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Sample records for antibody fragment scfv

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

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    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.

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

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    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

  3. Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv against human ICAM-1

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    H. Sun

    2014-07-01

    Full Text Available Intercellular adhesion molecule-1 (ICAM-1 is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

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

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    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.

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

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    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.

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

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    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.

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

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    Tombesi Marina

    2008-09-01

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

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

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    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).

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

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    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.

  10. Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support.

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    Hu, Xuejun; O'Hara, Liam; White, Simon; Magner, Edmond; Kane, Marian; Wall, J Gerard

    2007-03-01

    Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.

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

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    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.

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

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    Torsten Rülker

    Full Text Available 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.

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

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    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.

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

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    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.

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

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    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.

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

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

    2008-08-01

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

  17. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT

    2002-01-01

    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of s

  18. Construction of multiform scFv antibodies using linker peptide

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  19. 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.

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    John M Louis

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

  20. Engineered single chain antibody fragments for radioimmunotherapy

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    Huhalov, A.; Chester, K. A. [Cancer Research UK Imaging and Targeting Group Royal Free, London (United Kingdom). Department of Oncology; University College Medical School Royal Free Campus, London (United Kingdom)

    2004-12-01

    An ideal molecule to deliver radioimmunotherapy (RIT) would be target specific and have prolonged residence time at high concentrations in the tumour with rapid clearance from normal tissues. It would also be non-immunogenic. These features can be rationally introduced into recombinant antibody-based proteins using antibody engineering techniques. This reviews focuses on the use of antibody engineering in the design and development of RIT molecules which have single chain Fv (scFv) antibody fragments as building blocks.

  1. Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

    Institute of Scientific and Technical Information of China (English)

    Mei-Yun LIU; Wei HAN; Yan-Li DING; Tian-Hong ZHOU; Rui-Yang TIAN; Sheng-Li YANG; Hui LIU; Yi GONG

    2005-01-01

    B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

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

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    Fuchs, P; Breitling, F; Little, M; Dübel, S

    1997-06-01

    The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  4. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  5. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  6. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  7. Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv

    Directory of Open Access Journals (Sweden)

    Elias Waldir P

    2011-02-01

    Full Text Available Abstract Background Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC and enterohemorrhagic Escherichia coli (EHEC. Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667. In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv. Findings Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction, was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  10. Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

    DEFF Research Database (Denmark)

    Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.;

    2006-01-01

    Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage...... of the PAM1 mAb, we describe here the production of a phage-free, soluble scFv version of the PAM1 mAb (PAM1scFv). Using the new PAM1scFv probe, the occurrence of the HG epitope recognized can now be localized with high resolution within micro-domains of plant cell walls....

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

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

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

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

    Science.gov (United States)

    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.

  13. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

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

    Science.gov (United States)

    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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  16. Construction, Expression and In Vitro Biological Behaviors of Ig scFv Fragment in Patients with Chronic B Cell Leukemia

    Institute of Scientific and Technical Information of China (English)

    ZHU Lijuan; LIAO Wenjun; ZHU Huifen; LEI Ping; WANG Zhihua; SHAO Jingfang; ZHANG Yue; SHEN Guanxin

    2006-01-01

    The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDSPAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD=0.9921 ku),which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of IdHSP complex vaccine for B-CLL.

  17. Antibody Fragments as Probe in Biosensor Development

    Directory of Open Access Journals (Sweden)

    Serge Muyldermans

    2008-08-01

    Full Text Available Today’s proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

  18. Thermodynamic stability and flexibility characteristics of antibody fragment complexes.

    Science.gov (United States)

    Li, Tong; Verma, Deeptak; Tracka, Malgorzata B; Casas-Finet, Jose; Livesay, Dennis R; Jacobs, Donald J

    2014-01-01

    Free energy landscapes, backbone flexibility and residue-residue couplings for being co-rigid or co-flexible are calculated from the minimal Distance Constraint Model (mDCM) on an exploratory dataset consisting of VL, scFv and Fab antibody fragments. Experimental heat capacity curves are reproduced markedly well, and an analysis of quantitative stability/flexibility relationships (QSFR) is applied to a representative VL domain and several complexes in the scFv and Fab forms. Global flexibility in the denatured ensemble typically decreases in the larger complexes due to domain-domain interfaces. Slight decreases in global flexibility also occur in the native state of the larger fragments, but with a concurrent large increase in correlated flexibility. Typically, a VL fragment has more co-rigid residue pairs when isolated compared to the scFv and Fab forms, where correlated flexibility appears upon complex formation. This context dependence on residue- residue couplings in the VL domain across length scales of a complex is consistent with the evolutionary hypothesis of antibody maturation. In comparing two scFv mutants with similar thermodynamic stability, local and long-ranged changes in backbone flexibility are observed. In the case of anti-p24 HIV-1 Fab, a variety of QSFR metrics were found to be atypical, which includes comparatively greater co-flexibility in the VH domain and less co-flexibility in the VL domain. Interestingly, this fragment is the only example of a polyspecific antibody in our dataset. Finally, the mDCM method is extended to cases where thermodynamic data is incomplete, enabling high throughput QSFR studies on large numbers of antibody fragments and their complexes.

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

    Science.gov (United States)

    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.

  20. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

    Science.gov (United States)

    2016-08-01

    ECBC-TR-1395 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR...Thompson James Carney RESEARCH AND TECHNOLOGY DIRECTORATE Candice Warner Melody Zacharko EXCET, INC. Springfield, VA 22151-2110...4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv

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

    OpenAIRE

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

    2010-01-01

    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 Bece...

  2. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

    Directory of Open Access Journals (Sweden)

    Roopali eRajput

    2015-09-01

    Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

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

    Science.gov (United States)

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

    1997-09-15

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

  4. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Directory of Open Access Journals (Sweden)

    Safar Farajnia

    2013-02-01

    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    Oliinyk O. S.; Kaberniuk A. A.; Kolibo D. V.; Komisarenko S. V.

    2014-01-01

    Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv) antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized) human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, an...

  7. Engineered single-chain variable fragment antibody for immunodiagnosis of groundnut bud necrosis virus infection.

    Science.gov (United States)

    Maheshwari, Yogita; Vijayanandraj, S; Jain, R K; Mandal, Bikash

    2015-05-01

    Few studies have been done on engineered antibodies for diagnosis of tospovirus infections. The present study was undertaken to develop a single-chain variable fragment (scFv) for specific diagnosis of infection by groundnut bud necrosis virus (GBNV), the most prevalent serogroup IV tospovirus in India. Heavy chain (372 nucleotide [nt]) and light chain (363 nt) variable region clones obtained from a hybridoma were used to make an scFv construct that expressed a ~29-kDa protein in E. coli. The scFv specifically detected GBNV in field samples of cowpea, groundnut, mung bean, and tomato, and it did not recognize watermelon bud necrosis virus, a close relative of GBNV belonging to tospovirus serogroup IV. This study for the first time demonstrated the application of a functional scFv against a serogroup-IV tospovirus.

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

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    Saurabh Jain

    Full Text Available 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

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

    Science.gov (United States)

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    Science.gov (United States)

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

    2000-03-01

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

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

    Science.gov (United States)

    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.

  15. Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Hui LIU; Yan-Li DING; Wei HAN; Mei-Yun LIU; Rui-Yang TIAN; Sheng-Li YANG; Yi GONG

    2004-01-01

    Three single chain antibodies(scFv)against the proteins of severe acute respiratory syndrome coronavirus(SARS-CoV)were isolated by phage display from an scFv antibody library.Bio-panning was carried out against immobilized purified envelope(E)and nucleocapsid(N)proteins of SARS-CoV.Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA.Two of them,B 10 and C20,could recognize non-overlapping epitopes of the E protein according to the two-site binding test result.Clone A 17 could recognize N protein.The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ.The affinity(equilibrium dissociation constant,Kd)of SARS-CoV E protein was 5.7×10-8 M for B10 and 8.9×10-8 M for C20.The affinity of A17for N protein was 2.1 x 10-6 M.All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

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

    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明

    2002-01-01

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

  17. A novel human recombinant antibody fragment capable of neutralizing Mexican scorpion toxins.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Gurrola, Georgina B; Possani, Lourival D; Becerril, Baltazar

    2013-12-15

    Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    2014-10-01

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

  1. Improved Detection of Domoic Acid Using Covalently Immobilised Antibody Fragments

    Directory of Open Access Journals (Sweden)

    J. Gerard Wall

    2013-03-01

    Full Text Available Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.

  2. Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

  3. Production of monoclonal anti-GP Ⅱ b/Ⅲa scFv antibodies from scFv phage libraries%从scFv噬菌体抗体库中筛选抗GPⅡ b/Ⅲa单链抗体

    Institute of Scientific and Technical Information of China (English)

    夏红利; 谭最; 陈德杰; 乔建国; 邱仁峰

    2011-01-01

    目的 从scFv噬菌体库中获取人源化特异性Anti-GPⅡb/Ⅲa单克隆scFv抗体.方法 对Tomlinson I+J scFv文库进行3次淘洗,富集特异性的抗GPⅡb/Ⅲa抗体.通过酶联免疫吸附试验(ELISA)和双脱氧终止法基因测序及同源性对比,检测出人源化的抗GPⅡb/Ⅲa单克隆抗体.结果 在3次淘洗后,得到了抗GPⅡb/Ⅲa单克隆噬菌体抗体,阳性克隆的获取率在95.6%以上;ELISA和基因测序筛选出25种不同的全长抗GPⅡb/Ⅲa噬菌体抗体,这些基因序列与人免疫球蛋白可变区基因同源性达到89%以上;分泌性抗体ELISA检测提示这些抗体顺利表达了蛋白并特异性结合GPⅡb/Ⅲa,其中15种scFv对GPⅡb/Ⅲa有更强的阳性反应.结论 人源化的特异性抗-GPⅡb/Ⅲa scFv能通过噬菌体展示技术快速有效地获得.%Objective To screen monoclonal anti-GP Ⅱ b/Ⅲ a antibodies from scFv phage libraries and obtain specific monoclonal anti-GP Ⅱ b/Ⅲ a scFv. Methods Specific anti-GP Ⅱ b/Ⅲ a scFv antibodies were enriched by three rounds of selection from Tomlinson Ⅰ + J libraries. By using polyclonal and monoclonal phage enzyme linked immunosorbent assay ( ELISA) and gene sequencing by bideoxy chain termination, full-length specific monoclonal anti-GP Ⅱ b/Ⅲ a antibodies were picked out and their gene sequences were obtained. Results Twenty-five different full-length monoclonal scFv phage fragments were obtained after three rounds of panning and their gene sequences were identified, and positive rate of monoclones was above 95.6%; homology comparison with variable regions of human immunoglobulin gene showed the similarity was above 89%; the result of soluble scFv ELISA showed that these specific scFv could be expressed smoothly, and 15 full-length monoclonal scFv antibodies were stronger positive than the other in these scFv. Conclusion Antibody phage display was a rapid and effective method to obtain Anti-GP Ⅱ b/Ⅲ a scFv fragements.

  4. 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.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

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

  6. THE CONSTRUCTION AND EXPRESSION OF THE MURINE SCFV GENE IN E. COLI AGAINST HUMAN CERVICAL CANCER

    Institute of Scientific and Technical Information of China (English)

    Wang Ying; Chen Wei; Li Xu

    2006-01-01

    Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E. coli. Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E. coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E. coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E. coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.

  7. Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

    Science.gov (United States)

    Foerster, A; Hołowacz, I; Sunil Kumar, G B; Anandakumar, S; Wall, J G; Wawrzyńska, M; Paprocka, M; Kantor, A; Kraskiewicz, H; Olsztyńska-Janus, S; Hinder, S J; Bialy, D; Podbielska, H; Kopaczyńska, M

    2016-04-01

    Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs.

  8. In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences.

    Directory of Open Access Journals (Sweden)

    Angel M Cuesta

    Full Text Available There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed "trimerbody", comprises a single-chain antibody (scFv fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA, a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting.

  9. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody

    Science.gov (United States)

    2016-03-01

    ECBC-TR-1356 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR ANTIBODY CHARACTERIZATION...From - To) Oct 2010 – Sep 2012 4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization...Arlington, VA 22203-2114 10. SPONSOR/MONITOR’S ACRONYM(S) DARPA 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library.

    Science.gov (United States)

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon

    2015-10-01

    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

  14. Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin*

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Ledezma-Candanoza, Luis M.; Serrano-Posada, Hugo; Fernández-Taboada, Guillermo; Olamendi-Portugal, Timoteo; Rojas-Trejo, Sonia; Gómez-Ramírez, Ilse V.; Rudiño-Piñera, Enrique; Possani, Lourival D.; Becerril, Baltazar

    2016-01-01

    The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom. PMID:26589800

  15. Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Ledezma-Candanoza, Luis M; Serrano-Posada, Hugo; Fernández-Taboada, Guillermo; Olamendi-Portugal, Timoteo; Rojas-Trejo, Sonia; Gómez-Ramírez, Ilse V; Rudiño-Piñera, Enrique; Possani, Lourival D; Becerril, Baltazar

    2016-01-22

    The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

  16. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

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

    2015-08-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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).

