H

    2014-01-01

    To combine the CD27 stimulation inhibitory effect of blocking CD70 antibodies with an antibody-dependent cellular cytotoxicity (ADCC)-independent, cell death-inducing activity for targeting of CD70-expressing tumors, we evaluated here fusion proteins of the apoptosis-inducing TNF family member TRAIL and a single-chain variable fragment (scFv) derived from a high-affinity llama-derived anti-human CD70 antibody (lαhCD70). A fusion protein of scFv:lαhCD70 with TNC-TRAIL, a stabilized form of TRAIL, showed strongly enhanced apoptosis induction upon CD70 binding and furthermore efficiently interfered with CD70-CD27 interaction. Noteworthy, introduction of recently identified mutations that discriminate between TRAILR1 and TRAILR2 binding into the TRAIL part of scFv:lαhCD70-TNC-TRAIL resulted in TRAIL death receptor-specific fusion proteins with CD70-restricted activity. PMID:24481449

  12. 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

  13. 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.

  14. Preventive DNA vaccination against CEA-expressing tumors with anti-idiotypic scFv6.C4 DNA in CEA-expressing transgenic mice.

    PubMed

    Denapoli, Priscila M A; Zanetti, Bianca F; Dos Santos, Adara A; de Moraes, Jane Z; Han, Sang W

    2017-03-01

    Carcinoembryonic antigen (CEA) is expressed during embryonic life and in low level during adult life. Consequently, the CEA is recognized by the immune system as a self-antigen and thus CEA-expressing tumors are tolerated. Previously, we constructed a single chain variable fragment using the 6.C4 (scFv6.C4) hybridoma cell line, which gave rise to antibodies able to recognize CEA when C57/Bl6 mice were immunized. Here, the scFv6.C4 ability to prevent the CEA-expressing tumor growth was assessed in CEA-expressing transgenic mice CEA2682. CEA2682 mice immunized with the scFv6.C4 expressing plasmid vector (uP/PS-scFv6.C4) by electroporation gave rise to the CEA-specific AB3 antibody after the third immunization. Sera from immunized mice reacted with CEA-expressing human colorectal cell lines CO112, HCT-8, and LISP-1, as well as with murine melanoma B16F10 cells expressing CEA (B16F10-CEA). Cytotoxic T lymphocytes (CTL) from uP/PS-scFv6.C4 immunized mice lysed B16F10-CEA (56.7%) and B16F10 expressing scFv6.C4 (B16F10-scFv6.C4) (46.7%) cells, against CTL from uP-immunized mice (10%). After the last immunization, 5 × 10(5) B16F10-CEA cells were injected into the left flank. All mice immunized with the uP empty vector died within 40 days, but uP/PS-scFv6.C4 vaccinated mice (40%) remained free of tumor for more than 100 days. Splenocytes obtained from uP/PS-scFv6.C4 vaccinated mice showed higher T-cell proliferative activity than those from uP vaccinated mice. Collectively, DNA vaccination with the uP-PS/scFv6.C4 plasmid vector was able to give rise to specific humoral and cellular responses, which were sufficient to retard growth and/or eliminate the injected B16F10-CEA cells.

  15. 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). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Genetic modulation of the FV(Leiden)/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes.

    PubMed

    Segers, O; Simioni, P; Tormene, D; Bulato, C; Gavasso, S; Rosing, J; Castoldi, Elisabetta

    2012-01-01

    The factor (F)V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC-mediated inactivation and by impairing the APC-cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase-based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single-nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. In FV Leiden heterozygotes,VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G(K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV(Leiden)/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo-homozygotes, i.e. they carried a deleterious mutation on the non-Leiden allele. In FV Leiden heterozygotes, the prothrombinase-based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV(Leiden)/normal FV ratio in plasma.

  17. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    PubMed

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  18. 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

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

    PubMed

    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.

  20. 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.

  1. A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

    PubMed Central

    Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

    2017-01-01

    Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

  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 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

  3. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies.

    PubMed

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar

    2007-02-01

    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  4. 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.

  5. Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus.

    PubMed

    Zakri, Adel M; Ziegler, Angelika; Torrance, Lesley; Fischer, Rainer; Commandeur, Ulrich

    2010-03-01

    We report the establishment of a hybridoma cell line secreting the monoclonal antibody (mAb) HAV, which recognizes the coat (AV1) protein of tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus. The cell line was obtained following immunization of mice with purified recombinant AV1 fused to glutathione S-transferase (GST). A single-chain variable fragment (scFv-SAV) was assembled from hybridoma cDNA, but sequence analysis revealed a single nucleotide deletion causing a frame shift that resulted in a 21-residue N-terminal truncation. The missing nucleotide was restored by in vitro site-directed mutagenesis to create scFv-RWAV. The binding properties of mAb HAV and the corresponding scFvs were characterized by western blot, ELISA and surface plasmon resonance spectroscopy. MAb HAV bound to AV1 with nanomolar affinity but reacted neither with the N-terminal region of the protein nor with the GST fusion partner. This suggested that the antibody recognized a linear epitope in a region of the coat protein that is conserved among begomoviruses. Both scFvs retained the antigen specificity of mAb HAV, although the dissociation rate constant of scFv-RWAV was tenfold greater than that of scFv-SAV, showing the importance of restoring the 21 N-terminal amino acids.

  6. A Poly(Propyleneimine) Dendrimer-Based Polyplex-System for Single-Chain Antibody-Mediated Targeted Delivery and Cellular Uptake of SiRNA.

    PubMed

    Tietze, Stefanie; Schau, Isabell; Michen, Susanne; Ennen, Franka; Janke, Andreas; Schackert, Gabriele; Aigner, Achim; Appelhans, Dietmar; Temme, Achim

    2017-07-01

    Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection-disabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti-EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase-derived biotinylation acceptor (P-BAP) is bioconjugated to mal-PPI through a novel coupling strategy solely based on biotin-neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv-P-BAP, the generated EGFRvIII-specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor-mediated endocytosis. These results suggest that receptor-mediated uptake of otherwise noninternalized mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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.

  8. A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues.

    PubMed

    Yang, Ju-Lun; Liu, Du-Xian; Zhen, Shi-Jian; Zhou, Yun-Gang; Zhang, Dai-Jun; Yang, Li-Ying; Chen, Hao-Bing; Feng, Qiang

    2016-02-20

    The ras genes play an important role in the development and progression of human tumours. Neutralizing Ras proteins in the cytoplasm could be an effective approach to blocking ras signalling. In this study, we prepared anti-p21Ras single chain fragment variable antibody (scFv) and investigated its immunoreactivity with human tumours. The coding sequences of H-ras, K-ras, and N-ras were separately ligated into the vector pET-28a(+). Then, recombinant expressing plasmids were induced by IPTG for p21Ras expression in E. coli. Hybridoma cell lines producing anti-p21Ras monoclonal antibodies were isolated using wildtype p21Ras proteins as immunogens. Anti-p21Ras scFv antibody was prepared from the hybridoma by the phage scFv display method. The immunoreactivity of the anti-p21Ras monoclonal antibody and the scFv antibody was identified by ELISA and immunocytochemistry. We prokaryotically expressed wildtype H-p21Ras, K-p21Ras and N-p21Ras and generated the hybridoma cell line KGH-R1, producing anti-p21Ras monoclonal antibodies. It was demonstrated that KGH-R1 monoclonal antibody could recognize wildtype and mutated H-p21Ras, K-p21Ras and N-p21Ras in human tumour cell lines. In all 14 types of primary human cancer tissues tested, the monoclonal antibody presented strong immunoreactivity but showed weak or negative immunoreactivity in the corresponding normal tissues. Subsequently, we prepared anti-p21Ras scFv from hybridoma KGH-R1, which showed the same immunoreactivity as the original monoclonal antibody. Sequence analysis demonstrated that the nucleotides and amino acids of the scFv exhibited an approximately 50 % difference from the anti-p21Ras scFv reported previously. This study presents a novel anti-p21Ras scFv antibody. Our data suggest that the scFv may be useful for ras signalling blockage and may be a potential therapeutic antibody for ras-derived tumours.

  9. Multivalent Glycosylated Nanostructures for Ebola Virus Infection.

    PubMed

    Illescas, Beatriz M; Rojo, Javier; Delgado, Rafael; Martín, Nazario

    2017-04-10

    The infection of humans by lethal pathogens such as Ebola and other related viruses has not been properly addressed so far. In this context, a relevant question arises: what can chemistry do in the search for new strategies and approaches to solve this emergent problem? Although initially a variety of known chemical compounds - for other purposes - have been disappointingly tested against Ebola virus infection, more recently, specific molecules have been prepared. In this Perspective, we present a new approach directed to the design of efficient entry inhibitors to minimize the development of resistance by viral mutations. In particular, we focused on dendrimers as well as fullerene C60 - with a unique symmetrical and 3D globular structure - as biocompatible carbon platforms for the multivalent presentation of carbohydrates. The antiviral activity of these compounds in an Ebola pseudotyped infection model were in the low micromolar range for fullerenes with 12 and 36 mannoses. However, new tridecafullerenes - in which the central alkyne scaffold of [60]fullerene has been connected to 12 sugar-containing [60]fullerene units (total 120 mannoses)- exhibit an outstanding antiviral activity with IC50 in the subnanomolar range! The multivalent presentation of specific carbohydrates by using 3D fullerenes as controlled biocompatible carbon scaffolds represents a real advance being currently the most efficient molecules in vitro against Ebola virus infection. However, additional studies are needed to determine the optimized fullerene-based leads for practical applications.