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

    Science.gov (United States)

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

  20. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    Science.gov (United States)

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

  1. 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.

    Science.gov (United States)

    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.

  2. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur.

  3. Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

    Science.gov (United States)

    Säll, Anna; Sjöholm, Kristoffer; Waldemarson, Sofia; Happonen, Lotta; Karlsson, Christofer; Persson, Helena; Malmström, Johan

    2015-11-06

    Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

  4. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    Science.gov (United States)

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

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

    OpenAIRE

    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 th...

  6. Modulation of the CD40-CD40 ligand interaction using human anti-CD40 single-chain antibody fragments obtained from the n-CoDeR phage display library.

    Science.gov (United States)

    Ellmark, Peter; Ottosson, Camilla; Borrebaeck, Carl A K; Malmborg Hager, Ann-Christin; Furebring, Christina

    2002-08-01

    CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.

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

    Science.gov (United States)

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

    2016-10-01

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

  8. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  9. SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.

    Science.gov (United States)

    Niesen, Judith; Sack, Markus; Seidel, Melanie; Fendel, Rolf; Barth, Stefan; Fischer, Rainer; Stein, Christoph

    2016-08-17

    Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives.

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

    Science.gov (United States)

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

    2016-06-15

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

  11. 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.

    Science.gov (United States)

    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.

  12. Probing the soybean Bowman-Birk inhibitor using recombinant antibody fragments.

    Science.gov (United States)

    Muzard, Julien; Fields, Conor; O'Mahony, James John; Lee, Gil U

    2012-06-20

    The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman-Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (K(D)~1.10(-9) M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

  15. Screening and immunological identification of the human ScFv antibody against PSMA%全人源抗PSMA单链抗体的筛选及免疫活性鉴定

    Institute of Scientific and Technical Information of China (English)

    张才田; 刘金霞

    2011-01-01

    Objective To screen and identify a human single-chain variable fragment(scFv) antibody against prostate specific membrane antigen(PSMA) from a human scFv antibody library.Methods Using a synthetic PSMA peptide as the coating antigen, the antibody library was screened by five rounds of combining-eluting-amplification.The phage antibody against PSMA with high specificity was screened out from the human scFv antibody library and its binding ability to the antigen was tested by ELISA.The soluble antibody was produced by plasmids extracted from highly specific clones, whose binding ability to PSMA was further identified by Western blot and immunohistochemistry.The affinity constant of the soluble antibody was measured by non-competitive ELISA.Results The screened phage antibody was specific for PSMA by ELISA.The soluble antibody was also specific for PSMA, its molecular weight was about 30 kD by SDS-PAGE and its affinity constant was about 5.077 × 106 L/mol.Conclusions The screened scFv antibody is specific and has low immunogenicity.It can be further used in the target treatment of malignant tumors.%目的 从全人源单链噬菌体抗体库中筛选出抗前列腺特异性膜抗原(PSMA)特异性单链抗体并进行免疫活性鉴定.方法 以合成的PSMA多肽为抗原,经过五轮吸附-洗脱-扩增,从单链噬菌体抗体库中筛选出特异性抗PSMA噬菌体抗体,ELISA检测其抗原结合能力,并对特异性较强的克隆提取质粒,表达可溶性抗体.Western Blotting和免疫组织化学检测其抗原结合性,非竞争ELSIA法检测其亲和常数.结果 从单链噬菌体抗体库中筛选出的噬菌体抗体,经ELISA鉴定为抗PSMA的特异性噬菌体抗体.抗PSMA可溶性抗体相对分子质量约为3.0×104,与PSMA特异性结合,其亲和常数约为5.077×106L/mol.结论 所得全人源抗PSMA单链抗体保留完整抗体分子结合抗原的特异性,免疫原性弱,是肿瘤导向治疗的理想栽体.

  16. Yeast display of antibody fragments: a discovery and characterization platform

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael; Siegel, Robert W.

    2004-07-01

    This review will focus on some of the novel attributes of the yeast surface display platform for the discovery and characterization of novel affinity reagents, optimization of those reagents, and novel uses of the platform. This is not intended to serve as an exhaustive review on the broader topic of general scFv technologies (see Winter et al., 1994; Smith and Petrenko, 1997; Bradbury et al., 2003) Furthermore, the scFv format of antibodies are easily manipulated through molecular cloning into a number of other formats such IgG, Fab, diabodies and such, for use in down steam applications and the reader is encouraged to read ?IgG?, ?Fab?, or your favorite format whenever scFv is seen in this review. This review is presented in 5 parts; (1) description of yeast display and its components, (2) library types and construction methods, (3) screening approaches for non-immune libraries and benefits, (4) screening approaches for directed evolution, kinetic on and off rates and (5) epitope complementation binning of clones.

  17. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    Directory of Open Access Journals (Sweden)

    Tong Li

    2015-07-01

    Full Text Available The effects of somatic mutations that transform polyspecific germline (GL antibodies to affinity mature (AM antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM. We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab, and subsequently, the DCM was combined with molecular dynamics (MD simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  18. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    Science.gov (United States)

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

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

    Science.gov (United States)

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

    2012-01-01

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

  20. Dimerisation strategies for shark IgNAR single domain antibody fragments.

    Science.gov (United States)

    Simmons, David P; Abregu, Fiona A; Krishnan, Usha V; Proll, David F; Streltsov, Victor A; Doughty, Larissa; Hattarki, Meghan K; Nuttall, Stewart D

    2006-08-31

    Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (approximately 13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix-turn-helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (approximately 26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats.

  1. Construction of Human ScFv Phage Display Library against Ovarian Tumor

    Institute of Scientific and Technical Information of China (English)

    XIA Jinsong; BI Hao; YAO Qin; QU Shen; ZONG Yiqiang

    2006-01-01

    In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

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

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

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

  3. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    1992-08-01

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

  6. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    . Conclusion The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

  7. scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis.

    Science.gov (United States)

    Jannuzzi, Grasielle Pereira; Tavares, Aldo Henrique F P; Kaihami, Gilberto Hideo; de Almeida, José Roberto Fogaça; de Almeida, Sandro Rogério; Ferreira, Karen Spadari

    2015-01-01

    Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells.

  8. scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis.

    Directory of Open Access Journals (Sweden)

    Grasielle Pereira Jannuzzi

    Full Text Available Paracoccidioidomycosis (PCM, caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv that mimics the main antigen of P. brasiliensis (gp43 confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells.

  9. Considerations in producing preferentially reduced half-antibody fragments.

    Science.gov (United States)

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

  10. 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.

    Science.gov (United States)

    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.

  11. Preliminary radioimmunoimaging and biodistribution of 131iodine-labeled single-chain antibody fragment against progastrin-releasing peptide(31-98) in small cell lung cancer xenografts

    Institute of Scientific and Technical Information of China (English)

    Hong Zhihui; Shi Yizhen; Liu Zengli; Zhou Xiaolin; Yang Yi; Tang Jun

    2014-01-01

    Background Monoclonal antibodies (mAbs) such as DD3,raised against progastrin-releasing peptide(31-98) (ProGRP(31-98)) antigen,have been used to target small cell lung cancer (SCLC).However,as an intact mAb,DD3 is cleared slowly from the body,with an optimal radioimmunoimaging time of 72 hours.More recently,a singlechain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research.Thereby,it potentially increases the radioimmunoimaging efficacy.However,there have been few studies with this antibody fragment.The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of 1311I-anti-ProGRP(31-98)scFv in nude mice bearing SCLC xenografts.Methods Anti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry.131I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated.Similarly,the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated.After injection of 131I-anti-ProGRP(31-98) scFv,treated mice were imaged at 1,24,and 30 hours.Then the tumor/base ratios were calculated.Results ProGRP was highly expressed in NCI-H446 cells and xenograft tissue.The metabolism of 131I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes,respectively.The %ID/g of 131I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection,reaching a maximum of (5.38±0.92) %ID/g at 24 hours.Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours,with 24 hours proving optimal.Conclusion 131I-anti-ProGRP(31-98)scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid

  12. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

    Directory of Open Access Journals (Sweden)

    Philippe Thullier

    Full Text Available The lethal toxin (LT of Bacillus anthracis, composed of the protective antigen (PA and the lethal factor (LF, plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF to form the edema toxin (ET, which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236, of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260 was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-09

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

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

    Science.gov (United States)

    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.

  16. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

    Directory of Open Access Journals (Sweden)

    Man Cheng

    Full Text Available Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3. The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx. Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N protein of SARS-associated coronavirus (SCoV were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

  17. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

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    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

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

    Science.gov (United States)

    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.

  19. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

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

    Science.gov (United States)

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

    2016-01-01

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

  1. The Biochemical Properties of Antibodies and Their Fragments.

    Science.gov (United States)

    Hnasko, Robert M

    2015-01-01

    Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.

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

    Science.gov (United States)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    曹跃琼; 乔守怡; 袁有忠; 黄建生; 赵寿元

    1999-01-01

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

  4. Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Daniela Luz

    Full Text Available Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB bind to globotria(tetraosylceramide receptors (Gb3/Gb4 on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

  5. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  6. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  7. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  8. ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB′ FRAGMENT CONTAINING IMMUNOCONJUGATES

    Institute of Scientific and Technical Information of China (English)

    刘小云; 甄永苏

    2002-01-01

    Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.

  9. Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

    Directory of Open Access Journals (Sweden)

    Shukra M. Aavula

    2011-01-01

    Full Text Available Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

  10. Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

    Directory of Open Access Journals (Sweden)

    Hiroshi Hanagata

    2014-05-01

    Full Text Available Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production.

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

    Science.gov (United States)

    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.

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

    NARCIS (Netherlands)

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

    1999-01-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

  14. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  15. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

    Directory of Open Access Journals (Sweden)

    Katrin Spiesberger

    Full Text Available 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4.With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  16. PEGylation of antibody fragments for half-life extension.

    Science.gov (United States)

    Jevševar, Simona; Kusterle, Mateja; Kenig, Maja

    2012-01-01

    Antibody fragments (Fab's) represent important structure for creating new therapeutics. Compared to full antibodies Fab' fragments possess certain advantages, including higher mobility and tissue penetration, ability to bind antigen monovalently and lack of fragment crystallizable (Fc) region-mediated functions such as antibody-dependent cell mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The main drawback for the use of Fab's in clinical applications is associated with their short half-life in vivo, which is a consequence of no longer having the Fc region. To exert meaningful clinical effects, the half-life of Fab's need to be extended, which has been achieved by postproduction chemical attachment of polyethylene glycol (PEG) chain to protein using PEGylation technology. The most suitable approach employs PEG-maleimide attachment to cysteines, either to the free hinge cysteine or to C-terminal cysteines involved in interchain disulfide linkage of the heavy and light chain. Hence, protocols for mono-PEGylation of Fab via free cysteine in the hinge region and di-PEGylation of Fab via interchain disulfide bridge are provided in this chapter.

  17. Neutralisation and binding of VHS virus by monovalent antibody fragments

    DEFF Research Database (Denmark)

    Cupit, P.M.; Lorenzen, Niels; Strachan, G.

    2001-01-01

    appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial...... single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using...... the 3F1H10 variable heavy (VH) chain and variable light (V kappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured...

  18. 全人源抗鼻咽癌噬菌体单链抗体的筛选与鉴定%Screening and characterization of human scFv antibodies against nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    李艳东; 谢平丽; 王甲甲; 李跃辉; 胡锦跃; 李官成

    2009-01-01

    目的 从全人源抗鼻咽癌噬菌体抗体库中筛选特异性单链抗体(ScFv),并对其特异性进行鉴定.方法 通过噬菌体表面展示技术把ScFv表达在噬菌体表面,以鼻咽癌细胞作为抗原,用抗原递减法,通过"吸附-洗脱-扩增"过程筛选并富集特异性抗体,及ELISA筛选,获得特异阳性克隆进行免疫组化鉴定并测序.结果 通过对抗体库进行三轮正负淘洗和富集后,随机挑选4212个克隆进行ELISA,发现3个克隆对CNE2呈强阳性反应,而与人正常细胞系HUVEC等呈弱阳性反应或不反应.对克隆HNSAO33进一步进行免疫细胞化学验证,结果与ELISA反应一致;免疫组织化学鉴定表明克隆HNSAO33与鼻咽癌组织和鼻咽组织阳性率的差别有统计学意义.结论 通过淘选富集、ELISA和免疫化学鉴定获得特异性较强的噬菌体克隆,为鼻咽癌发病机制的研究和临床诊断以及治疗奠定了基础.%Objective To screen the anti-nasopharyngeal carcinoma scFv from a human anti-nasopharyngeal carcinoma single-chain phage antibody library, and identify its characteristics. Methods The single-chain phage antibody library was subjected to three rounds of positive and negative cell panning and enrichment, and then it was selected by ELISA. The binding specificity of phage antibodies with naso-pharyngeal carcinoma cells was confirmed by immunohistochemistry. Results After panning, enrichment and testing by ELISA, 3 phage an-tibody clones reacting with CNE2 more strongly than HUVEC and NP69 were picked out from 4212 clones. One clone, HNSAO33, was fur-ther analyzed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. HNSAO33 spe-cifically reacted to nasopharyngeal carcinoma cells in most human nasopharyngeal carcinoma tissue sections except a few human normal naso-pharyngeal tissue sections. The distinction of positive rates was of a great statistical significance. Conclusion ELISA

  19. The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A monoclonal antibody, LC-l, recognizing lung cancer associated common antigens was obtained in authors' laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A-1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.