  10. Multivalent and Multipathogen Viral Vector Vaccines

    PubMed Central

    Borrow, Ray; Blanchard, Thomas J.

    2016-01-01

    ABSTRACT The presentation and delivery of antigens are crucial for inducing immunity and, desirably, lifelong protection. Recombinant viral vectors—proven safe and successful in veterinary vaccine applications—are ideal shuttles to deliver foreign proteins to induce an immune response with protective antibody levels by mimicking natural infection. Some examples of viral vectors are adenoviruses, measles virus, or poxviruses. The required attributes to qualify as a vaccine vector are as follows: stable insertion of coding sequences into the genome, induction of a protective immune response, a proven safety record, and the potential for large-scale production. The need to develop new vaccines for infectious diseases, increase vaccine accessibility, reduce health costs, and simplify overloaded immunization schedules has driven the idea to combine antigens from the same or various pathogens. To protect effectively, some vaccines require multiple antigens of one pathogen or different pathogen serotypes/serogroups in combination (multivalent or polyvalent vaccines). Future multivalent vaccine candidates are likely to be required for complex diseases like malaria and HIV. Other novel strategies propose an antigen combination of different pathogens to protect against several diseases at once (multidisease or multipathogen vaccines). PMID:27535837

  11. Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

    DTIC Science & Technology

    2011-01-01

    Appli- cations of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnology Adv 27, 502–520. 6. Denzin , L. K...4-20. J Biol Chem 266, 14095–14103. Transfected Cell Microarrays 137 7. Denzin , L. K., Gulliver, G. A., Voss, E. W., Jr. (1993) Mutational analysis of

  12. Modelling of reversible single chain polymer self-assembly: from the polymer towards the protein limit.

    PubMed

    Danilov, Denis; Barner-Kowollik, Christopher; Wenzel, Wolfgang

    2015-04-07

    The thermodynamic properties of reversible single chain polymer self-assembly are characterized by all-atom simulations. The ensemble of closed chains collapses from multiple conformations for long chains to nearly unique conformations for shorter chains, suggesting that the engineered polymers can fold into stable unique conformations at moderate temperatures.

  13. Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains.

    PubMed

    Lakowitz, Antonia; Krull, Rainer; Biedendieck, Rebekka

    2017-01-23

    Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L(-1)) and highest space-time-yield (8 mg L(-1) h(-1)) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody

  14. Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

    PubMed Central

    Khan, Lubina; Kumar, Rajesh; Thiruvengadam, Ramachandran; Parray, Hilal Ahmad; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Aggarwal, Heena; Mohata, Madhav; Hussain, Abdul Wahid; Das, Raksha; Varadarajan, Raghavan; Bhattacharya, Jayanta; Vajpayee, Madhu; Murugavel, K. G.; Solomon, Suniti; Sinha, Subrata; Luthra, Kalpana

    2017-01-01

    More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design. PMID:28332627

  15. 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

  16. Comparing solvophobic and multivalent induced collapse in polyelectrolyte brushes

    DOE PAGES

    Jackson, Nicholas E.; Brettmann, Blair K.; Vishwanath, Venkatram; ...

    2017-02-03

    Here, coarse-grained molecular dynamics enhanced by free-energy sampling methods is used to examine the roles of solvophobicity and multivalent salts on polyelectrolyte brush collapse. Specifically, we demonstrate that while ostensibly similar, solvophobic collapsed brushes and multivalent-ion collapsed brushes exhibit distinct mechanistic and structural features. Notably, multivalent-induced heterogeneous brush collapse is observed under good solvent polymer backbone conditions, demonstrating that the mechanism of multivalent collapse is not contingent upon a solvophobic backbone. Umbrella sampling of the potential of mean-force (PMF) between two individual brush strands confirms this analysis, revealing starkly different PMFs under solvophobic and multivalent conditions, suggesting the role ofmore » multivalent “bridging” as the discriminating feature in trivalent collapse. Structurally, multivalent ions show a propensity for nucleating order within collapsed brushes, whereas poor-solvent collapsed brushes are more disordered; this difference is traced to the existence of a metastable PMF minimum for poor solvent conditions, and a global PMF minimum for trivalent systems, under experimentally relevant conditions.« less

  17. 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.

  18. 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

  19. Using multivalent adenoviral vectors for HIV vaccination.

    PubMed

    Gu, Linlin; Li, Zan C; Krendelchtchikov, Alexandre; Krendelchtchikova, Valentina; Wu, Hongju; Matthews, Qiana L

    2013-01-01

    Adenoviral vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. For effective vaccine development it is often necessary to express or present multiple antigens to the immune system to elicit an optimal vaccine as observed preclinically with mosaic/polyvalent HIV vaccines or malaria vaccines. Due to the wide flexibility of Ad vectors they are an ideal platform for expressing large amounts of antigen and/or polyvalent mosaic antigens. Ad vectors that display antigens on their capsid surface can elicit a robust humoral immune response, the "antigen capsid-incorporation" strategy. The adenoviral hexon protein has been utilized to display peptides in the majority of vaccine strategies involving capsid incorporation. Based on our abilities to manipulate hexon HVR2 and HVR5, we sought to manipulate HVR1 in the context of HIV antigen display for the first time ever. More importantly, peptide incorporation within HVR1 was utilized in combination with other HVRs, thus creating multivalent vectors. To date this is the first report where dual antigens are displayed within one Ad hexon particle. These vectors utilize HVR1 as an incorporation site for a seven amino acid region of the HIV glycoprotein 41, in combination with six Histidine incorporation within HVR2 or HVR5. Our study illustrates that these multivalent antigen vectors are viable and can present HIV antigen as well as His6 within one Ad virion particle. Furthermore, mouse immunizations with these vectors demonstrate that these vectors can elicit a HIV and His6 epitope-specific humoral immune response.

  20. 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.

  1. Single chain folding of synthetic polymers by covalent and non-covalent interactions: current status and future perspectives.

    PubMed

    Altintas, Ozcan; Barner-Kowollik, Christopher

    2012-06-14

    The present feature article highlights the preparation of polymeric nanoparticles and initial attempts towards mimicking the structure of natural biomacromolecules by single chain folding of well-defined linear polymers through covalent and non-covalent interactions. Initially, the discussion focuses on the synthesis and characterization of single chain self-folded structures by non-covalent interactions. The second part of the article summarizes the folding of single chain polymers by means of covalent interactions into nanoparticle systems. The current state of the art in the field of single chain folding indicates that covalent-bond-driven nanoparticle preparation is well advanced, while the first encouraging steps towards building reversible single chain folding systems by the use of mutually orthogonal hydrogen-bonding motifs have been made. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Coacervation with surfactants: From single-chain surfactants to gemini surfactants.

    PubMed

    Zhao, Weiwei; Wang, Yilin

    2017-01-01

    Coacervation is a spontaneous process during which a colloidal dispersion separates into two immiscible liquid phases: a colloid-rich liquid phase in equilibrium with a diluted phase. Coacervation is usually divided into simple coacervation and complex coacervation according to the number of components. Surfactant-based coacervation normally contains traditional single-chain surfactants. With the development of surfactants, gemini surfactants with two amphiphilic moieties have been applied to form coacervation. This review summarizes the development of simple coacervation and complex coacervation in the systems of single-chain surfactants and gemini surfactants. Simple coacervation in surfactant solutions with additives or at elevated temperature and complex coacervation in surfactant/polymer mixtures by changing charge densities, molecular weight, ionic strength, pH, or temperature are reviewed. The comparison between gemini surfactants and corresponding monomeric single-chain surfactants reveals that the unique structures of gemini surfactants endow them with higher propensity to generate coacervation. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.

    PubMed

    Li, J; Cook, R; Dede, K; Chaiken, I

    1996-01-26

    Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

  4. Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer.

    PubMed

    Chen, Hongwei; Wang, Liya; Yu, Qiqi; Qian, Weiping; Tiwari, Diana; Yi, Hong; Wang, Andrew Y; Huang, Jing; Yang, Lily; Mao, Hui

    2013-01-01

    Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs.

  5. Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

    PubMed Central

    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

  6. Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine.

    PubMed

    Moghaddam, Amir; Borgen, Tine; Stacy, John; Kausmally, Louise; Simonsen, Bjørg; Marvik, Ole J; Brekke, Ole Henrik; Braunagel, Michael

    2003-09-01

    Use of phage display of recombinant antibodies and large repertoire naïve antibody libraries for identifying antibodies of high specificity has been extensively reported. Nevertheless, there have been few reported antibodies to haptens that have originated from naïve antibody libraries with potential use in diagnostics. We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naïve antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM). The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display). The antibodies recognise 6MAM with affinities of 1-3x10(-7) M with no crossreactivity to morphine. These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs. To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine.

  7. Immunodetection of the "brown" spider (Loxosceles intermedia) dermonecrotoxin with an scFv-alkaline phosphatase fusion protein.

    PubMed

    Jiacomini, Isabella; Silva, Sabrina K; Aubrey, Nicolas; Muzard, Julien; Chavez-Olortegui, Carlos; De Moura, Juliana; Billiald, Philippe; Alvarenga, Larissa M

    2016-05-01

    Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  8. Targeted Delivery of Cisplatin to Lung Cancer Using ScFvEGFR-Heparin-Cisplatin Nanoparticles

    PubMed Central

    Peng, Xiang-Hong; Wang, Yiqing; Huang, Donghai; Wang, Yuxiang; Shin, Hyung Juc.; Chen, Zhengjia; Spewak, Micheal B; Mao, Hui; Wang, Xu; Wang, Ying; Chen, Zhuo (Georgia); Nie, Shuming; Shin, Dong M.