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

    Science.gov (United States)

    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.

  1. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    Science.gov (United States)

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  2. Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

    Directory of Open Access Journals (Sweden)

    Mai Sabine

    2008-01-01

    Full Text Available Abstract Background Expression of intracellular antibodies (intrabodies has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer. Results The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9 reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system. Conclusion The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.

  3. Purification of human monoclonal antibodies and their fragments.

    Science.gov (United States)

    Müller-Späth, Thomas; Morbidelli, Massimo

    2014-01-01

    This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have been proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.

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

    Directory of Open Access Journals (Sweden)

    Accardi Luisa

    2007-01-01

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

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

    Science.gov (United States)

    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.

  6. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies

    Directory of Open Access Journals (Sweden)

    Stefanie Claudia Pohl

    2012-04-01

    Full Text Available Bivalent single chain (scFv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4 revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Froyen, G; Billiau, A

    1997-01-01

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

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

    Science.gov (United States)

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

    1999-05-01

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

  10. 一种特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达方案%Strategy for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin

    Institute of Scientific and Technical Information of China (English)

    饶瑜; 钟利桥; 张凤; 张晓华; 戴和平

    2011-01-01

    We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.%为了实现特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达,将不能以可溶性蛋白形式表达的、只能以噬菌体展示形式特异性识别斑马鱼卵黄蛋白原的单链抗体F5的基因,克隆到pET 32a载体并转化入大肠杆菌ori DE3中.结果表明,通过诱导表达,可获得可溶性的并且仍特异性识别斑马鱼卵黄蛋白原的单链抗体32a-F5.噬菌体展示单链抗体不能可溶性表达是噬菌体展示技术应用中常见的问题,该方法提供了一种表达可溶性单链抗体的可行性方案.

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

    Science.gov (United States)

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

    2015-08-01

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

  12. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    2005-01-01

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain

  13. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, T.P.

    2003-12-31

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with {sup 99m}Tc and {sup 188}Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic

  14. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2011-12-01

    Full Text Available Abstract Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY, the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the sc

  15. Single chain Fab (scFab fragment

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    Brenneis Mariam

    2007-03-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

    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(-).

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  18. Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

    NARCIS (Netherlands)

    Dolk, E.; Vliet, C. van; Perez, J.M.J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L.G.J.; Verrips, T.

    2005-01-01

    In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear

  19. Recombinant anti-human melanoma antibodies are versatile molecules.

    Science.gov (United States)

    Neri, D; Natali, P G; Petrul, H; Soldani, P; Nicotra, M R; Vola, R; Rivella, A; Creighton, A M; Neri, P; Mariani, M

    1996-08-01

    The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

  20. Preparation of single chain variable fragment of MG7 mAb by phage display technology

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Yong-Zhan Nie; Dai-Ming Fan; Xue-Yong Zhang

    2001-01-01

    AIM To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS mRNA was isolated from MG7-producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOⅢ of highly expressing MG7binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigenbinding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE. RESULTS The VH, VL and ScFv DNAs were about 340bp,320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATO Ⅲ cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATO Ⅲ cells.SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32. CONCLUSION The MG7 ScFv was successfully produced by phage antibody technology, which may

  1. 抗镰刀菌单链抗体在大肠杆菌中可溶性表达条件的研究%A Study on the Optimization of Condition for Soluble Expression of Fusarium-specific scFv Antibody in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    胡祖权; 李和平; 吴平; 廖玉才; 张静柏

    2015-01-01

    The objectives of the work is to optimize the conditions for inducing expression, and obtain the soluble and high-yield expression of a Fusarium-specific single-chain variable fragment(scFv)antibody in the periplasmic space of Escherichia coli XL1-Blue. The recombinant plasmid containing a Fusarium-specific scFv antibody FvSG7 was transferred into E. coli XL1-Blue. After the culture medium for inducing selected, the soluble expression level and activity of FvSG7 antibody were analyzed by Western blot and ELISA detection for studying the influence of temperature, IPTG concentration and induction time on expression level. The maximum productivity of soluble FvSG7 antibody was obtained after induction at 25℃for 2 h, with a final concentration of 0.1 mmol/L β-D-thiogalactopyranoside(IPTG)and the cultured bacteria growing to the OD600 value of 0.5. In conclusion, the soluble expression of FvSG7 antibody in the periplasm of E. coli XL1-Blue increased significantly by optimizing the expression’s conditions of induction temperature, IPTG concentration and induction time.%通过优化诱导表达条件,实现抗镰刀菌单链抗体在大肠杆菌周质中高效可溶性表达。将抗镰刀菌单链抗体FvSG7转化到大肠杆菌XL1-Blue中,确定诱导表达培养基,诱导温度、诱导剂IPTG的浓度和诱导时间条件下培养重组大肠杆菌,通过Western杂交和ELISA检测分析单链抗体的可溶性表达情况以及抗体的活性。重组大肠杆菌培养至OD600nm为0.5时加入终浓度为0.1 mmol/L IPTG,25℃诱导表达2 h可获得最大量的可溶性单链抗体。通过对诱导温度、IPTG浓度和诱导时间等表达条件的优化,可以显著提高FvSG7抗体在大肠杆菌XL1-Blue周质中的表达量。

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

    NARCIS (Netherlands)

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The a

  3. A simple vector system to improve performance and utilisation of recombinant antibodies

    Directory of Open Access Journals (Sweden)

    Vincent Karen J

    2006-12-01

    Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

  4. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    Science.gov (United States)

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  5. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    Science.gov (United States)

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

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

    Science.gov (United States)

    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. Characterization of single chain antibody targets through yeast two hybrid

    Directory of Open Access Journals (Sweden)

    Vielemeyer Ole

    2010-08-01

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

  8. Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

    Science.gov (United States)

    Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Brockmann, Eeva-Christine; Azhayev, Alex; Wang, Qi; Vehniäinen, Markus; Lamminmäki, Urpo

    2013-10-01

    In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    OpenAIRE

    2013-01-01

    Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raj...

  11. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    Science.gov (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  12. Expression and bioactivity identification of soluble MG7 scFv

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Bo-Rong Pan; Dai-Ming Fan; Xue-Yong Zhang

    2002-01-01

    AIM: To examine the molecular mass and identify thebioactivity of MG7 scFv for its application as a targetingmediator in gene therapy of gastric cancer.METHODS: Two strongly positive recombinant phage clonesscreened from MG7 recombinant phage antibody library wereseparately transfected into E. coli TG1. Plasmid wasisolated from the transfected E. coli TG1 and digested byEcoR Ⅰ and Hind Ⅲ to examine the length of exogenousscFv gene. Then, the positive recombinant phage cloneswere individually transfected into E. coli HB2151. Thetransfectant was cultured and induced by IPTG. Perplasmicextracts was prepared from the induced transfectant byosmotic shock. ELISA was used to examine the antigen-binding affinity of the soluble MG7 scFv. Immunodottingassay was adopted to evaluate the yield of soluble MG7 scFvproduced by transfected E. coli HB2151. Western blot wasused to examine the molecular mass of MG7 scFv. Finally,the nucleotide sequence of MG7 scFv was examined by DNAsequencing.RESULTS: Two positive recombinant phage clones werefound to contain the exogenous scFv gene. ELISA showedthat MG7 scFv had strong antigen-binding affinity.Immuodotting assay showed that transfected E. coli HB2151could successfully produce the soluble MG7 scFv with highyield via induction by IPTG. The molecular mass of MG7scFv was 30 kDa by western blot. DNA sequencingdemonstrated that the VH and VL genes of MG7 scFv were363bp and 321 bp, respectively.CONCLUSION: We have successfully developed the solubleMG7 scFv which possessed strong antigen-binding affinity.

  13. Improved production and function of llama heavy chain antibody fragments by molecular evolution

    NARCIS (Netherlands)

    Linden, van der R.H.; Geus, de B.; Frenken, G.J.; Peters, H.; Verrips, C.T.

    2000-01-01

    The aim of this study was to improve production level of llama heavy chain antibody fragments (V (HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V (HH) fragments specific for the azo-dye reactive red-6. In

  14. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.

    2007-01-01

    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms a

  15. Generation of recombinant alpaca VHH antibody fragments for the detection of the mycotoxin ochratoxin A

    NARCIS (Netherlands)

    Houwelingen, van A.M.M.L.; Saeger, de T.; Rusanova, T.; Waalwijk, C.; Beekwilder, M.J.

    2008-01-01

    To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin o

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

    Science.gov (United States)

    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.

  17. Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

    Science.gov (United States)

    Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

    2014-01-01

    Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Karen S Ferreira

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  2. Stirred batch crystallization of a therapeutic antibody fragment.

    Science.gov (United States)

    Hebel, Dirk; Huber, Sabine; Stanislawski, Bernd; Hekmat, Dariusch

    2013-07-20

    Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10μL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.

  3. Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity.

    Science.gov (United States)

    Lee, Seung-Hyun; Park, Dong-Woon; Sung, Eun-Sil; Park, Hye-Ran; Kim, Jin-Kyoo; Kim, Yong-Sung

    2010-01-01

    Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.

  4. Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

    DEFF Research Database (Denmark)

    Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten

    2011-01-01

    We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

  5. Chemical Chaperones Increasing Expression Level of Soluble Single-chain Fv Antibody(scFv2F3)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) is a selenoenzyme that protects the biomembrane and other cellular components against oxidative damage. The selenium-containing single chain Fv fragment of monoclonal antibody 2F3 (SescFv2F3 ) is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. Se-scFv2F3 is generated by the chemical modification of the single chain Fy fragment of monoclonal antibody 2F3 ( scFv2F3 ), which can be expressed in E. coli. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates.The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.

  6. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga

    2014-08-01

    Full Text Available Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.

  7. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J

    2016-01-01

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform...... reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples...

  8. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  9. Utility of recombinant Fragment C for assessment of anti-tetanus antibodies in plasma

    Science.gov (United States)

    Ramakrishnan, Girija; Pedersen, Karl; Guenette, Denis; Sink, Joyce; Haque, Rashidul; Petri, William A.; Herbein, Joel; Gilchrist, Carol A.

    2016-01-01

    Anti-tetanus antibodies in biological samples are typically detected using an ELISA based on toxoided tetanus neurotoxin as antigen. We demonstrate that recombinantly produced Fragment C of the toxin heavy chain (rFragC) is an effective alternative antigen for assessment of tetanus- immune status in plasma samples. PMID:25749462

  10. Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides

    NARCIS (Netherlands)

    Harmsen, M.M.; Fijten, H.P.D.

    2012-01-01

    We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to

  11. Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

    Science.gov (United States)

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

  12. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Luis Mario Rodríguez-Martínez

    Full Text Available Current Ebola virus (EBOV detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV proteins. In particular, several monoclonal antibodies (mAbs have been described that bind the capsid glycoprotein (GP of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude and they are easily and economically produced in bacterial cultures.Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.

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

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

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

  14. Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.

    Science.gov (United States)

    Billah, Md Morsaline; Hodges, Christopher S; Hays, Henry C W; Millner, P A

    2010-11-01

    The successful construction of an immunosensor depends on having an effective procedure for immobilising the bio-recognition element to the transducer surface. In the present study, an amino-terminated 4-aminothiophenol (ATP) self-assembled monolayer (SAM) was modified with heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate to couple reduced anti-myoglobin half-antibody fragments. The disulphide groups present in the hinge region of IgG molecules were selectively cleaved by 2-mercaptoethylamine to produce reduced half-antibody fragments with free sulphydryl groups. The maleimide terminated 4-ATP SAM modified surface was coupled to these reduced antibody fragments to produce highly oriented immobilization of the half-antibody via its Fc domain and to allow free access to the Fv bindings sites. This represents an improvement by comparison with biotin/avidin mediated IgG attachment which is essentially randomly oriented. Functional immunosensors were able to detect myoglobin in both phosphate buffered saline and whole serum over the range of concentrations from 10(-13)M to 10(-6)M, and order of magnitude better than avidin/biotin linked immunosensors. In addition, atomic force microscopy (AFM) was carried out to elucidate the nanotopology of the immunosensor surface at different stages of fabrication; the images demonstrate that half antibodies bind as described and show structural changes on subsequent antigen binding.