    2011-01-01

    The clinical application of cis-diamminedichloroplatinum(II) (DDP, cisplatin) for cancer therapy is limited by its non-specific biodistribution and severe side effects. Here, we have developed EGFR-targeted heparin-DDP (EHDDP) nanoparticles for tumor targeted delivery of DDP. This nanoparticle delivery system possesses the following unique properties: i) the succinic anhydride-modified heparin is biocompatible and biodegradable with no anticoagulant activity; ii) the single chain variable fragment anti-EGFR antibody (ScFvEGFR) was conjugated to the nanoparticles as an EGFR-targeting ligand. Our results showed that EHDDP nanoparticles can significantly increase the intracellular concentrations of DDP and Pt-DNA adducts in EGFR-expressing non-small cell lung cancer H292 cells via an EGFR-mediated pathway. Compared to the free DDP, significantly prolonged blood circulation time and improved pharmacokinetics and biodistribution of Pt were observed after systemic delivery of the EHDDP nanoparticles. The new EHDDP nanoparticle delivery system significantly enhanced antitumor activity of DDP without weight loss or damage to the kidney and spleen in nude mice bearing H292 cell tumors. PMID:22032622

  9. 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

  10. Understanding the contribution of disulphide bridges to the folding and misfolding of an anti-Aβ scFv.

    PubMed

    Montoliu-Gaya, Laia; Martínez, Jose C; Villegas, Sandra

    2017-03-24

    ScFv-h3D6 is a single chain variable fragment that precludes Aβ peptide-induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway to the worm-like pathway. Production of scFv molecules is not a straightforward procedure because of the occurrence of disulphide scrambled conformations generated in the refolding process. Here, we separately removed the disulphide bond of each domain and solved the scrambling problem; and then, we intended to compensate the loss of thermodynamic stability by adding three C-terminal elongation mutations previously described to stabilize the native fold of scFv-h3D6. Such stabilization occurred through stabilization of the intermediate state in the folding pathway and destabilization of a different, β-rich, intermediate state driving to worm-like fibrils. Elimination of the disulphide bridge of the less stable domain, VL , deeply compromised the yield and increased the aggregation tendency, but elimination of the disulphide bridge of the more stable domain, VH , solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm-like morphology to an amyloid one. This was so because a partially unfolded intermediate driving to amyloid aggregation was present, instead of the β-rich intermediate driving to worm-like fibrils. When combining with the elongation mutants, stabilization of the partially unfolded intermediate driving to amyloid fibrils was the only effect observed. Therefore, the same mutations drove to completely different scenarios depending on the presence of disulphide bridges and this illustrates the relevance of such linkages in the stability of different intermediate states for folding and misfolding. This article is protected by copyright. All rights reserved.

  11. Synthetic Multivalent Antifungal Peptides Effective against Fungi

    PubMed Central

    Li, Jianguo; Nandhakumar, Muruganantham; Aung, Thet Tun; Goh, Eunice; Chang, Jamie Ya Ting; Saraswathi, Padhmanaban; Tang, Charles; Safie, Siti Radiah Binte; Lin, Lim Yih; Riezman, Howard; Lei, Zhou; Verma, Chandra S.; Beuerman, Roger W.

    2014-01-01

    Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides. PMID:24498363

  12. Multivalent Induction of Biodegradative Threonine Deaminase

    PubMed Central

    Yui, Yoshiki; Watanabe, Yasuyoshi; Ito, Seiji; Shizuta, Yutaka; Hayaishi, Osamu

    1977-01-01

    To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term “multivalent induction” has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids. PMID:334736

  13. 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).

  14. Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity

    PubMed Central

    Tsutsumi, Yasuo; Onda, Masanori; Nagata, Satoshi; Lee, Byungkook; Kreitman, Robert J.; Pastan, Ira

    2000-01-01

    Chemical modification of proteins with polyethylene glycol (PEGylation) can increase plasma half-lives, stability, and therapeutic potency. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of anti-Tac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the IL-2 receptor α subunit fused to a 38-kDa fragment of Pseudomonas exotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then site-specifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activities in vitro but superior stability at 37°C in mouse serum, a 5- to 8-fold increase in plasma half-lives in mice, and a 3- to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans. PMID:10890891

  15. 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.

  16. 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.

  17. Nano-magnetic materials: spin crossover compounds vs. single molecule magnets vs. single chain magnets.

    PubMed

    Brooker, Sally; Kitchen, Jonathan A

    2009-09-28

    Brief introductions to spin crossover (SCO), single molecule magnetism (SMM) and single chain magnetism (SCM) are provided. Each section is illustrated by selected examples that have contributed significantly to the development of these fields, including recent efforts to produce materials (films, attachment to surfaces etc.). The advantages and disadvantages of each class of magnetically interesting compound are considered, along with the key challenges that remain to be overcome before such compounds can be used commercially as nanocomponents. This invited perspective article is intended to be easily comprehensible to non-specialists in the field.

  18. Multivalent interaction based carbohydrate biosensors for signal amplification

    PubMed Central

    Wang, Yanyan; Chalagalla, Srinivas; Li, Tiehai; Sun, Xue-long; Zhao, Wei; Wang, Peng; Zeng, Xiangqun

    2010-01-01

    Multivalent interaction between boronic acids immobilized on Quartz Crystal Microbalance (QCM) sensor surface and the carbohydrates modified Au - nanoparticle (AuNP) has been demonstrated for the development of a sensitive carbohydrate biosensor. Briefly, a boronic acid - containing polymer (boropolymer) as multivalent carbohydrate receptor was oriented immobilized on the cysteamine coated electrode through isourea bond formation. Carbohydrates were conjugated to AuNPs to generate a multivalent carbohydrates moiety to amplify the response signal. Thus, the binding of the carbohydrate conjugated AuNPs to the boropolymer surface are multivalent which could simultaneously increase the binding affinity and specificity. We systematically studied the binding between five carbohydrate conjugated AuNPs and the boropolymer. Our studies show that the associate constant (Ka) was in the order of fucose < glucose < mannose < galactose < maltose. A linear response in the range from 23 µM to 3.83 mM was observed for mannose conjugated AuNPs and the boropolymer recognition elements, with the lower detection limit of 1.5 µM for the carbohydrate analytes. Furthermore, the multivalent binding between carbohydrates and boronic acids are reversible and allow the regeneration of boropolymer surface by using 1M acetic acid so as to sequentially capture and release the carbohydrate analytes. PMID:20863680

  19. Multivalent nanoparticles bind the retinal and choroidal vasculature.

    PubMed

    Hennig, Robert; Ohlmann, Andreas; Staffel, Janina; Pollinger, Klaus; Haunberger, Alexandra; Breunig, Miriam; Schweda, Frank; Tamm, Ernst R; Goepferich, Achim

    2015-12-28

    The angiotensin II receptor type 1 (AT1R), which is expressed in blood vessels of the posterior eye, is of paramount significance in the pathogenesis of severe ocular diseases such as diabetic retinopathy and age-related macular degeneration. However, small molecule angiotensin receptor blockers (ARBs) have not proven to be a significant therapeutic success. We report here on a nanoparticle system consisting of ARB molecules presented in a multivalent fashion on the surface of quantum dots (Qdots). As a result of the multivalent receptor binding, nanoparticles targeted cells with high AT1R expression and inhibited their angiotensin receptor signaling with an IC50 of 3.8 nM while showing only minor association to cells with low AT1R expression. After intravenous injection into the tail vein of mice, multivalent nanoparticles accumulated in retinal and choroidal blood vessels of the posterior eye. At the same time, multivalent ligand display doubled the Qdot concentration in the blood vessels compared to non-targeted Qdots. Remarkably, ARB-targeted Qdots showed no pronounced accumulation in AT1R-expressing off-target tissues such as the kidney. Following systemic application, this multivalent targeting approach has the potential to amplify AT1R blockade in the eye and concomitantly deliver a therapeutic payload into ocular lesions.

  20. 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.

  1. Localization of single-chain interruptions in bacteriophage T5 DNA I. Electron microscopic studies.

    PubMed Central

    Scheible, P P; Rhoades, E A; Rhoades, M

    1977-01-01

    Bacteriophage T5 DNA was examined in an electron microscope after limited digestion with exonuclease III from Escherichia coli. The effect of the exonuclease treatment was to convert each naturally occurring single-chain interruption in T5 DNA into a short segment of single-stranded DNA. The locations of these segments were determined for T5st(+) DNA, T5st(0) DNA, and fragments of T5st(0) DNA generated by EcoRI restriction endonuclease. The results indicate that single-chain interruptions occurr in a variable, but nonrandom, manner in T5 DNA. T5st(+) DNA has four principal interruptions located at sites approximately 7.9, 18.5, 32.6, and 64.8% from one end of the molecule. Interruptions occur at these sites in 80 to 90% of the population. A large number of additional sites, located primarily at the ends of the DNA, contain interruptions at lower frequencies. The average number of interruptions per genome, as determined by this method, is 8. A similar distribution of breaks occurs in T5st(0) DNA, except that the 32.6% site is missing. At least one of the principal interruptions is reproducibly located within an interval of 0.2% of the entire DNA. Images PMID:330881

  2. humMR1, a highly specific humanized single chain antibody for targeting EGFRvIII.

    PubMed

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

    2014-02-01

    Production of an efficient humanized single chain antibody is reported here to specifically target EGFRvIII, a truncated receptor expressed in a wide variety of human cancers. CDR loops of MR1, a phage display-derived murine single chain antibody developed against this mutant receptor, were grafted on human frameworks that had been selected based on similarity to MR1 in terms of two distinct parameters, variable domain protein sequence and CDR canonical structures. Moreover, two point mutations were introduced in CDR-H2 and CDR-H3 loops of the humanized antibody to destroy its cross-reactivity to wild-type EGFR. The resultant antibody, referred to as humMR1, was found by MTT assay, ELISA and western blot techniques to be highly specific for EGFRvIII. The affinity of this antibody for EGFRvIII-specific 14-amino acid synthetic peptide and HC2 cells were measured to be 1.87 × 10(10) and 2.17 × 10(10)/M respectively. This humanized antibody leads to 78.5% inhibition in proliferation of EGFRvIII-overexpressing cells.

  3. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease.

    PubMed

    Grizot, Sylvestre; Smith, Julianne; Daboussi, Fayza; Prieto, Jesús; Redondo, Pilar; Merino, Nekane; Villate, Maider; Thomas, Séverine; Lemaire, Laetitia; Montoya, Guillermo; Blanco, Francisco J; Pâques, Frédéric; Duchateau, Philippe

    2009-09-01

    Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches.

  4. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease

    PubMed Central

    Grizot, Sylvestre; Smith, Julianne; Daboussi, Fayza; Prieto, Jesús; Redondo, Pilar; Merino, Nekane; Villate, Maider; Thomas, Séverine; Lemaire, Laetitia; Montoya, Guillermo; Blanco, Francisco J.; Pâques, Frédéric; Duchateau, Philippe

    2009-01-01

    Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches. PMID:19584299

  5. Characterization of a single-chain T-cell receptor expressed in Escherichia coli.

    PubMed

    Hoo, W F; Lacy, M J; Denzin, L K; Voss, E W; Hardman, K D; Kranz, D M

    1992-05-15

    Despite progress in defining the nature of major histocompatibility complex products that are recognized by the T-cell antigen receptor, the binding properties and structure of the receptor have not been solved. The primary problem has been the difficulty in obtaining sufficient quantities of active receptor. In this report we show that a single-chain T-cell receptor gene can be expressed in Escherichia coli. The protein consists of the variable (V) regions of the alpha and beta chains (V alpha and V beta) encoded by the cytotoxic T-lymphocyte clone 2C (a H-2b anti-H-2d alloreactive cell line) linked by a 25-amino acid flexible peptide. Solubilized extracts that contain the 27-kDa V alpha 3V beta 8 protein are positive in solid-phase immunoassays with the anti-V beta 8 antibody KJ16 and the anti-clonotypic antibody 1B2. Approximately 1% of the protein can be specifically purified on a 1B2-conjugated column. These results indicate that a fraction of the protein is able to fold into a native conformation and that single-chain proteins should be useful not only as immunogens for eliciting anti-T-cell receptor antibodies but in the study of T-cell receptor structure and function.

  6. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

    PubMed

    Bedzyk, W D; Weidner, K M; Denzin, L K; Johnson, L S; Hardman, K D; Pantoliano, M W; Asel, E D; Voss, E W

    1990-10-25

    Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

  7. 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. © 2014 Wiley Periodicals, Inc.

  8. Expression of bioactive single-chain murine IL-12 in transgenic plants.

    PubMed

    Liu, Jianyun; Dolan, Maureen C; Reidy, Michael; Cramer, Carole L

    2008-06-01

    Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic. However, its clinical application is limited in part by lack of an effective bioproduction system for this complex heterodimeric glycoprotein. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. To test the potential of plants to effectively produce bioactive IL-12, we developed transgenic tobacco plant lines and derived root cultures yielding high levels of mouse IL-12 (MuIL-12). Functional IL-12 is a heterodimer consisting of two disulfide-linked subunits, p35 and p40. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of MuIL-12. Plant-derived single-chain MuIL-12 was characterized and purified for in vitro bioactivity assays. Our results demonstrated precise cleavage of the endogenous mouse p40 signal peptide in plants as well as addition of N-linked glycans. Plant-derived MuIL-12 triggered induction of interferon-gamma (IFN-gamma) secretion from mouse splenocytes and stimulated splenocyte proliferation with comparable activities to those observed for commercially available animal cell-derived MuIL-12. These studies indicate that plants produce fully functional MuIL-12 at levels compatible with commercial production and may serve as an effective bioproduction platform for bioactive IL-12s from other species for human or veterinary vaccine and therapeutic applications.

  9. Expression of a functional single-chain antibody via Corynebacterium pseudodiphtheriticum.

    PubMed

    Sundaram, R K; Hurwitz, I; Matthews, S; Hoy, E; Kurapati, S; Crawford, C; Sundaram, P; Durvasula, R V

    2008-07-01

    Antibody-based therapeutics are effective against conditions ranging from acute infections to malignancy. They may prove crucial in combating bioterrorism and responding to drug-resistant and emerging pathogens. At present the cost of producing therapeutic monoclonal antibodies is between $1,000 to $6,000 per gram. The need to administer antibodies parenterally at frequent intervals further drives the cost of this treatment. Here we present an antibody delivery system, termed paratransgenesis, with the potential to overcome these limitations. The paratransgenic approach involves genetically transforming a commensal or symbiont bacterium to express foreign molecules that target pathogens. We describe transformation of Corynebacterium pseudodiptheriticum, a commensal bacterium found in the human respiratory tract, to express a murine single-chain antibody binding progesterone. The antibody was functional and bound specifically to progesterone in a concentration-dependent manner. This marker antibody system is the precursor to development of expression systems producing recombinant humanized single-chain antibodies. Studies are in progress evaluating fitness, transgene stablility, and pathogenecity of the genetically engineered C. pseudodiptheriticum. We anticipate developing a repertoire of expressed molecules targeting infectious agents and surface epitopes of pulmonary mass lesions. If expression systems for anti-pathogen molecules in C. pseudodiptheriticum and other respiratory commensal bacteria can be optimized, these bacteria have the potential for a range of therapeutic and prophylactic applications.

  10. A Brief Review on Multivalent Intercalation Batteries with Aqueous Electrolytes

    PubMed Central

    Guduru, Ramesh K.; Icaza, Juan C.

    2016-01-01

    Rapidly growing global demand for high energy density rechargeable batteries has driven the research toward developing new chemistries and battery systems beyond Li-ion batteries. Due to the advantages of delivering more than one electron and giving more charge capacity, the multivalent systems have gained considerable attention. At the same time, affordability, ease of fabrication and safety aspects have also directed researchers to focus on aqueous electrolyte based multivalent intercalation batteries. There have been a decent number of publications disclosing capabilities and challenges of several multivalent battery systems in aqueous electrolytes, and while considering an increasing interest in this area, here, we present a brief overview of their recent progress, including electrode chemistries, functionalities and challenges. PMID:28344298

  11. A Brief Review on Multivalent Intercalation Batteries with Aqueous Electrolytes.

    PubMed

    Guduru, Ramesh K; Icaza, Juan C

    2016-02-26

    Rapidly growing global demand for high energy density rechargeable batteries has driven the research toward developing new chemistries and battery systems beyond Li-ion batteries. Due to the advantages of delivering more than one electron and giving more charge capacity, the multivalent systems have gained considerable attention. At the same time, affordability, ease of fabrication and safety aspects have also directed researchers to focus on aqueous electrolyte based multivalent intercalation batteries. There have been a decent number of publications disclosing capabilities and challenges of several multivalent battery systems in aqueous electrolytes, and while considering an increasing interest in this area, here, we present a brief overview of their recent progress, including electrode chemistries, functionalities and challenges.

  12. Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

    PubMed

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

    2013-11-19

    A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) μg/mL, 1000-fold more sensitive than that reported previously (1 μg/mL). The fusion protein was able to detect fungal concentrations below 1 μg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 μg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

  13. Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.

    PubMed

    Wang, Welson Wen-Shang; Das, Dipankar; McQuarrie, Stephen A; Suresh, Mavanur R

    2007-03-01

    We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread.

  14. Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

    PubMed

    Denzin, L K; Voss, E W

    1992-05-05

    In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active

  15. 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

  16. 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

  17. Tailoring single chain polymer nanoparticle thermo-mechanical behavior by cross-link density.

    PubMed

    Bae, Suwon; Galant, Or; Diesendruck, Charles E; Silberstein, Meredith N

    2017-04-12

    Single chain polymer nanoparticles (SCPNs) are formed from intrachain cross-linking of a single polymer chain, making SCPN distinct from other polymer nanoparticles for which the shape is predefined before polymerization. The degree of cross-linking in large part determines the internal architecture of the SCPNs and therefore their mechanical and thermomechanical properties. Here, we use molecular dynamics (MD) simulations to study thermomechanical behavior of individual SCPNs with different underlying structures by varying the ratio of cross-linking and the degree of polymerization. We characterize the particles in terms of shape, structure, glass transition temperature, mobility, and stress response to compressive loading. The results indicate that the constituent monomers of SCPNs become less mobile as the degree of cross-linking is increased corresponding to lower diffusivity and higher stress at a given temperature.

  18. 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.

  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.