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

    Science.gov (United States)

    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.

  16. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

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

    Science.gov (United States)

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

    2009-01-01

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

  18. Isolation of anti-T cell receptor scFv mutants by yeast surface display.

    Science.gov (United States)

    Kieke, M C; Cho, B K; Boder, E T; Kranz, D M; Wittrup, K D

    1997-11-01

    Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

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

    Science.gov (United States)

    Iyer, Arun K; Su, Yang; Feng, Jinjin; Lan, Xiaoli; Zhu, Xiaodong; Liu, Yue; Gao, Dongwei; Seo, Youngho; Vanbrocklin, Henry F; 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.

  20. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    Science.gov (United States)

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

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

    Science.gov (United States)

    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.

  2. Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Imanaka Tadayuki

    2002-09-01

    Full Text Available Abstract Background Hep27 monoclonal (Hep27 Mab is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102. We attempted to produce a single-chain fragment (scFv, a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. Results The sequences encoding the variable regions of heavy (VH and light (VL chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa. Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab. Conclusion This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.

  3. Cholestatic liver disease after rituximab and adalimumab and the possible role of cross-reacting antibodies to Fab 2 fragments.

    Directory of Open Access Journals (Sweden)

    Joerg Latus

    Full Text Available BACKGROUND: Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA-associated granulomatosis with polyangiitis (GPA who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. METHODS: Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. RESULTS: Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients' sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. CONCLUSION: This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects

  4. Renal toxicity of radiolabeled peptides and antibody fragments: mechanisms, impact on radionuclide therapy, and strategies for prevention.

    NARCIS (Netherlands)

    Vegt, E. de; Jong, M. de; Wetzels, J.F.M.; Masereeuw, R.; Melis, M.; Oyen, W.J.G.; Gotthardt, M.; Boerman, O.C.

    2010-01-01

    Peptide-receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogs such as octreotide is an effective therapy against neuroendocrine tumors. Other radiolabeled peptides and antibody fragments are under investigation. Most of these compounds are cleared through the kidneys and reabso

  5. Renal toxicity of radiolabeled peptides and antibody fragments: Mechanisms, impact on radionuclide therapy, and strategies for prevention

    NARCIS (Netherlands)

    E. Vegt (Erik); M. de Jong (Marion); J.F.M. Wetzels (Jack); R. Masereeuw (Rosalinde); M.L. Melis (Marleen); W.J. Oyen (Wim); M. Gotthardt (Martin); O.C. Boerman (Otto)

    2010-01-01

    textabstractPeptide-receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogs such as octreotide is an effective therapy against neuroendocrine tumors. Other radiolabeled peptides and antibody fragments are under investigation. Most of these compounds are cleared through the kidney

  6. High efficiency recovery and epitope specific sorting of an scFv yeast display library

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, Robert W.; Coleman, James R.; Miller, Keith D.; Feldhaus, Michael

    2004-03-01

    In order to more productively utilize the rich source of antigen specific reagents present in the previously described non-immune scFv yeast display library (Feldhaus et al., 2003) one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi MacsTm system to recover greater than 90% of the antigen specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope specific scFv. Combining these two improvements in library screening allowed isolation and characterization of 3 epitope specific scFv (including a previously uncharacterized epitope) to a 6 kd protein, epidermal growth factor EGF.

  7. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model

    Directory of Open Access Journals (Sweden)

    Ji-Guo Su

    2015-12-01

    Full Text Available Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM. Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF of the residues, as well as the MSF in the internal distance (MSFID between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

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

    Directory of Open Access Journals (Sweden)

    Benevolo Maria

    2006-09-01

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

  9. Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

    Directory of Open Access Journals (Sweden)

    Phelps Amanda L

    2009-11-01

    Full Text Available Abstract Background There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV, as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology. Results In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge. Conclusion A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans. Crown Copyright © 2009

  10. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

    Directory of Open Access Journals (Sweden)

    Keith R Miller

    Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

  11. Functional improvement of antibody fragments using a novel phage coat protein III fusion system

    DEFF Research Database (Denmark)

    Jensen, Kim Bak; Larsen, Martin; Pedersen, Jesper Søndergaard;

    2002-01-01

    Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance...... of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria......(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity....

  12. 细胞表达流感病毒抗体CR6261VH与scFv对流感病毒感染的影响%Effect of antibody CR6261 VH and scFv expression on influenza virus infection

    Institute of Scientific and Technical Information of China (English)

    李燕; 李秀岚; 严景华

    2011-01-01

    The emerging CR6261 antibody could neutralize several subtype influenza virus with high affinity, whose VH domain binds to the HA protein conserved domain. Therefore, it has drawn much attention as a potential broad-spectrum therapeutic antibody against influenza virus. In this study, we constructed the eukaryotic expression vectors pCR6261VH, pCR6261VH-GFP and pCR6261scFv and screened the monoclonal cell lines that could stably express CR6261VH, CR6261VH-GFP and CR6261scFv on the cell membrane. After influenza virus infecting the stable cell lines, the titers of viruses were tested by hemagglutination inhibition test. The result shows that the titers of viruses in CR6261scFv and CR6261VH-GFP stable expression cell lines decreased and there was no obvious discrimination between the CR6261VH expression cell line and the negative control, suggesting that CR6261VH and CR6261scFv expressing on the cell membrane could partly inhibit the virus infection. Though the effective inhibition strategy is undergoing, our research will provide new clues for the breeding of anti influenza transgenic animals.%由于新发现的流感病毒抗体CR6261可以中和多种亚型的流感病毒,而且是其VH区结合于流感病毒蛋白HA的保守区,因此该抗体有望成为一种广谱的流感治疗性抗体而备受关注.通过构建真核表达栽体pCR6261VH、pCR6261VH-GFP、pCR6261 scFv,成功地筛选到在细胞膜上稳定表达CR6261 VH、CR6261VH-GFP与CR6261 scFv的单克隆细胞系.用流感病毒去感染稳定细胞系,间隔12 h取上清检测病毒血凝效价.结果表明:稳定表达CR6261 scFv和CR6261VH-GFP细胞系能够降低病毒的血凝效价,而表达CR6261VH细胞系的血凝效价与对照无显著差异.说明CR6261VH与CR6261scFv在细胞膜上表达能够部分抑制病毒侵染细胞.尽管更有效的抑制策略还在研究中,但本研究将为抗流感转基因动物的育种提供新的思路.

  13. Generation, use, and validation of receptor-selective antibodies.

    Science.gov (United States)

    Mackrill, John J

    2004-01-01

    Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GPCR size, abundance, distribution, turnover, modification, interaction with other proteins, and functional properties. In this chapter, techniques for the generation and characterization of receptor-selective antibodies are described. Two protocols are given for the generation of antibodies: (1) development of polyclonal antibodies (PAbs) against synthetic peptides corresponding to a specific site within a GPCR and (2) selection of synthetic single-chain fragment variable (scFv) monoclonal antibodies (MAbs) from libraries expressed on the surface of bacteriophage. Immunoblot and enzyme-linked immunosorbent assays for characterization of the selectivity and affinity of such antibodies are described. Finally, methods are given for improvement of the titer and specificity of PAbs.

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

    Directory of Open Access Journals (Sweden)

    Dübel Stefan

    2007-11-01

    Full Text Available Abstract 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-18

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

  16. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    Science.gov (United States)

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  17. [Digitoxin poisoning: reversing ventricular fibrillation with Fab fragments of anti-digoxin antibody].

    Science.gov (United States)

    Domart, Y; Bismuth, C; Schermann, J M; Abuaf, N; Pontal, P G; Baud, F; Bolo, A; Gailliot, M; Fournier, P E

    1982-12-25

    Purified Fab fragments of ovine anti-digoxin antibodies (Wellcome Foundation) were used to treat a patient who attempted suicide by absorbing 10 mg of digitoxin (serum concentration 265 micrograms/l). The poor prognosis, as assessed clinically and from serum potassium levels (7.5 mEq/l), seemed to warrant such a treatment. The weak (6.85%) cross-reactivity elicited in vitro between the anti-digoxin antibodies and digitoxin was compensated by increasing the doses, but improvement was observed with 3.6 g, i.e. about half the effective dosage initially considered. The criteria of effectiveness were clinical, electrocardiographic (reversal of the ventricular fibrillation), biochemical (simultaneous and opposite changes in extra- and intracellular potassium levels, suggesting that ATPase inhibition by digitalis is a reversible process) and toxicological: there was an increase in digitoxin serum levels suggesting displacement of the drug from tissue sites to plasma and other extracellular compartments where the Fab fragments are distributed, and Fab-bound digitoxin appeared fairly rapidly in the urine, which suggested shunting of the normal hepatic metabolic pathway.

  18. Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

    Directory of Open Access Journals (Sweden)

    Boechat L.H.B.

    1999-01-01

    Full Text Available Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis and non-autoimmune (subacute thyroiditis disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

  19. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    Science.gov (United States)

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  20. 肿瘤干细胞表面标志物CD133单链抗体基因的分离与鉴定%Identification and characterization of single chain antibody of cancer stem cell surface marker CD133 scFv CD133

    Institute of Scientific and Technical Information of China (English)

    韩瑜; 周炳喜; 张延瑞; 韩双印

    2011-01-01

    Objective To identify,construct and express scFv CD133,verify its biological function.Methods VL and VH were isolated from hybridoma of mAb CD133 by using antibody engineering technology.Its DNA sequencing and CDR were determined.scFv CD133 was then cloned into pET32a,transformed into Origami,induced by IPTG,purified by Ni2+-NTA His resin.Its affinity and specificity were tested by NH4SCN elution and ELISA.Results The size of VL and VH of scFv CD133 was 339 bp and 342 bp,which coded 113 and 114 amino acid separately.Its VL belonged to mouse Igκ chain and VH belonged to mouse IgG heavy chain subtype I.The molecular weight of scFv CD133 was about 27 × 103 which was testified by SDSPAGE and Western blot.Its affinity and specificity were also verified.Conclusion scFv CD133 has been successfully constructed and expressed in Origami,which could supply basis for target therapy of CD+133 cancer stem cell.%目的 分离、构建和表达抗人CD133单链抗体(scFvCD133),测定其生物学活性.方法 用抗体工程技术从抗人CD133单克隆抗体(mAb)杂交瘤细胞中分离抗体可变区基因(VL和VH),测定DNA序列并确定抗体互补决定区(CDR);将scFv CD133基因克隆至pET32a中,转化Origami菌株,IPTG诱导,Ni2+-NTA His树脂纯化单链抗体,梯度硫氰酸盐洗脱法和酶联免疫吸附(ELISA)法检测其亲和性和特异性.结果 scFvCD133基因VL和VH长度分别为339 bp和342 bp,各编码113和114个氨基酸,其中VL隶属于小鼠Ig κ轻链,VH隶属于小鼠Ig重链Ⅰ亚类.scFvCD133经SDS-PAGE和Western blot 分析证明相对分子质量为27 × 103,体外实验具有一定的亲和性和特异性.结论 获得scFv CD133基因,为CD+133肿瘤干细胞的靶向治疗奠定了基础.

  1. Crystal Structure of the Fab Fragment of an Anti-factor IX Antibody 10C12

    Institute of Scientific and Technical Information of China (English)

    SHI Xiao-Li; ZENG Tu; HUANG Ming-Dong

    2008-01-01

    10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C 12.

  2. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

    Directory of Open Access Journals (Sweden)

    Stewart Nuttall

    2013-01-01

    Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

  3. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2011-02-15

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  4. Fibrotic remodeling of the extracellular matrix through a novel (engineered, dual-function antibody reactive to a cryptic epitope on the N-terminal 30 kDa fragment of fibronectin.

    Directory of Open Access Journals (Sweden)

    Maryada Sharma

    Full Text Available Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM, leading to tissue contraction and impaired function of the organ. Fibronectin (Fn is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR, a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE; fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv termed Fn52RGDS, which acts in two ways: i binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii interacts with the cell surface receptors, ie., integrins (through an attached "RGD" sequence tag, and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis--migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling. The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.

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

    Science.gov (United States)

    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.

  6. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    Science.gov (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

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

    Science.gov (United States)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  9. A nucleic-acid hydrolyzing single chain antibody confers resistance to DNA virus infection in hela cells and C57BL/6 mice.

    Science.gov (United States)

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

    2014-06-01

    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 (SCH07072) [corrected] 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.