  20. Comparative reduction/oxidation studies with single chain des-(B30) insulin and porcine proinsulin.

    PubMed

    Markussen, J

    1985-04-01

    The single chain des-(B30) insulin molecule (SCI) has been reduced and reoxidized together with porcine proinsulin (PPI). Yields of correctly folded and reoxidized SCI and PPI were analyzed by HPLC. The concentrations of both proteins were 10(-3) M during reduction and 10(-5) M during oxidation. The pH during reoxidation was varied from 8.6 to 9.2 and the temperature from 4 to 37 degrees. Under all conditions tested, the recovery of SCI was substantially higher than that of PPI. The recoveries peaked after 24-72 h. It is suggested that the "miniproinsulin" SCI folds correctly up more efficiently than porcine proinsulin, resulting in higher yields of reoxidized SCI.

  1. Single-ion and single-chain magnetism in triangular spin-chain oxides

    NASA Astrophysics Data System (ADS)

    Seikh, Md. Motin; Caignaert, Vincent; Perez, Olivier; Raveau, Bernard; Hardy, Vincent

    2017-05-01

    S r4 -xC axM n2Co O9 oxides (x =0 and x =2 ) are found to exhibit magnetic responses typical of single-chain magnets (SCMs) and single-ion magnets (SIMs), two features generally investigated in coordination polymers or complexes. The compound x =0 appears to be a genuine SCM, in that blocking effects associated with slow spin dynamics yield remanence and coercivity in the absence of long-range ordering (LRO). In addition, SIM signatures of nearly identical nature are detected in both compounds, coexisting with SCM in x =0 and with LRO in x =2 . It is also observed that a SCM response can be recovered in x =2 after application of magnetic field. These results suggest that purely inorganic systems could play a valuable role in the topical issue of the interplay among SIM, SCM, and LRO phenomena in low-dimensional magnetism.

  2. Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation.

    PubMed

    Ivanov, Ivan; Matafonov, Anton; Sun, Mao-Fu; Cheng, Qiufang; Dickeson, S Kent; Verhamme, Ingrid M; Emsley, Jonas; Gailani, David

    2017-03-16

    When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

  3. Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications.

    PubMed

    Hua, Qing-xin; Nakagawa, Satoe H; Jia, Wenhua; Huang, Kun; Phillips, Nelson B; Hu, Shi-quan; Weiss, Michael A

    2008-05-23

    Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 +/- 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (DeltaDeltaG(u) = 0.7 +/- 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 +/- 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val(A3) at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 alpha-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.

  4. Novel designs of single-chain MHC I/peptide complex for the magnetosome display system.

    PubMed

    Honda, Toru; Maeda, Yoshiaki; Yasuda, Takayuki; Tanaka, Tsuyoshi; Matsunaga, Tadashi; Yoshino, Tomoko

    2015-02-01

    The magnetic nanoparticles displaying the class I major histocompatibility complex (MHC I) were biologically synthesized using the magnetotactic bacterium Magnetospirillum magneticum AMB-1. Expression level and antigen peptide (HER263-71)-binding capability of the MHC I were evaluated on bacterial magnetic particles (BacMPs, also known as magnetosomes). Furthermore, the single-chain complexes of MHC I and HER263-71 were de novo designed for the magnetosome display system in order to improve the interaction between MHC I and HER263-71. Two types of the fusion arrangements were tested, and one of the complexes was estimated to fold into the correct conformation at the level of over 70%. In addition to the high folding ratio, an advantage of this system is that any refolding processes were not required even though the N-terminus of HER263-71 peptide is not free, which conventional bacterial expression systems have never demonstrated. The as-prepared single-chain MHC I/HER263-71 complex-displaying BacMPs (MHC I/HER2-BacMPs) specifically interacted with, and magnetically separated the HER263-71-induced cells, suggesting that the native T-cell receptor could recognize the engineered MHC I/HER2 complex on the BacMPs. By optimizing the magnetic sorting method, the MHC I/HER2-BacMPs developed in this study would be useful in immunotherapeutic applications. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids.

    PubMed

    Pungente, Michael D; Jubeli, Emile; Øpstad, Christer L; Al-Kawaz, Mais; Barakat, Nour; Ibrahim, Tarek; Abdul Khalique, Nada; Raju, Liji; Jones, Rachel; Leopold, Philip L; Sliwka, Hans-Richard; Partali, Vassilia

    2012-03-16

    The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

  6. 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.

  7. 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.

  8. An ScFv Intrabody Against the Non-Amyloid Component of Alpha Synuclein Reduces Intracellular Aggregation and Toxicity

    PubMed Central

    Lynch, Sandra M.; Zhou, Chun; Messer, Anne

    2008-01-01

    Summary Prevention of abnormal misfolding and aggregation of alpha-synuclein (α-syn) protein in vulnerable neurons should be a viable therapeutic strategy for reducing pathogenesis in Parkinson’s disease (PD). The non-amyloid component (NAC) region of α-syn shows strong tendencies to form β-sheet structures, and deletion of this region has been shown to reduce aggregation and toxicity in vitro and in vivo. Binding of a molecular species to this region may mimic effects of such deletions. Single-chain Fv antibodies (scFvs) retain the binding specificity of antibodies, and when genetically manipulated to create high-diversity libraries, allow in vitro selection against peptides. Accordingly, we used a yeast surface display library of an entire naïve repertoire of human scFv antibodies to select for binding to a NAC peptide. Candidate scFvs (after transfer to mammalian expression vectors), were screened for viability in a neuronal cell line by transient co-transfection with A53T mutant α-syn. This provided a ranking of the protective efficacies of the initial panel of intracellular antibodies (intrabodies). High steady-state expression levels and apparent conformational epitope binding appeared more important than in vitro affinity in these assays. None of the scFvs selected matched the sequences of previously-reported anti- α-syn scFvs. A stable cell line expressing the most effective intrabody, NAC32, showed highly significant reductions of abnormal aggregation in two separate models. Recently, intrabodies have shown promising anti-aggregation and neuroprotective effects against misfolded mutant huntingtin protein. The NAC32 study extends such work significantly, utilizing information about the pathogenic capacity of a specific α-syn region to offer a new generation of in vitro-derived antibody fragments, both for further engineering as direct therapeutics and as tools for rational drug design for PD. PMID:18237741

  9. 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

  10. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    PubMed

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

  11. 77 FR 35850 - Safety Zone; F/V Deep Sea, Penn Cove, WA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-15

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone; F/V Deep Sea, Penn Cove, WA AGENCY: Coast... the Fishing Vessel (F/V) Deep Sea, located in Penn Cove, WA. This action is necessary to ensure the... materials associated with the sunken F/V Deep Sea. B. Basis and Purpose On the evening of May 13, 2012,...

  12. 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

    Kaku, Yoshihiro; Noguchi, Akira; Okutani, Akiko; Inoue, Satoshi; Tanabayashi, Kiyoshi; Yamamoto, Yoshie; Hotta, Akitoyo; Suzuki, Michio; Sugiura, Naoko; Yamada, Akio

    2012-09-04

    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). 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. 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 advantageous for the rapid development of

  13. Light quality regulates flowering in FvFT1/FvTFL1 dependent manner in the woodland strawberry Fragaria vesca

    PubMed Central

    Rantanen, Marja; Kurokura, Takeshi; Mouhu, Katriina; Pinho, Paulo; Tetri, Eino; Halonen, Liisa; Palonen, Pauliina; Elomaa, Paula; Hytönen, Timo

    2014-01-01

    Control of flowering in the perennial model, the woodland strawberry (Fragaria vesca L.), involves distinct molecular mechanisms that result in contrasting photoperiodic flowering responses and growth cycles in different accessions. The F. vesca homolog of TERMINAL FLOWER1 (FvTFL1) functions as a key floral repressor that causes short-day (SD) requirement of flowering and seasonal flowering habit in the SD strawberry. In contrast, perpetual flowering F. vesca accessions lacking functional FvTFL1 show FLOWERING LOCUS T (FvFT1)-dependent early flowering specifically under long-days (LD). We show here that the end-of-day far-red (FR) and blue (B) light activate the expression of FvFT1 and the F. vesca homolog of SUPPRESSOR OF THE OVEREXPRESSION OF CONSTANS (FvSOC1) in both SD and LD strawberries, whereas low expression levels are detected in red (R) and SD treatments. By using transgenic lines, we demonstrate that FvFT1 advances flowering under FR and B treatments compared to R and SD treatments in the LD strawberry, and that FvSOC1 is specifically needed for the B light response. In the SD strawberry, flowering responses to these light quality treatments are reversed due to up-regulation of the floral repressor FvTFL1 in parallel with FvFT1 and FvSOC1. Our data highlights the central role of FvFT1 in the light quality dependent flower induction in the LD strawberry and demonstrates that FvTFL1 reverses not only photoperiodic requirements but also light quality effects on flower induction in the SD strawberry. PMID:24966865

  14. Light quality regulates flowering in FvFT1/FvTFL1 dependent manner in the woodland strawberry Fragaria vesca.

    PubMed

    Rantanen, Marja; Kurokura, Takeshi; Mouhu, Katriina; Pinho, Paulo; Tetri, Eino; Halonen, Liisa; Palonen, Pauliina; Elomaa, Paula; Hytönen, Timo

    2014-01-01

    Control of flowering in the perennial model, the woodland strawberry (Fragaria vesca L.), involves distinct molecular mechanisms that result in contrasting photoperiodic flowering responses and growth cycles in different accessions. The F. vesca homolog of TERMINAL FLOWER1 (FvTFL1) functions as a key floral repressor that causes short-day (SD) requirement of flowering and seasonal flowering habit in the SD strawberry. In contrast, perpetual flowering F. vesca accessions lacking functional FvTFL1 show FLOWERING LOCUS T (FvFT1)-dependent early flowering specifically under long-days (LD). We show here that the end-of-day far-red (FR) and blue (B) light activate the expression of FvFT1 and the F. vesca homolog of SUPPRESSOR OF THE OVEREXPRESSION OF CONSTANS (FvSOC1) in both SD and LD strawberries, whereas low expression levels are detected in red (R) and SD treatments. By using transgenic lines, we demonstrate that FvFT1 advances flowering under FR and B treatments compared to R and SD treatments in the LD strawberry, and that FvSOC1 is specifically needed for the B light response. In the SD strawberry, flowering responses to these light quality treatments are reversed due to up-regulation of the floral repressor FvTFL1 in parallel with FvFT1 and FvSOC1. Our data highlights the central role of FvFT1 in the light quality dependent flower induction in the LD strawberry and demonstrates that FvTFL1 reverses not only photoperiodic requirements but also light quality effects on flower induction in the SD strawberry.

  15. Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins

    PubMed Central

    2005-01-01

    scAvd (single-chain avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken avidin. The DNA construct encoding scAvd contains four circularly permuted avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type avidin, which contains four identical avidin monomers, scAvd enables each one of the four avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences. PMID:16092919

  16. Simulation guided design of globular single-chain nanoparticles by tuning the solvent quality.

    PubMed

    Lo Verso, Federica; Pomposo, José A; Colmenero, Juan; Moreno, Angel J

    2015-02-04

    The control of primary and further structures of individual folded/collapsed synthetic polymers has received significant attention in recent years. However, the synthesis of single-chain nanoparticles (SCNPs) showing a compact, globular conformation in solution has turned out so far to be highly elusive. By means of simulations, we propose two methods for obtaining globular SCNPs in solution. The first synthesis route is performed in the bad solvent, with the precursor anchored to a surface. In the second route we use a random copolymer precursor with unreactive solvophilic and reactive solvophobic units, which form a single core-shell structure. Both protocols prevent intermolecular cross-linking. After recovering good solvent conditions, the swollen nanoparticles maintain their globular character. The proposed methods are experimentally realizable and do not require specific sequence control of the precursors. Our results pave the way for the synthesis via solvent-assisted design of a new generation of globular soft nanoparticles mimicking global conformations of native proteins in solution.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  18. 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

  19. Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

    PubMed

    Hojo, Keiko; Hossain, Mohammed Akhter; Tailhades, Julien; Shabanpoor, Fazel; Wong, Lilian L L; Ong-Pålsson, Emma E K; Kastman, Hanna E; Ma, Sherie; Gundlach, Andrew L; Rosengren, K Johan; Wade, John D; Bathgate, Ross A D

    2016-08-25

    Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

  20. 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.

  1. Rational Design of Single-Chain Polymeric Nanoparticles That Kill Planktonic and Biofilm Bacteria.

    PubMed

    Nguyen, Thuy-Khanh; Lam, Shu Jie; Ho, Kitty K K; Kumar, Naresh; Qiao, Greg G; Egan, Suhelen; Boyer, Cyrille; Wong, Edgar H H

    2017-02-08

    Infections caused by multidrug-resistant bacteria are on the rise and, therefore, new antimicrobial agents are required to prevent the onset of a postantibiotic era. In this study, we develop new antimicrobial compounds in the form of single-chain polymeric nanoparticles (SCPNs) that exhibit excellent antimicrobial activity against Gram-negative bacteria (e.g., Pseudomonas aeruginosa) at micromolar concentrations (e.g., 1.4 μM) and remarkably kill ≥99.99% of both planktonic cells and biofilm within an hour. Linear random copolymers, which comprise oligoethylene glycol (OEG), hydrophobic, and amine groups, undergo self-folding in aqueous systems due to intramolecular hydrophobic interactions to yield these SCPNs. By systematically varying the hydrophobicity of the polymer, we can tune the extent of cell membrane wall disruption, which in turn governs the antimicrobial activity and rate of resistance acquisition in bacteria. We also show that the incorporation of OEG groups into the polymer design is essential in preventing complexation with proteins in biological medium, thereby maintaining the antimicrobial efficacy of the compound even in in vivo mimicking conditions. In comparison to the last-resort antibiotic colistin, our lead agents have a higher therapeutic index (by ca. 2-3 times) and hence better biocompatibility. We believe that the SCPNs developed here have potential for clinical applications and the information pertaining to their structure-activity relationship will be valuable toward the general design of synthetic antimicrobial (macro)molecules.

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

    PubMed

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

    2016-09-23

    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.

  3. 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

  4. A light-induced spin crossover actuated single-chain magnet

    NASA Astrophysics Data System (ADS)

    Liu, Tao; Zheng, Hui; Kang, Soonchul; Shiota, Yoshihito; Hayami, Shinya; Mito, Masaki; Sato, Osamu; Yoshizawa, Kazunari; Kanegawa, Shinji; Duan, Chunying

    2013-11-01

    Both spin-crossover complexes and molecular nanomagnets display bistable magnetic states, potentially behaving as elementary binary units for information storage. It is a challenge to introduce spin-crossover units into molecular nanomagnets to switch the bistable state of the nanomagnets through external stimuli-tuned spin crossover. Here we report an iron(II) spin-crossover unit and paramagnetic iron(III) ions that are incorporated into a well-isolated double-zigzag chain. The chain exhibits thermally induced reversible spin-crossover and light-induced excited spin-state trapping at the iron(II) sites. Single-chain magnet behaviour is actuated accompanying the synergy between light-induced excited spin-state trapping at the iron(II) sites and ferromagnetic interactions between the photoinduced high-spin iron(II) and low-spin iron(III) ions in the chain. The result provides a strategy to switch the bistable state of molecular nanomagnets using external stimuli such as light and heat, with the potential to erase and write information at a molecular level.

  5. In situ product recovery of single-chain antibodies in a membrane bioreactor.

    PubMed

    Meier, Kristina; Carstensen, Frederike; Scheeren, Christoph; Regestein, Lars; Wessling, Matthias; Büchs, Jochen

    2014-08-01

    The demand for biopharmaceuticals, such as monoclonal antibodies, has risen significantly over the last years. To be competitive, continuous production processes that yield consistent product quality and an economic advantage are desirable. In this study, an in situ product recovery process is described, involving use of submerged membranes to recover single-chain antibodies from a continuous fermentation of Hansenula polymorpha yeast cells.Reverse-flow diafiltration (RFD) was applied to prevent cake layer formation. Optimal flux ranges for this process could be identified by a systematic flux step method. The RFD process was optimized, preventing mixing of permeate and unreacted substrate: the space-time yield of antibodies using RFD could be tripled. Increase of the fouling related transmembrane pressure was below 45 Pa min(-1) for all applied dilution rates, indicating that the filtration process was stable. The membrane as well as the feeding mode of RFD did not influence cell viability nor product concentration. A wide range of dilution rates was successfully tested, demonstrating that this process is suitable for industrial applications.

  6. Identification of peptide mimotopes of gp96 using single-chain antibody library.

    PubMed

    Shanmugam, Arulkumaran; Suriano, Robert; Goswami, Neha; Chaudhuri, Devyani; Ashok, Badithe T; Rajoria, Shilpi; George, Andrea L; Mittelman, Abraham; Tiwari, Raj K

    2011-03-01

    Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96-peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund's adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4(+) and CD8(+)) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.

  7. Single-chain self-folding of synthetic polymers induced by metal-ligand complexation.

    PubMed

    Willenbacher, Johannes; Altintas, Ozcan; Roesky, Peter W; Barner-Kowollik, Christopher

    2014-01-01

    The controlled folding of a single polymer chain is for the first time realized by metal- complexation. α,ω-Bromine functional linear polymers are prepared via activators regenerated by electron transfer (ARGET) ATRP (M¯n,SEC = 5900 g mol(-1) , Đ = 1.07 and 12 000 g mol(-1) , Đ = 1.06) and the end groups of the polymers are subsequently converted to azide functionalities. A copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is carried out in the presence of a novel triphenylphosphine ligand and the polymers to afford homotelechelic bis-triphenylphosphine polymeric-macroligands (MLs) (M¯n,SEC = 6600 g mol(-1) , Đ = 1.07, and 12 800 g mol(-1) , Đ = 1.06). Single-chain metal complexes (SCMCs) are formed in the presence of Pd(II) ions in highly diluted solution at ambient temperature. The results derived via (1) H and (31) P{(1) H} NMR experiments, SEC, and DLS unambiguously evidence the efficient formation of SCMCs via metal ligand complexation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Effects of Metal Blending in Random Bimetallic Single-Chain Magnets: Synergetic, Antagonistic, or Innocent.

    PubMed

    Wang, Yan-Qin; Yue, Qi; Gao, En-Qing

    2017-01-18

    A family of isomorphous three-dimensional metal-organic frameworks based on bimetallic (FeCo, FeNi, and CoNi) chains with random metal sites have been prepared and magnetically characterized. The solid-solution-type bimetallic materials inherit intrachain ferromagnetic interactions and single-chain-magnet (SCM) behaviors from the homometallic parent materials. Interestingly, different composition dependence of magnetic behaviors has been found. The Fe(II)1-x Ni(II)x series (0≤x≤1) show an innocent composition dependence, where the blocking temperature of slow relaxation decreases monotonically as Fe(II) is replaced by less anisotropic Ni(II) . The Fe(II)1-x Co(II)x series show an unexpected antagonistic blending effect on slow relaxation: blending Fe(II) and Co(II) tends to depress the spin dynamics, and the bimetallic materials with intermediate composition show significantly lower blocking temperature than both Fe(II) and Co(II) materials. This is quite the opposite of what happens in the Co1-x Nix series, where Co(II) and Ni(II) seem to have a synergetic effect so that slow relaxation in bimetallic systems can be promoted to higher temperature than both Co(II) and Ni(II) materials. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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.