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

    Institute of Scientific and Technical Information of China (English)

    Jiong Cai; Fang Li; Shizhen Wang

    2008-01-01

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

  11. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael (BATTELLE (PACIFIC NW LAB)); Siegel, Robert W.(BATTELLE (PACIFIC NW LAB)); Opresko, Lee (BATTELLE (PACIFIC NW LAB)); Coleman, James R.(BATTELLE (PACIFIC NW LAB)); Feldhaus, Jane M.(BATTELLE (PACIFIC NW LAB)); Yeung, Yik A.(Massachusetts Institute Of Tec); Cochran, Jennifer R.(Massachusetts Institute Of Tec); Heinzelman, Peter (Massachusetts Institute Of Tec); Colby, David (Massachusetts Institute Of Tec); Swers, Jeffrey (Massachusetts Institute Of Tec); Graff, Christilyn (Massachusetts Institute Of Tec); Wiley, H Steven (BATTELLE (PACIFIC NW LAB)); Wittrup, K D.(Massachusetts Institute Of Tec)

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

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

    Science.gov (United States)

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

    2016-04-20

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

  13. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    Science.gov (United States)

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M [Los Alamos National Laboratory; Velappan, Nileena [Los Alamos National Laboratory; Schmidt, Jurgen G [Los Alamos National Laboratory

    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. Electrochemical detection of vascular endothelial growth factors (VEGFs) using VEGF antibody fragments modified Au NPs/ITO electrode.

    Science.gov (United States)

    Kim, Gang-Il; Kim, Kyung-Woo; Oh, Min-Kyu; Sung, Yun-Mo

    2010-03-15

    A new electrochemical technique for the detection of vascular endothelial growth factors (VEGFs) as a cancer-related biomarker is presented in this paper. Gold nanoparticles (Au NPs) were self-assembled onto an indium tin oxide (ITO) electrode to prepare a modified sandwich type electrochemical immunoassay platform. VEGF antibodies were cleaved into two half-fragments by 2-mercaptoethylamine-HCl (2-MEA) and the fragments were immobilized onto the Au NP substrates by their thiol groups. Through this strategy, randomly oriented attachment of antibodies was prevented which frequently occurs in a general use of whole antibody and reduces the number of available sites for the attachment of target molecules. VEGF target molecules were applied to the immunoelectrodes and they combined with the antibody fragments covering the Au NP electrode, forming antigen-antibody complexes. Then, ferrocene-tagged antibodies, which release electrons under a proper applied potential, were added to the system and they combined with the VEGF molecules pre-attached to the antibody fragments. The redox current of ferrocene measured by the differential pulse voltammetry (DPV) increased almost linearly from 1.27 x 10(-4) to 4.17 x 10(-4)A according to the increase in the concentration of the VEGF target molecules from 100 to 600 pg/ml. The measured current values represent the concentration of the VEGF since they are proportional to the number of ferrocene molecules which is in turn proportional to the concentration of VEGF target molecules. Using this modified sandwich immunoassay with the Au NP/ITO electrode, VEGFs as low as 100 pg/ml were detected with high specificity.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

    Institute of Scientific and Technical Information of China (English)

    WANG Cheng; MU Ying; L(U) Shao-wu; LUO Gui-min; WAN Pei; GONG Ping-sheng; L(U) Li-min; XU Ya-wei; ZHAO Yang; HE Bo; ZHAO Gang; YAN Gang-lin

    2011-01-01

    In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv),a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis,three-dimensional(3D)model building,binding-site analysis and docking.The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+),then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC).The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method,thus,the human Se-scFv with GPX activity was obtained.The GPX activity of the Se-scFv protein was characterized.Compared with other Se-scFv,the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione.It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

  19. In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments

    NARCIS (Netherlands)

    F. Aladin (Farah); A.W.C. Einerhand (Sandra); J. Bouma (Janneke); S. Bezemer (Sandra); P. Hermans (Pim); D. Wolvers (Danielle); K. Bellamy (Kate); L.G.J. Frenken (Leon); J. Gray (Jim); M. Iturriza-Gómara (Miren)

    2012-01-01

    textabstractRotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (refe

  20. Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast

    NARCIS (Netherlands)

    Harmsen, M.M.; Smits, C.B.; Geus, de B.

    2002-01-01

    We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with

  1. Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins

    NARCIS (Netherlands)

    Harmsen, M.M.; Solt, van C.B.; Fijten, H.P.D.; Setten, van M.C.

    2005-01-01

    The therapeutic parenteral application of llama single-domain antibody fragments (VHHs) is hampered by their small size, resulting in a fast elimination from the body. Here we describe a method to increase the serum half-life of VHHs in pigs by fusion to another VHH binding to porcine immunoglobulin

  2. ISOLATION OF ENDOTOXIN-SPECIFIC ANTIBODIES BY SELECTION OF AN SINGLE CHAIN PHAGE ANTIBODY LIBRARY

    Institute of Scientific and Technical Information of China (English)

    陈鸣; 俞丽丽; 张雪; 府伟灵

    2002-01-01

    Objective: To isolate murine anti endotoxin single chain phage antibody from a constructed library. Methods: Total RNA was firstly extracted from murine splenic cells and mRNA was reverse-transcribed into cDNA. Then the designed primers were used to amplify the variable region genes of the heavy and light chain (VH, VL) with polymerase chain reaction. The linker was used to assemble the VH and VL into ScFv, and the NotI and SfiI restriction enzymes were used to digest the ScFv in order to ligate into the pCANTAB5E phagemid vector that was already digested with the same restriction enzymes. The ligated vector was then introduced into competent E.coli TG1 cells to construct a single-chain phage antibody library. After rescued with M13KO7 helper phage, recombinant phages displaying ScFv fragments were harvested from the supernatant and selected with endotoxin. The enriched positive clones were reinfected into TG1 cells. Finally, 190 clones were randomly selected to detect the anti endotoxin antibody with indirect ELISA. Results: The titer of anti endotoxin in murine sera was 1:12,800. The concentration of total RNA was 12.38 μg/ml. 1.9×107 clones were obtained after transformed into TG1. 3×104 colonies were gotten after one round panning. Two positive colonies were confirmed with indirect ELISA among 190 randomly selected colonies. Conclusion: A 1.9×107 murine anti endotoxin single chain phage antibody library was successfully constructed. Two anti endotoxin antibodies were obtained from the library.

  3. Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

  4. A Co-expression System Based on Phage and Phagemid to Select Cognate Antibody-antigen Pairs in vivo

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A modified selectively-infective phage (SIP) is developed to facilitate the selection of interacting antibody-antigen pairs from a large single-chain antibody (scFv) library in vivo. The system is constructed with a modified helper phage M13KO7 and phagemid pCANTAB 5 E. The antigen fused to the C-terminal of N1-N2 domain and the scFv to the N-terminal of CT domain of the gIIIp of filamentous phage are encoded on the phage and phagemid vectors respectively. The phages produced by co-transformants restore infectivity via interaction between antigen and antibody fusions in the cell periplasm. In a model system, the scFv fragment of the anti-hemagglutinin 17/9 antibody and its corresponding antigen are detected in the presence of a 105 fold excess of a non-interacting control pairs, which demonstrates this system to be very sensitive and facile to screen a large single-chain antibody library.

  5. The Purification of Natural and Recombinant Peptide Antibodies by Affinity Chromatographic Strategies.

    Science.gov (United States)

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    The purification of peptide antibodies (e.g., IgG, IgY, scFv, and Fab) are described in this chapter. Affinity chromatographic purification, a very convenient and effective antibody purification strategy, is used to isolate peptide antibodies based on specific binding, i.e., binding of the antibody to a column on which its specific ligand is immobilized with subsequent elution of the purified antibody. In addition, the application of purification methods based on the use of proteins A, G, and L, each of which bind to specific domains on an antibody/fragment, or the use of specific tags (e.g., histidine and biotin) attached to antibodies or antigens are also described.

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

    Science.gov (United States)

    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.

  7. Construction and identification of anti-ENR phage display scFv libraries%抗恩诺沙星噬菌体单链抗体库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    温凯; 沈建忠; 孟辉; 吴聪明; 王战辉; 张素霞

    2011-01-01

    本研究以兽药恩诺沙星为对象,构建噬菌体抗体库,为低成本、快速制备目的抗体提供新的途径.以恩诺沙星-鸡卵清蛋白(ENR-OVA)为免疫原对Balb/c小鼠进行免疫,取其脾细胞提取总RNA,并分别扩增全套抗体轻、重链基因,通过重叠延伸PCR技术,以编码柔性多肽(Gly3Ser)4的基因为接头,将轻重链基因组装为完整的scFv基因,将之克隆入pCANTAB5E载体,转化大肠杆菌XL1-Blue,以辅助噬菌体M13KO7对其进行超感染,构建噬菌体抗体库并进行富集、筛选和鉴定;构建了库容量约为2.2×106的抗恩诺沙星噬菌体单链抗体库,并筛选出26株阳性克隆,为表达单链抗体、建立免疫检测方法奠定基础.%This study focuses on construction and identification of immunized phage display library from splenocytes of hyperimmunized BALB/C mice for screening and isolation of scFv fragment against ENR, an alternative method to produce antibodies for veterinary drug residues detection. The total RNA was isolated from splenocytes of a BALB/C mouse hyperimmunized with the Enrofloxacin conjugated to chicken OVA. Variable light and heavy domains of the immunoglobulin genes were amplified by PCR and assembled to produce full-length single-chain Fv(scFV) by overlap extension PCR using a linker primer containing flexible polypeptide-(Gly3Ser)4. The scFv DNA fragment was ligated into phagemid vector pCANTAB5E and electroporated into E. coli XL1-Blue cells. The transformed cells were rescued by M13KO7 helper phage and phage libraries were constructed. The size of antibody libraries is 2.2 × 106. Following the construction of phage display scFv libraries, the recombinant phage displaying scFv were enriched and identified. There are 26 clones against ENR generated in this study.

  8. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  9. Structural models of antibody variable fragments: A method for investigating binding mechanisms

    Science.gov (United States)

    Petit, Samuel; Brard, Frédéric; Coquerel, Gérard; Perez, Guy; Tron, François

    1998-03-01

    The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that anti-nucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nculeosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

  10. A 10-RESIDUE FRAGMENT OF AN ANTIBODY (MINI-ANTIBODY) DIRECTED AGAINST LYSOZYME AS LIGAND IN IMMUNOAFFINITY CHROMATOGRAPHY

    NARCIS (Netherlands)

    WELLING, GW; VANGORKUM, J; DAMHOF, RA; DRIJFHOUT, JW; BLOEMHOFF, W; WELLINGWESTER, S

    1991-01-01

    The interaction between an antibody molecule and a protein antigen is an example of "natural" protein modelling. Amino acids of the antigen-binding site consisting of three hypervariable segments (L1, L2, L3) of the light (L) and three (H1, H2, H3) of the heavy (H) chain of an antibody molecule inte

  11. Screening of Human Antibody Fab Fragment against HBsAg and the Construction of its dsFv Form

    Directory of Open Access Journals (Sweden)

    Leili Jia, Jiyun Yu, Hongbin Song, Xuelin Liu, Weina Ma, Yuanyong Xu, Chuanfu Zhang, Shicun Dong, Qiao Li

    2008-01-01

    Full Text Available The objective of this study was to pursue the techniques involving the screening of the human antibody Fab fragment against hepatitis B virus surface antigen (HBsAg and the construction of its disulfide-stabilized Fv fragment (dsFv. The phage antibody Fab fragments against HBsAg were screened from the human combinatorial immunoglobulin library. Sequence analysis revealed that its heavy chain gene was complete, but the light chain gene was lost. To improve the affinity of the antibody by chain shuffling, a human antibody light chain gene repertoire was generated by reverse transcriptase-polymerase chain reaction (RT-PCR from the human peripheral blood lymphocytes. A phage antibody sub-library was then constructed by inserting the light chain gene repertoire into the phagmid that contained the Fd gene. Five clones with appreciably higher absorbance than that of the original clone were obtained, which indicated that the affinity of the light chain-shuffled phage antibodies was improved. Then, the mutated genes of dsFv against HBsAg were constructed by using PCR-based point mutagenesis method. Purified VH and VL proteins were folded into a 25-kDa protein, designated as anti-HBsAg dsFv. ELISA and competition ELISA revealed that the dsFv maintained the specificity of the Fab by binding to HBsAg, even through with a lower binding activity. These results have facilitated the undertaking of further functional analyses of the constructed dsFv, and may therefore provide an improved technique for the production and application of dsFvs against HBsAg.

  12. A new type of pseudothrombocytopenia: EDTA-mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody.

    Science.gov (United States)

    Christopoulos, C G; Machin, S J

    1994-07-01

    In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA-anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti-platelet antibodies.