  10. New human single chain anti-idiotypic antibody against benzo[a]pyrene

    PubMed Central

    Ustinov, Valentin A.; Morozova, Vera V.; Tikunova, Nina V.; Glushkov, Andrey N.

    2017-01-01

    The nal¨ve library from the lymphocytes of healthy humans was screened by murine single-stranded idiotypic antibodies against benzo[a]pyrene (pSh). The phage clone which contained of anti-idiotypic antibody against benzo[a]pyrene, designated as A4, was chosen for further work because of highly specific to pSh. The available protein databases were searched. The A4 amino acid sequence was unique and 76% identical to a sequence in antibody against interferon g. The A4 protein was expressed in bacteria and purified by two different methods: His-tagged A4 and CBD-fusion A4. Both the A4 bound to pSh and also to the human single chain idiotypic antibody against the benzo[a]pyrene (T72) by ELISA. The Kd values of A4 for pSh and T72 were very close: 4.44 × 10-7 M and 5.71 × 10-7M, respectively. A4 was a competitor with benzo[a]pyrene for binding sites of both idiotypic pSh and T72 in competitive ELISA. Thus, A4 was a high affinity anti-idiotypic against benzo[a]pyrene which recognised pSh and T72 active sites. PMID:28860930

  11. Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

    PubMed Central

    Goffinet, Marine; Chinestra, Patrick; Lajoie-Mazenc, Isabelle; Medale-Giamarchi, Claire; Favre, Gilles; Faye, Jean-Charles

    2008-01-01

    Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in

  12. Lamellar self-assemblies of single-chain amphiphiles and sterols and their derived liposomes: distinct compositions and distinct properties.

    PubMed

    Cui, Zhong-Kai; Lafleur, Michel

    2014-02-01

    Typically, single-chain amphiphiles and sterols do not form fluid lamellar phases once hydrated individually. Most of the single-chain amphiphiles form actually micelles in aqueous environments, while sterols display a very limited solubility in water. However, under certain conditions, mixtures of single-chain amphiphiles and sterols lead to the formation of stable fluid bilayers. Over the past decade, several of these systems leading to fluid lamellar self-assemblies have been identified and this article reviews the current knowledge relative to these non-phospholipid bilayers made of single-chain amphiphiles and sterols. It presents an integrated view about the molecular features that are required for their stability, the properties they share, and the origin of these characteristics. It was also shown that these lamellar systems could lead to the formation of unilamellar vesicles, similar to phospholipid based liposomes. These vesicles display distinct properties that make them potentially appealing for technological applications; they display a limited permeability, they are stable, they are formed with molecules that are relatively chemically inert (and relatively cheap), and they can be readily functionalized. The features of these distinct liposomes and their technological applications are reviewed. Finally, the putative biological implications of these non-phospholipid fluid bilayers are also discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Functional expression and affinity selection of single-chain cro by phage display: isolation of novel DNA-binding proteins.

    PubMed

    Nilsson, M T; Mossing, M C; Widersten, M

    2000-07-01

    A robust selection system affording phage display of the DNA-binding helix-turn-helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage lambda Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.

  14. Construction, characterization, and selected site-specific mutagenesis of an anti-single-stranded DNA single-chain autoantibody.

    PubMed

    Rumbley, C A; Denzin, L K; Yantz, L; Tetin, S Y; Voss, E W

    1993-06-25

    Single-chain antibodies are comprised of immunoglobulin light and heavy chain variable domains joined through a polypeptide linker. A single-chain autoantibody, containing the 14-amino acid 212-polypeptide linker (GSTSGSGKSSEGKG), was constructed based on the light and heavy chain variable region gene sequences of anti-single-stranded DNA autoantibody BV04-01 (IgG2b, kappa). Following protein expression in Escherichia coli, denaturation, refolding, and affinity purification, single-chain autoantibody 04-01 binding with single-stranded DNA and poly(dT) was characterized in solid-phase and solution-phase assays. Homopolymer ligand binding results demonstrated that single-chain autoantibody 04-01 possessed anti-DNA binding properties similar to BV04-01 IgG and Fab fragments. Based on x-ray crystallographic analyses of BV04-01, site-specific mutagenesis studies were conducted on 2 residues (L32Tyr and H100aTrp) involved in aromatic stacking interactions with the middle thymidine of a (dT)3 ligand.

  15. 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.

  16. 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

    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.

  17. Genetically engineered colorimetric single-chain antibody fusion protein for rapid diagnosis of rabies virus.

    PubMed

    Mousli, M; Turki, I; Kharmachi, H; Dellagi, K

    2008-01-01

    The most widely used test for rabies diagnostics is the fluorescent antibody test, which is recommended by both the World Health Organization and the World Organisation for Animal Health (OIE). This test may be used directly on a smear, and can also be used to confirm the presence of rabies antigen in cell culture or in brain tissue for diagnosis. The colorimetric enzymes are usually coupled to an antibody by chemical means using cross-linking reagents. However, such non-specific procedures lead to heterogeneous conjugates, sometimes with reduced activity and specificity. To bypass these problems, genetic engineering has provided a way to create chimeric bifunctional molecules in which the variable domains of an antibody are genetically linked to unrelated protein tracers. In this study, we describe the successful production of a bifunctional chimeric protein based on alkaline phosphatase-fused anti-rabies virus glycoprotein scFv antibody fragment. We also report the antigen binding properties and the alkaline phosphatase activity of the recombinant conjugate protein. We established its value as a novel in vitro tool for detecting the rabies virus in brain smear in a one-step procedure; it presents a similar sensitivity and specificity to that obtained using standard reagents.

  18. 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.

  19. FV-MHMM: A Discussion on Weighting Schemes.

    NASA Astrophysics Data System (ADS)

    Franc, J.; Gerald, D.; Jeannin, L.; Egermann, P.; Masson, R.

    2016-12-01

    Upscaling or homogenization techniques consist in finding block-equivalentor equivalent upscaled properties on a coarse grid from heterogeneousproperties defined on an underlying fine grid. However, this couldbecome costly and resource consuming. Harder et al., 2013, have developeda Multiscale Hybrid-Mixed Method (MHMM) of upscaling to treat Darcytype equations on heterogeneous fields formulated using a finite elementmethod. Recently, Franc et al. 2016, has extended this method of upscalingto finite volume formulation (FV-MHMM). Although convergence refiningLagrange multipliers space has been observed, numerical artefactscan occur while trapping numerically the flow in regions of low permeability. This work will present the development of the method along with theresults obtained from its classical formulation. Then, two weightingschemes and their benefits on the FV-MHMM method will be presented insome simple random permeability cases. Next example will involve alarger heterogeneous 2D permeability field extracted from the 10thSPE test case. Eventually, multiphase flow will be addressed asan extension of this single phase flow method. An elliptic pressureequation solved on the coarse grid via FV-MHMM will be sequentiallycoupled with a hyperbolic saturation equation on the fine grid. Theimproved accuracy thanks to the weighting scheme will be measuredcompared to a finite volume fine grid solution. References: Harder, C., Paredes, D. and Valentin, F., A family of multiscalehybrid-mixed finite element methods for the Darcy equation with roughcoefficients, Journal of Computational Physics, 2013. Franc J., Debenest G., Jeannin L., Egermann P. and Masson R., FV-MHMMfor reservoir modelling ECMOR XV-15th European Conference on the Mathematicsof Oil Recovery, 2015.

  20. Switch-like surface binding of competing multivalent particles

    NASA Astrophysics Data System (ADS)

    Tito, N. B.; Frenkel, D.

    2016-10-01

    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.

  1. 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.

  2. 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.

  3. A multivalent approach to drug discovery for novel antibiotics.

    PubMed

    Long, Daniel D; Aggen, James B; Christensen, Burton G; Judice, J Kevin; Hegde, Sharath S; Kaniga, Koné; Krause, Kevin M; Linsell, Martin S; Moran, Edmund J; Pace, John L

    2008-10-01

    The design, synthesis and antibacterial activity of novel glycopeptide/beta-lactam heterodimers is reported. Employing a multivalent approach to drug discovery, vancomycin and cephalosporin synthons, A and B respectively, were chemically linked to yield heterodimer antibiotics. These novel compounds were designed to inhibit Gram-positive bacterial cell wall biosynthesis by simultaneously targeting the principal cellular targets of both glycopeptides and beta-lactams. The antibiotics 8a-f displayed remarkable potency against a wide range of Gram-positive organisms including methicillin-resistant Staphylococcus aureus (MRSA). Compound 8e demonstrated excellent bactericidal activity against MRSA (ATCC 33591) and initial evidence supports a multivalent mechanism of action for this important new class of antibiotic.