  13. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Jun [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Xiang, Jun-Jian, E-mail: txjj@jnu.edu.cn [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China); Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Li, Dan [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Deng, Ning; Wang, Hong; Gong, Yi-Ping [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China)

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  14. Application of {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroomi [Kyoto Prefectural Univ. of Medicine (Japan)

    1999-06-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with {sup 99m}Tc, preventing depression of the antigen-binding activity. {sup 99m}Tc-labeled monoclonal antibody A7, {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7, {sup 99m}Tc-labeled normal mouse IgG and {sup 99m}Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  15. Digoxin-specific antibody fragments and a calcium antagonist for reversal of digoxin-induced mesenteric vasoconstriction.

    Science.gov (United States)

    Hess, T; Scholtysik, G; Salzmann, R; Riesen, W

    1983-10-01

    The effect of digoxin-specific antibody fragments on glycoside-induced mesenteric vasoconstriction were investigated. Digoxin caused a sustained contraction of strips of isolated feline mesenteric artery lasting for several hours, while in anaesthetized cats it produced a significant decrease in blood flow and increase in resistance in the mesenteric artery. In-vitro, digoxin's contractile effect was inhibited by 'prophylactic' addition of antibody to the organ bath, but the clinical use for prophylaxis is not a practical proposition. When the antibodies were added with the contraction of the arterial strip in response to digoxin already established, the tone of the preparation decreased significantly over 3 h, but the effect of the glycoside was not fully reversible. In-vivo, control animals not treated with antibodies developed arrhythmias, mesenteric blood flow fell by more than 50% and resistance increased by more than 80% relative to the initial values. These animals died of ventricular fibrillation before the end of the experiment. Animals treated with digoxin-specific antibody fragments after receiving digoxin injections showed no further decrease in mesenteric blood flow and 90 min after the last dose of digoxin, the flow was recovering and mesenteric resistance decreasing. Furthermore, all the animals that had received antibodies remained in sinus rhythm to the end of the experiment. In view of the latent period to onset of action of the antibodies, valuable time may be lost in impaired mesenteric blood flow. To bridge the gap or, indeed, as primary treatment, calcium antagonists merit consideration; in our experiments mesenteric vasoconstriction was abolished within a few minutes by application of the dihydropyridine calcium antagonist 4-(2,1,3-benzo-oxadiazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic aid, diethyl ester (PY 108-068).

  16. PREPARATION OF IMMUNOGEN AND PURIFICA¬TION OF HIGH AFFINITY AND SPECIFICITY FAB FRAGMENT OF ANTI-DIGOXIN POLYCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    M. Pour-Amir

    2000-01-01

    Full Text Available In this study we produced and purified a high titer of specific and high affin¬ity Fab fragments of anti-digoxin antibody. Immunization of rabbits with a conju¬gate of the cardiac glycoside digoxin, coupled by a periodate oxidation method to the amino group of lysine in bovine serum albumin resulted in the production of this type of high titer digoxin-specific antibodies with exceptionally high affinity (109 L/mol and specificity in immune response. Increase in titer was found in steps of purification ending up with the highest titer for Fab fragment to be at 1.75 ug of purified Fab (for 50% binding of I25I-digoxin. High specificity for antigenic determinants of the steroid nucleus of digoxin was observed such that much less cross-reaction with digoxin (2.3% and no cross-reaction with ouabaine, estradiol, Cortisol, progesterone and testosterone were detected.

  17. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

    DEFF Research Database (Denmark)

    Encinas, P.; Gomez-Casado, E.; Grandes, Fregeneda

    2011-01-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using...... infections. The viral frgs approach might also be used with other fish species and/or viruses....

  18. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Chatelier's principle.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc., La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect.

  19. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  20. Efficacy of anti-CD20 chi- meric Fab′fragment on pro- liferation of B lymphoma cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HI47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab¢ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab¢ fragment was expressed in E. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab¢ fragment inhibited binding of HI47 to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab¢ fragment inhibited the proliferation of Daudi cells, IC50 = 69 mg/mL. Affinity of chimeric anti-CD20 Fab¢ fragment was determined, Ka was about 8.9′108 (mol/L)-1.

  1. Production and radioimmunoimaging of novel fully human phage display recombinant antibodies and growth inhibition of lung adenocarcinoma cell line overexpressing Prx I.

    Science.gov (United States)

    Luo, Yi; Pang, Hua; Li, Shujie; Cao, Hui; Peng, Zhiping; Fan, Chunbo; Li, Shaolin

    2009-07-01

    The Peroxiredoxin I (Prx I) is a member of the Peroxiredoxin family, which is overexpressed in many diverse tumor types and is an anti-apoptosis protein for tumor cell proliferation and survival. Therapeutic strategies targeting the Prx I may therefore be effective broad-spectrum anticancer agents. We constructed a phage display single-chain variable fragment (scFv) antibody library and sieve out the fully human, lung adenocarcinoma-sepcific monoclonal antibodies. The selection on Prx I was performed using above-mentioned lung adenocarcinoma-sepcific monoclonal antibodies with high affinity to Prx I overexpressing lung adenocarcinoma cells. The candidate scFv sequences, based on enzyme-linked immunosorbent assay (ELISA) screening data, were chosen for soluble expression, and a 30 kDa band was observed on polyacrylamide gel electrophoresis as predicted. The purified antibodies were characterized by immunoblotting and showed high specificity to Prx I-overexpressing lung adenocarcinoma cells A549. Radioimmunoimaging was taken to evaluate specificity and distribution of antibodies in vivo. The radiolocalization index (RI) of tumor/serum and tumor/muscle gradually increased, reaching its peak (4.06 +/- 0.13 and 5.17 +/- 0.97, respectively) at 48 h postadministration. Single photon emission computed tomography (SPECT) imaging showed the radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. The internalized scFv resulted in antibody-mediated cell apoptosis and downregulation of Prx I expression. These results demonstrate that the scFv possesses strong antitumor activity on lung adenocarcinoma and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on Prx I for growth and survival.

  2. Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

    Institute of Scientific and Technical Information of China (English)

    LU Min; GONG Xing-guo; YU Hong; LI Jian-yong

    2005-01-01

    Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-I gene, the variable regions of heavy chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl were connected to single chain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP)and introduced into E. coli JMi09. The fusion protein induced by IPTG (Isopropylthiogalactoside) was about 57000 on a 10%SDS-PAGE gel (10% Sds Polyacrylamide Gel Electrophoresis), and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.

  3. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity.

    Science.gov (United States)

    Bai, Xuelian; Kim, Jihye; Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo

    2015-01-01

    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications.

  4. Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

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

    Science.gov (United States)

    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.

  6. Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.

    Science.gov (United States)

    Suharni; Nomura, Yayoi; Arakawa, Takatoshi; Hino, Tomoya; Abe, Hitomi; Nakada-Nakura, Yoshiko; Sato, Yumi; Iwanari, Hiroko; Shiroishi, Mitsunori; Asada, Hidetsugu; Shimamura, Tatsuro; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So; Nomura, Norimichi

    2014-12-01

    The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

  7. Human IgG1 Cγ1 Domain Is Crucial for the Bioactivity of the Engineered Anti-CD20 Antibodies

    Institute of Scientific and Technical Information of China (English)

    Shusheng Geng; Jiannan Feng; Yan Li; Xianjiang Kang; Yingxun Sun; Xin Gu; Ying Huang; Hong Chang; Beifen Shen

    2007-01-01

    In this study, we discussed the necessity of human IgG1 Cγ1 domain for recombinant antibody using computeraided homology modeling method and experimental studies. The heavy (VH) and light (VL) chain variable regions of 1-28, a murine IgM-type anti-CD20 mAb, were ligated by linker peptide (Gly4Ser)3 to form the single-chain Fv fragment (scFv). Then, the engineered antibody (LH1-3) was generated by fusing scFv with the entire IgG1 heavy constant regions. The 3-D structure of LH1-3 was modeled using computer-aided homology modeling method and the binding activity of LH1-3 was evaluated theoretically. Compared to the 3-D structure of the Fv fragment of the parent antibody, the conformation of the active pocket of LH1-3 was remained because of the rigid support of Cγ1.Further experimental results of flow cytometry showed that the engineered anti-CD20 antibody possessed specifically binding activity to CD20-expressing target cells. The anti-CD20 antibody fragments could also mediate complement-dependent cytotoxicity (CDC) of human B-lymphoid cell lines. Our study highlights some interests and advantages of a methodology based on the homology modeling and analysis of molecular structural properties.

  8. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    Science.gov (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  9. Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

    Directory of Open Access Journals (Sweden)

    Macek Jeanette

    2009-09-01

    Full Text Available Abstract Background Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Results Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability. Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. Conclusion The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

  10. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik

    2009-01-01

    was successfully developed for the detection of scFvs binding to different alpha(s1)-casein fragments inside a cheese matrix by CLSM. To our knowledge, this is the first demonstrated immunofluorescent labeling method for in situ analysis of proteolysis phenomena in the cheese matrix. Additionally, this technique......A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward...

  11. Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding.

    Directory of Open Access Journals (Sweden)

    Alfred Lammens

    Full Text Available The tumor necrosis factor-like weak inducer of apoptosis (TWEAK is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.

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

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

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

  13. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    Science.gov (United States)

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  14. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    Science.gov (United States)

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  15. Dynamic force spectroscopy of parallel individual mucin1-antibody bonds

    Energy Technology Data Exchange (ETDEWEB)

    Sulchek, T A; Friddle, R W; Langry, K; Lau, E; Albrecht, H; Ratto, T; DeNardo, S; Colvin, M E; Noy, A

    2005-05-02

    We used atomic force microscopy (AFM) to measure the binding forces between Mucin1 (MUC1) peptide and a single chain antibody fragment (scFv) selected from a scFv library screened against MUC1. This binding interaction is central to the design of the molecules for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from non-specific interactions by tethering the antibody and MUC1 molecules to the AFM tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the bond rupture events corresponding to different number of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for mono-, bi-, and tri-valent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for mono and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.

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

    Science.gov (United States)

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

    2016-10-01

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

  17. Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

    Science.gov (United States)

    Chaisemartin, L; Chinestra, P; Favre, G; Blonski, C; Faye, J C

    2009-05-20

    The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

  18. Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences.

    Science.gov (United States)

    Hochleitner, K; Thomale, J; Nikitin AYu; Rajewsky, M F

    1991-08-25

    We have established a sensitive, monoclonal antibody (Mab)-based procedure permitting the selective enrichment of sequences containing the miscoding alkylation product O6-ethylguanine (O6-EtGua) from mammalian DNA. H5 rat hepatoma cells were reacted with the N-nitroso carcinogen N-ethyl-N-nitrosourea in vitro, to give overall levels of greater than or equal to 25 O6-EtGua residues per diploid genome (corresponding to O6-EtGua/guanine molar ratios of greater than or equal to 10(-8). For analysis, enzymatically restricted DNA from these cells is incubated with an antibody specific for O6-ethyl-2'-deoxyguanosine, the resulting Mab-DNA complexes are separated from (O6-EtGua)-free fragments by filtration through a nitrocellulose (NC) membrane, and the DNA is recovered from the filter-bound complexes quantitatively. The efficiency of Mab binding to DNA fragments containing O6-EtGua is constant over a range of O6-EtGua/guanine molar ratios between 10(-5) and 10(-8). (O6-EtGua)-containing restriction fragments encompassing known gene sequences (e.g., the immunoglobulin E heavy chain gene of H5 rat hepatoma cells used as a model in this study) are subsequently amplified by PCR and quantified by slot-blot hybridisation. The content and distribution of a specific carcinogen-DNA adduct in defined sequences of genomic DNA can thus be analyzed as well as the kinetics of intragenomic (toposelective) repair of any DNA lesion for which a suitable Mab is available.

  19. Selection of Human Antibody Fragments Which Bind Novel Breast Tumor Antigens

    Science.gov (United States)

    1998-09-01

    6157-6162, 1998. 32-38 Appendix 2: Adams et al. Brit. J. Cancer 77: 1405-1412, 1998. 39-47 Appendix 3: Becerril et al. Biochem. Biophys. Res. Comm...James D. Marks M.D., Ph.D. Appendix 3 page (48) Towards selection of internalizing antibodies from phage libraries Baltazar Becerril , Marie-Alix Poul and

  20. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    Science.gov (United States)

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  1. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    Science.gov (United States)

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods.

  2. Transfer of engineered biophysical properties between different antibody formats and expression systems.

    Science.gov (United States)

    Schaefer, Jonas V; Plückthun, Andreas

    2012-10-01

    Recombinant antibodies and their derivatives are receiving ever increasing attention for many applications. Nevertheless, they differ widely in biophysical properties, from stable monomers to metastable aggregation-prone mixtures of oligomers. Previous work from our laboratory presented the combination of structure-based analysis with family consensus alignments as being able to improve the properties of immunoglobulin variable domains. We had identified a series of mutations in the variable domains that greatly influenced both the stability and the expression level of single-chain Fv (scFv) fragments produced in the periplasm of Escherichia coli. We now investigated whether these effects are transferable to Fab fragments and immunoglobulin G (IgG) produced in bacteria, Pichia pastoris, and mammalian cells. Taken together, our data indicate that engineered mutations can increase functional expression levels only for periplasmic expression in prokaryotes. In contrast, stability against thermal and denaturant-induced unfolding is improved by the same mutations in all formats tested, including scFv, Fab and IgG, independent of the expression system. The mutations in V(H) also influenced the structural homogeneity of full-length IgG, and the reducibility of the distant C(H)1-C(L) inter-chain disulfide bond. These results confirm the potential of structure-based protein engineering in the context of full-length IgGs and the transferability of stability improvements discovered with smaller antibody fragments.

  3. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    Science.gov (United States)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  4. Targeted elimination of activated hepatic stellate cells by an anti-epidermal growth factor-receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL)

    NARCIS (Netherlands)

    Arabpour, Mohammad; Poelstra, Klaas; Helfrich, Wijnand; Bremer, Edwin; Haisma, Hidde J.

    2014-01-01

    BackgroundProgressive liver fibrosis is the result of chronic liver injury and is characterized by the excessive accumulation of extracellular matrix that may result in liver failure. Activated hepatic stellate cells are known to play a central role in this process and their elimination is a crucial

  5. Novel impedimetric immunosensor for the detection and quantitation of Adenovirus using reduced antibody fragments immobilized onto a conducting copolymer surface.

    Science.gov (United States)

    Caygill, Rebecca L; Hodges, Christopher S; Holmes, Joanne L; Higson, Séamus P J; Blair, G Eric; Millner, Paul A

    2012-02-15

    The number of Adenovirus (Ad) infections detected in immunocompromised people has increased due to the number of patients receiving transplants, as well as the HIV pandemic. Ads cause life-threatening diseases specific to the infected organs of immunocompromised hosts, with discontinuation of immunosuppressive agents necessary to prevent morbidity. The methodology in this paper has been employed to develop a novel impedimetric based assay platform to detect and quantify human Ads, which is comparable in performance to current methods, such as ELISA and PCR, but is also less expensive and faster. Novel immunosensors have been fabricated using polyclonal antibodies raised against a human Ad (Ad5) capsid protein, which were selectively cleaved into antibody fragments by 2-mercaptoethylamine. The fragments were immobilized onto a functionalized conducting copolymer matrix comprising polyaniline and 2-aminobenzylamine. Fully fabricated sensors were incubated with two immunologically distinct serotypes of Ad, Ad5 and Ad3, with between 10 and 10(12)virus particles/mL prior to sensor interrogation. Electrochemical impedance spectroscopy was used to measure the charge transfer resistance of the sensors over a range of frequencies from 25 kHz to 0.1 Hz. Our data demonstrate that the immunosensors specifically detect, and differentiate between, closely related human Ad serotypes with a limit of detection of 10(3)virus particles/mL. In addition, atomic force microscopy was applied to study the sensor surface nanostructure. Future work looks to test virus containing clinical samples but this could be a viable and valuable alternative for point-of-care virus detection and quantification.

  6. Uptake of /sup 99m/Tc labelled (Fab')/sub 2/ fragments of monoclonal antibody 225. 28S by a benign ocular naevus

    Energy Technology Data Exchange (ETDEWEB)

    Bomanji, J.; Granowska, M.; Britton, K.E.; Hungerford, J.L.

    1988-06-01

    Malignant melanoma is one of the most common primary intraocular neoplasms. Recently, /sup 99m/Tc radiolabelled (Fab')/sub 2/ fragments of monoclonal antibody 225.28S raised against cutaneous melanomas have been used for imaging uveal melanomas. We report here a case where uptake of radiolabelled antibody was observed in a choroidal melanoma of the right eye and a benign choroidal naevus of the left.

  7. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  8. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    Science.gov (United States)

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  11. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    Science.gov (United States)

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression.

  12. High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.

    Science.gov (United States)

    Papalia, Giuseppe A; Baer, Mark; Luehrsen, Kenneth; Nordin, Helena; Flynn, Peter; Myszka, David G

    2006-12-01

    A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

  13. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Xiong Hairong [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Medicine, Wuhan (China); Lee, Robert J. [Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH (United States); Haura, Eric B. [Thoracic Oncology and Experimental Therapeutics Programs, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Edwards, John G. [Apeliotus Technologies, Inc., Atlanta, GA (United States); Dynan, William S. [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Li Shuyi, E-mail: sli@georgiahealth.edu [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Apeliotus Technologies, Inc., Atlanta, GA (United States)

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

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

    Institute of Scientific and Technical Information of China (English)

    王维刚[1; 张胜华[2; 李毅[3; 徐琳娜[4; 周京华[5; 甄永苏[6

    2000-01-01

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

  15. COMPARISON OF FOUR METHODS TO GENERATE IMMUNOREACTIVE FRAGMENTS OF A MURINE MONOCLONAL ANTIBODY OC859 AGAINST HUMAN OVARIAN EPITHELIAL CANCER ANTIGEN

    Institute of Scientific and Technical Information of China (English)

    邹颖; 卞美璐; 杨子义; 连利娟; 刘文淑; 许秀英

    1995-01-01

    In the present study,four different proteases (pepsin,papain,bromelain and ficin) were screened with a murine monoclonal antibody OC859,in order to verify whether different digestion procedures could improve yield and stability of the F(ab')2 or Fab fragments.The yields of F(ab')2 or Fab fragments from digestion with pepsin,papain,bromelain and ficin were respectively 20.3+/-2.0%,50.5%+/-5.0%,74.4+/-2.7% and 82.8+/-10.2% of the theoretical maximum.Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10+/-5%,36+/-5%,60+/-6% and 75+/-6% of the intact OC859 IgG.These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.

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

    Institute of Scientific and Technical Information of China (English)

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

    1997-01-01

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

  17. Production, purification, and characterization of scFv TNF ligand fusion proteins.

    Science.gov (United States)

    Fick, Andrea; Wyzgol, Agnes; Wajant, Harald

    2012-01-01

    Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins.

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

    NARCIS (Netherlands)

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

    2005-01-01

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

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

    NARCIS (Netherlands)

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

    2005-01-01

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

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

    Science.gov (United States)

    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.

  1. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  2. Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.

    Science.gov (United States)

    Massa, Sam; Xavier, Catarina; De Vos, Jens; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Devoogdt, Nick

    2014-05-21

    Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

  3. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

    Science.gov (United States)

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

    2013-01-01

    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  4. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    Science.gov (United States)

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-01

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  5. Co-expression of anti-rotavirus proteins (llama VHH antibody fragments in Lactobacillus: development and functionality of vectors containing two expression cassettes in tandem.

    Directory of Open Access Journals (Sweden)

    Gökçe Günaydın

    Full Text Available Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3 as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11, by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules.

  6. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    Science.gov (United States)

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  7. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  8. Development of Antibody-Coated Magnetite Nanoparticles for Biomarker Immobilization

    Directory of Open Access Journals (Sweden)

    Christian Chapa Gonzalez

    2014-01-01

    Full Text Available Magnetic nanoparticles (MNPs have great potential in biomedical applications because of their magnetic response offers the possibility to direct them to specific areas and target biological entities. Magnetic separation of biomolecules is one of the most important applications of MNPs because their versatility in detecting cancer biomarkers. However, the effectiveness of this method depends on many factors, including the type of functionalization onto MNPs. Therefore, in this study, magnetite nanoparticles have been developed in order to separate the 5′-nucleotidase enzyme (5eNT. The 5eNT is used as a bio-indicator for diagnosing diseases such as hepatic ischaemia, liver tumor, and hepatotoxic drugs damage. Magnetic nanoparticles were covered in a core/shell type with silica, aminosilane, and a double shell of silica-aminosilane. A ScFv (fragment antibody and anti-CD73 antibody were attached to the coated nanoparticles in order to separate the enzyme. The magnetic separation of this enzyme with fragment antibody was found to be 28% higher than anti-CD73 antibody and the enzyme adsorption was improved with the double shell due to the increased length of the polymeric chain. Magnetite nanoparticles with a double shell (silica-aminosilane were also found to be more sensitive than magnetite with a single shell in the detection of biomarkers.

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

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

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

  10. Complete regression of a guinea pig hepatocarcinoma by immunotherapy with "tumor-immune" RNA or antibody to fibrin fragment E.

    Science.gov (United States)

    Schlager, S I; Dray, S

    1976-01-01

    Two novel immunotherapeutic regimens were developed for a uniformly lethal, intradermally growing transplantable ascites variant (line 10) of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. In an apparently tumor-specific immunotherapy model, 32 guinea pigs were cured by the injection into the tumor area, five or seven days after tumor challenge, of syngeneic or xenogeneic RNA extracts obtained from lymphoid tissues of line 10-immune strain 2 guinea pigs or rhesus monkeys, as part of a total regimen which included syngeneic nonsensitive peritoneal exudate cells injected prior to, and tumor-specific antigen injected after, the RNA. In another immunotherapy model, not tumor-specific, 18 strain 2 guinea pigs were cured by the injection into the tumor area, 6 and 16 days after tumor challenge, of antibody specific for fibrin fragment E (FFE), an essential component in the formation of a fibrin matrix considered to be important in tumor development. When therapy was delayed to 12 days in the RNA test system, or to 16 days in the anti-FFE test system, complete abrogation of the tumors did not occur. The long-term survival of the 50 successfully treated animals and their immunity to further tumor challenge indicated that both immunotherapeutic procedures had systemic effects. To test this further, line 10 cells were injected intradermally simultaneously at two sites and only one site was treated. When the one tumor location was treated with anti-FFE, complete regression of the treated tumor and a 30% retardation in the development of the untreated tumor were observed. When this tumor location was treated with the RNA regimen, complete regression of the tumors occurred at both the treated and the untreated sites. Optimal conditions for both immunotherapeutic models and their combination have yet to be establshed. Nonetheless, both immunotherapeutic regimens were more effective than any other immunotherapy thus far reported for this tumor, including the use

  11. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments.

    Science.gov (United States)

    Encinas, P; Gomez-Casado, E; Fregeneda-Grandes; Olesen, N J; Lorenzen, N; Estepa, A; Coll, J M

    2011-03-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

  12. A fusogenic dengue virus-derived peptide enhances antitumor efficacy of an antibody-ribonuclease fusion protein targeting the EGF receptor.

    Science.gov (United States)

    Kiesgen, Stefan; Liebers, Nora; Cremer, Martin; Arnold, Ulrich; Weber, Tobias; Keller, Armin; Herold-Mende, Christel; Dyckhoff, Gerhard; Jäger, Dirk; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

    2014-10-01

    Due to its frequent overexpression in a variety of solid tumors the epidermal growth factor receptor (EGFR) is a well-established target for therapeutic interventions in epithelial cancers. In order to target EGFR in head and neck cancer, we have generated a ribonuclease (RNase) fusion protein comprising a humanized anti-EGFR antibody single-chain Fv fragment (scFv) and Ranpirnase, an RNase from Rana pipiens. Fusion of Ranpirnase to the N-terminus of the scFv via a flexible glycine-serine linker (G4S)3 resulted in very poor cytotoxicity of the fusion protein. As endosomal accumulation and lysosomal degradation have been reported to diminish the antitumor efficacy of ribonuclease or toxin-based immunoagents, we explored a fusion peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. This peptide was introduced as a linker between Ranpirnase and the scFv moiety. The modified immunoRNase exhibited exceptionally high cytotoxicity toward EGFR-expressing head and neck cell lines without affecting specificity. These results indicate that endosomal entrapment needs to be considered for Ranpirnase-based immunoagents and might be overcome by the use of tailored transduction domains from viral proteins.

  13. A novel assay for monitoring internalization of nanocarrier coupled antibodies

    Directory of Open Access Journals (Sweden)

    Pickering Edward M

    2006-10-01

    Full Text Available Abstract Background Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. Results The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv (F5 antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol-linked lipid (DSPE-PEG-NTA-Ni was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. Conclusion The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.