  4. Multivalent Molecules as Modulators of RNA Granule Size and Composition.

    PubMed

    Falkenberg, Cibele Vieira; Carson, John H; Blinov, Michael L

    2017-02-24

    RNA granules are ensembles of specific RNA and protein molecules that mediate localized translation in eukaryotic cells. The mechanisms for formation and selectivity of RNA granules are unknown. Here we present a model for assembly of one type of RNA granule based on experimentally measured binding interactions among three core multivalent molecular components necessary for such assembly: specific RNA molecules that contain a cis-acting sequence called the A2 response element (A2RE), hnRNP A2 proteins that bind specifically (with high affinity) to A2RE sequences or nonspecifically (with lower affinity) to other RNA sequences, and heptavalent protein cytoskeleton-associated protein 5 (CKAP5, an alternative name for TOG protein) that binds both hnRNP A2 molecules and RNA. Non-A2RE RNA molecules (RNA without the A2RE sequence) that may be recruited to the granules through nonspecific interactions are also considered in the model. Modeling multivalent molecular interactions in granules is challenging because of combinatorial complexity in the number of potential molecular complexes among these core components and dynamic changes in granule composition and structure in response to changes in local intracellular environment. We use a hybrid modeling approach (deterministic-stochastic-statistical) that is appropriate when the overall compositions of multimolecular ensembles are of greater importance than the specific interactions among individual molecular components. Modeling studies titrating the concentrations of various granule components and varying effective site pair affinities and RNA valency demonstrate that interactions between multivalent components (TOG and RNA) are modulated by a bivalent adaptor molecule (hnRNP A2). Formation and disruption of granules, as well as RNA selectivity in granule composition are regulated by distinct concentration regimes of A2. Our results suggest that granule assembly is tightly controlled by multivalent molecular interactions

  5. Multivalent Presentation of Antihantavirus Peptides on Nanoparticles Enhances Infection Blockade▿

    PubMed Central

    Hall, Pamela R.; Hjelle, Brian; Brown, David C.; Ye, Chunyan; Bondu-Hawkins, Virginie; Kilpatrick, Kathleen A.; Larson, Richard S.

    2008-01-01

    Viral entry into susceptible host cells typically results from multivalent interactions between viral surface proteins and host entry receptors. In the case of Sin Nombre virus (SNV), a New World hantavirus that causes hantavirus cardiopulmonary syndrome, infection involves the interaction between viral membrane surface glycoproteins and the human integrin αvβ3. Currently, there are no therapeutic agents available which specifically target SNV. To address this problem, we used phage display selection of cyclic nonapeptides to identify peptides that bound SNV and specifically prevented SNV infection in vitro. We synthesized cyclic nonapeptides based on peptide sequences of phage demonstrating the strongest inhibition of infection, and in all cases, the isolated peptides were less effective at blocking infection (9.0% to 27.6% inhibition) than were the same peptides presented by phage (74.0% to 82.6% inhibition). Since peptides presented by the phage were pentavalent, we determined whether the identified peptides would show greater inhibition if presented in a multivalent format. We used carboxyl linkages to conjugate selected cyclic peptides to multivalent nanoparticles and tested infection inhibition. Two of the peptides, CLVRNLAWC and CQATTARNC, showed inhibition that was improved over that of the free format when presented on nanoparticles at a 4:1 nanoparticle-to-virus ratio (9.0% to 32.5% and 27.6% to 37.6%, respectively), with CQATTARNC inhibition surpassing 50% when nanoparticles were used at a 20:1 ratio versus virus. These data illustrate that multivalent inhibitors may disrupt polyvalent protein-protein interactions, such as those utilized for viral infection of host cells, and may represent a useful therapeutic approach. PMID:18391034

  6. Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases.

    PubMed

    Touisni, Nadia; Kanfar, Nasreddine; Ulrich, Sébastien; Dumy, Pascal; Supuran, Claudiu T; Mehdi, Ahmad; Winum, Jean-Yves

    2015-07-13

    Invited for the cover of this issue are Jean-Yves Winum and co-workers at University of Montpellier (France) and University of Florence (Italy). The image depicts the multivalency approach applied to zinc metalloenzyme carbonic anhydrases. Read the full text of the article at 10.1002/chem.201501037. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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

  8. 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.

  9. 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. Copyright 2010 Elsevier B.V. All rights reserved.

  10. 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

  11. Ligand Characteristics Important to Avidity Interactions of Multivalent Nanoparticles.

    PubMed

    Li, Ming-Hsin; Zong, Hong; Leroueil, Pascale R; Choi, Seok Ki; Baker, James R

    2017-04-11

    Multivalent interactions involve the engagement of multiple ligand-receptor pairs and are important in synthetic biology as design paradigms for targeted nanoparticles (NPs). However, little is known about the specific ligand parameters important to multivalent interactions. We employed a series of oligonucleotides as ligands conjugated to dendrimers as nanoparticles, and used complementary oligonucleotides on a functionalized SPR surface to measure binding. We compared the effect of ligand affinity to ligand number on the avidity characteristics of functionalized NPs. Changing the ligand affinity, either by changing the temperature of the system or by substitution non-complementary base pairs into the oligonucleotides, had little effect on multivalent interaction; the overall avidity, number of ligands required for avidity per particle and the number of particles showing avidity did not significantly change. We then made NP conjugates with the same oligonucleotide using an efficient copper-free click chemistry that resulted in essentially all of the NP in the population exceeding the threshold ligand value. The particles exceeding the threshold ligand number again demonstrated high avidity interactions. This work validates the concept of a threshold ligand valence and suggests that the number of ligands per nanoparticle is the defining factor in achieving high avidity interactions.

  12. Designing super selectivity in multivalent nano-particle binding.

    PubMed

    Martinez-Veracoechea, Francisco J; Frenkel, Daan

    2011-07-05

    A key challenge in nano-science is to design ligand-coated nano-particles that can bind selectively to surfaces that display the cognate receptors above a threshold (surface) concentration. Nano-particles that bind monovalently to a target surface do not discriminate sharply between surfaces with high and low receptor coverage. In contrast, "multivalent" nano-particles that can bind to a larger number of ligands simultaneously, display regimes of "super selectivity" where the fraction of bound particles varies sharply with the receptor concentration. We present numerical simulations that show that multivalent nano-particles can be designed such that they approach the "on-off" binding behavior ideal for receptor-concentration selective targeting. We propose a simple analytical model that accounts for the super selective behavior of multivalent nano-particles. The model shows that the super selectivity is due to the fact that the number of distinct ligand-receptor binding arrangements increases in a highly nonlinear way with receptor coverage. Somewhat counterintuitively, our study shows that selectivity can be improved by making the individual ligand-receptor bonds weaker. We propose a simple rule of thumb to predict the conditions under which super selectivity can be achieved. We validate our model predictions against the Monte Carlo simulations.

  13. 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.

  14. 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

  15. Mechanistic Insight into Heptosyltransferase Inhibition by using Kdo Multivalent Glycoclusters.

    PubMed

    Tikad, Abdellatif; Fu, Huixiao; Sevrain, Charlotte M; Laurent, Sophie; Nierengarten, Jean-François; Vincent, Stéphane P

    2016-09-05

    The synthesis of unprecedented multimeric Kdo glycoclusters based on fullerene and calix[4]arene central scaffolds is reported. The compounds were used to study the mechanism and scope of multivalent glycosyltransferase inhibition. Multimeric mannosides based on porphyrin and pillar[5]arenes were also generated in a controlled manner. Twelve glycoclusters and their monomeric ligands were thus assayed against heptosyltransferase WaaC, which is an important bacterial glycosyltransferase that is involved in lipopolysaccharide biosynthesis. It was first found that all the multimers interact solely with the acceptor binding site of the enzyme even when the multimeric ligands mimic the heptose donor. Second, the novel Kdo glycofullerenes displayed very potent inhibition (Ki =0.14 μm for the best inhibitor); an inhibition level rarely observed with glycosyltransferases. Although the observed "multivalent effects" (i.e., the enhancement of affinity of a ligand when presented in a multimeric fashion) were in general modest, a dramatic effect of the central scaffold on the inhibition level was evidenced: the fullerene and the porphyrin scaffolds being by far superior to the calix- and pillar-arenes. We could also show, by dynamic light scattering analysis, that the best inhibitor had the propensity to form aggregates with the heptosyltransferase. This aggregative property may contribute to the global multivalent enzyme inhibition, but probably do not constitute the main origin of inhibition.

  16. 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).

  17. 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

  18. Secondary batteries with multivalent ions for energy storage.

    PubMed

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

    2015-09-14

    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. A novel anti-PSMA human scFv has the potential to be used as a diagnostic tool in prostate cancer

    PubMed Central

    Han, Yueheng; Wei, Ming; Han, Sen; Lin, Ruihe; Sun, Ziyong; Yang, Fa; Jiao, Dian; Xie, Pin; Zhang, Lingling; Yang, An-Gang; Zhao, Aizhi; Wen, Weihong; Qin, Weijun

    2016-01-01

    Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer related death in men. The early diagnosis and treatment of PCa are still challenging due to the lack of efficient tumor targeting agents in traditional managements. Prostate specific membrane antigen (PSMA) is highly expressed in PCa, while only has limited expression in other organs, providing an ideal target for the diagnosis and therapy of PCa. The antibody library technique has opened the avenue for the discovery of novel antibodies to be used in the diagnosis and therapy of cancer. In this paper, by screening a large yeast display naive human single chain antibody fragment (scFv) library, we obtained a high affinity scFv targeting PSMA, called gy1. The gy1 scFv was expressed in E.coli and purified via a C terminal 6His tag. The binding affinity of gy1 was shown to be at the nanomolar level and gy1 can specifically bind with PSMA positive cancer cells, and binding triggers its rapid internalization through the endosome-lysosome pathway. The specific targeting of gy1 to PSMA positive tumor tissues was also evaluated in vivo. We showed that the IRDye800CW labeled gy1 can efficiently target and specifically distribute in PSMA positive tumor tissues after being injected into xenograft nude mice. This study indicated that the novel antibody gy1 could be used as a great tool for the development of PSMA targeted imaging and therapy agents for PCa. PMID:27448970