  14. Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

    Science.gov (United States)

    Okazaki, Fumiyoshi; Aoki, Jun-ichi; Tabuchi, Soichiro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2012-10-01

    We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

  15. Immunoscintigraphy in the detection of tuberculosis with radiolabelled antibody fragment against Mycobacterium bovis bacillus Calmette-Guerin. A preliminary study in a rabbit model

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J.D.; Park, C.Y.; Yoo, H.S.; Lee, J.T. (Yonsei Univ., Seoul (Korea, Republic of). Dept. of Diagnostic Radiology); Shin, K.H. (Yonsei Univ., Seoul (Korea, Republic of). Dept. of Orthopedic Surgery); Cho, S.N.; Shin, J.S. (Yonsei Univ., Seoul (Korea, Republic of). Dept. of Microbiology); Lee, M.G. (Yonsei Univ., Seoul (Korea, Republic of). Dept. of Dermatology); Yang, W.I. (Yonsei Univ., Seoul (Korea, Republic of). Dept. of Pathology); Awh, O.D. (Korea Atomic Energy Research Inst., Seoul (Korea, Republic of). Dept. of Reactor Isotopes)

    1992-12-01

    Immunoscintigraphy with radiolabelled monoclonal antibodies is widely used to detect solid tumours, but only a few trials have been carried out concerning the specific in vivo localization of an inflammatory process. The purpose of this study was to investigate the detectability of tuberculous foci utilizing this method with radiolabelled bacille Colmette-Guerin (BCG)-specific F(ab')[sub 2] in rabbits. All of the tuberculous lesions (n=8) were clearly visualized on serial scintigraphy for up to 48 h after injection of the antibody. Immunohistochemical and Ziel-Neelson staining of the tuberculous lesions confirmed the presence of the tuberculous antigens and bacilli. It failed to demonstrate any sustained retention of the BCG-specific antibody fragment in the control group with syphilitic orchitis (n=2). Therefore, the specific in vivo localization of tuberculosis is feasible by immunoscintigraphy. (orig.).

  16. Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity.

    Science.gov (United States)

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

    2016-07-01

    The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.

  17. Whole-body imaging of HER2/neu-overexpressing tumors using scFv-antibody conjugated quantum dots

    Science.gov (United States)

    Balalaeva, Irina V.; Zdobnova, Tatiana A.; Brilkina, Anna A.; Krutova, Irina M.; Stremovskiy, Oleg A.; Lebedenko, Elena N.; Vodeneev, Vladimir V.; Turchin, Ilya V.; Deyev, Sergey M.

    2010-02-01

    Semiconductor quantum dots (QDs) are widely used in different fields of bioscience and biotechnology due to their unique optical properties. QDs can be used as fluorescent markers for optical detection and monitoring of deeply located tumors in vivo after specific labeling achieved by conjugating of QDs with targeting molecules. In this work the possibilities of intravital tumor labeling with QDs and subsequent in vivo tumor imaging were estimated. The experiments were run on immunodeficient nu/nu mice bearing human breast carcinoma SKBR-3, overexpressing surface protein HER2/neu. We used quantum dots Qdot 705 ITK (Invitrogen, USA) linked to anti-HER2/neu 4D5 scFv antibody. Antibody scFv fragments as a targeting agent for directed delivery of fluorophores possess significant advantages over full-size antibodies due to their small size, lower cross-reactivity and immunogenicity. QDs were bound to 4D5 scFv by barnase-barstar system (bn-bst) analogous to the streptavidin-biotidin system. Whole-body images were obtained using diffuse fluorescence tomography (DFT) setup with low-frequency modulation and transilluminative configuration of scanning, created at the Institute of Applied Physics of RAS, Russia). DFT-results were confirmed ex vivo by confocal microscopy. We report the results of in vivo whole-body tumor imaging with QDs complexes as contrasting agents. Intravital images of QDs-labeled tumors were obtained using specific tumor cells targeting and fluorescence transilluminative imaging method, while "passive" QD-labeling failed to mark effectively the tumor.

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

    Science.gov (United States)

    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.

  19. Identification of MCAM/CD146 as the target antigen of a human monoclonal antibody that recognizes both epithelioid and sarcomatoid types of mesothelioma.

    Science.gov (United States)

    Bidlingmaier, Scott; He, Jiang; Wang, Yong; An, Feng; Feng, Jinjin; Barbone, Dario; Gao, Dongwei; Franc, Ben; Broaddus, V Courtney; Liu, Bin

    2009-02-15

    The prognosis for patients diagnosed with mesothelioma is generally poor, and currently available treatments are usually ineffective. Therapies that specifically target tumor cells hold much promise for the treatment of cancers that are resistant to current approaches. We have previously selected phage antibody display libraries on mesothelioma cell lines to identify a panel of internalizing human single chain (scFv) antibodies that target mesothelioma-associated, clinically represented cell surface antigens and further exploited the internalizing function of these scFvs to specifically deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells. Here, we report the identification of MCAM/MUC18/CD146 as the surface antigen bound by one of the mesothelioma-targeting scFvs using a novel cloning strategy based on yeast surface human proteome display. Immunohistochemical analysis of mesothelioma tissue microarrays confirmed that MCAM is widely expressed by both epithelioid and sarcomatous types of mesothelioma tumor cells in situ but not by normal mesothelial cells. In addition, quantum dot-labeled anti-MCAM scFv targets primary meosthelioma cells in tumor fragment spheroids cultured ex vivo. As the first step in evaluating the therapeutic potential of MCAM-targeting antibodies, we performed single-photon emission computed tomography studies using the anti-MCAM scFv and found that it recognizes mesothelioma organotypic xenografts in vivo. The combination of phage antibody library selection on tumor cells and rapid target antigen identification by screening the yeast surface-displayed human proteome could be a powerful method for mapping the targetable tumor cell surface epitope space.

  20. Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.

    Science.gov (United States)

    Park, Jun T; Bradbury, Lisa; Kragl, Frank J; Lukens, Dennis C; Valdes, James J

    2006-01-01

    A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

  1. Thermally induced degradation pathways of three different antibody-based drug development candidates.

    Science.gov (United States)

    Fincke, Anja; Winter, Jonas; Bunte, Thomas; Olbrich, Carsten

    2014-10-01

    Protein-based medicinal products are prone to undergo a variety of chemical and physical degradation pathways. One of the most important exogenous stress condition to consider during manufacturing, transport and storage processes is temperature, because antibody-based therapeutics are only stable in a limited temperature range. In this study, three different formats of antibody-based molecules (IgG1, a bispecific scFv and a fab fragment) were exposed to thermal stress conditions occurring during transport and storage. For evaluation, an analytical platform was developed for the detection and characterization of relevant degradation pathways of different antibody-based therapeutics. The effect of thermal stress conditions on the stability of the three antibody-based formats was therefore investigated using visual inspection, different spectroscopic measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electrophoresis, asymmetric flow field-flow fractionation (AF4) and surface plasmon resonance technology (SPR). In summary, thermal stress led to heterogeneous chemical and physical degradation pathways of all three antibody-based formats used. In addition, identical exogenous stress conditions resulted in different kinds and levels of aggregates and fragmentation products. This knowledge is fundamental for a systematic and successful stabilization of protein-based therapeutics by the use of formulation additives.

  2. {sup 68}Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours

    Energy Technology Data Exchange (ETDEWEB)

    Eder, Matthias; Eisenhut, Michael [German Cancer Research Center, Radiopharmaceutical Chemistry, Heidelberg (Germany); Knackmuss, Stefan; Gall, Fabrice Le; Reusch, Uwe; Little, Melvyn [Affimed Therapeutics AG, Heidelberg (Germany); Rybin, Vladimir [European Molecular Biology Laboratory, Heidelberg (Germany); Haberkorn, Uwe; Mier, Walter [University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany)

    2010-07-15

    Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope {sup 68}Ga. In order to combine the promising tumour targeting properties of antibodies with {sup 68}Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naive antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N{sup '}-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N{sup '}-diacetic acid (HBED-CC) to enable {sup 68}Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments. The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to {sup 68}Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation. The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the {sup 68}Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the

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

    Directory of Open Access Journals (Sweden)

    Christiane Y Ozaki

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    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.

  6. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Miles, Luke A. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Crespi, Gabriela A. N. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Wycherley, Kaye [WEHI Biotechnology Centre, La Trobe R& D Park, Bundoora, Victoria 3086 (Australia); Ascher, David B. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Barnham, Kevin J.; Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Beyreuther, Konrad [ZMBH, University of Heidelberg, Heidelberg (Germany); Masters, Colin L. [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); McKinstry, William J., E-mail: wjmckinstry@hotmail.com [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Medicine (St Vincent’s Hospital), The University of Melbourne, 41 Victoria Parade, Fitzroy 3065 (Australia)

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  7. Antibody fragments directed against different portions of the human neural cell adhesion molecule L1 act as inhibitors or activators of L1 function.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs, named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig domains 1-4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn domains 1-3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H(2O(2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1-4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1-3 trigger L1 functions of cultured neuroblastoma cells.

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

    Science.gov (United States)

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

    2012-02-24

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

  9. The effects of N-terminal insertion into VSV-G of an scFv peptide

    Directory of Open Access Journals (Sweden)

    Piechaczyk Marc

    2006-09-01

    Full Text Available Abstract Recombinant retroviruses, including lentiviruses, are the most widely used vectors for both in vitro and in vivo stable gene transfer. However, the inability to selectively deliver transgenes into cells of interest limits the use of this technology. Due to its wide tropism, stability and ability to pseudotype a range of viral vectors, vesicular stomatitis virus G protein (VSV-G is the most commonly used pseudotyping protein. Here, we attempted to engineer this protein for targeting purposes. Chimaeric VSV-G proteins were constructed by linking a cell-directing single-chain antibody (scFv to its N-terminal. We show that the chimaeric VSV-G molecules can integrate into retroviral and lentiviral particles. HIV-1 particles pseudotyped with VSV-G linked to an scFv against human Major Histocompatibility Complex class I (MHC-I bind strongly and specifically to human cells. Also, this novel molecule preferentially drives lentiviral transduction of human cells, although the titre is considerably lower that viruses pseudotyped with VSV-G. This is likely due to the inefficient fusion activity of the modified protein. To our knowledge, this is the first report where VSV-G was successfully engineered to include a large (253 amino acids exogenous peptide and where attempts were made to change the infection profile of VSV-G pseudotyped vectors.

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

    Science.gov (United States)

    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.

  11. 引入易错PCR和DNA-shuffling技术构建单链抗体库%The introduction of error-prone PCR and DNA-shuffling technology to build mono-chain antibody library

    Institute of Scientific and Technical Information of China (English)

    杨浩; 杨青平; 查成喜; 韩跃武

    2012-01-01

    目的 引入易错PCR和DNA-shuffling技术构建高库容量的单链抗体库.方法 收集不同年龄、性别、健康状态的人的静脉血各5 mL,提取单个核细胞总RNA,反转录为cDNA,PCR扩增VH和VL基因,应用易错PCR对VH和VL基因进行突变,同时用DNA-shuffling技术对全长单链抗体基因进行体外改组,获得的分子与T载体连接,转化E.coli DH5α,选取阳性克隆,经菌落PCR后酶切鉴定抗体库多样性,计算分析单链抗体库容量.结果 成功构建了库容量为4.37×1013的单链抗体库,经酶切鉴定,初步判定抗体库多样性良好.结论 引入易错PCR和DNA-shuffling技术,成功构建了单链抗体库,为后续筛选高亲和力抗体奠定了实验基础.%Objective To construct a mono-storage capacity single-chain antibody (scFv) library using error-prone PCR and DNA-shuffling technologies. Methods Peripheral blood was collected from different age, gender and healthy people. Total RNA was extracted from human peripheral blood mononuclear cells and reversely transcribed to cDNA by RT-PCR. VH and VL genes were amplified by PCR and mutated by error-prone PCR. The full-length ScFv fragments were recombinated with the DNA-shuffling technology in vitro, and then connected with T vector and transformed into E. Coli DH5α. Antibody library diversity was identified by restriction enzyme digestion after colony PCR, and then the capacity of scFv was calculated. Results The 4. 37 x 1013 capacity and better diversity ScFv library was constructed by error-prone PCR and DNA-shuffling techniques. Conclusions The scFv library with high-storage capacity and genetic diversity has been constructed, which laid a foundation of selection high-affinity antibodies.

  12. An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

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    Urszula Jarocka

    2014-08-01

    Full Text Available This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii immobilization of antibody-binding fragments (Fab’ of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab’ fragments and hemagglutinin (HA variants have been explored with electrochemical impedance spectroscopy (EIS in the presence of [Fe(CN6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

  13. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

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    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G. (Purdue); (WU-MED); (Crucell)

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  14. Positron Emission Tomographic Imaging of Iodine 124 Anti–Prostate Stem Cell Antigen–Engineered Antibody Fragments in LAPC-9 Tumor–Bearing Severe Combined Immunodeficiency Mice

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    Jeffrey V. Leyton

    2013-05-01

    Full Text Available The humanized antibody (hu1G8 has been shown to localize to prostate stem cell antigen (PSCA and image PSCA-positive xenografts. We previously constructed hu1G8 anti-PSCA antibody fragments and tested them for tumor targeting and the ability to image prostate cancer at early and late time points postinjection by positron emission tomography (PET. We now then compare the PET imaging and the radioactivity accumulation properties in prostate cancer tumors and nontarget tissues to determine the superior 124I-labeled hu1G8 antibody format. 124I-labeled diabody, minibody, scFv-Fc, scFv-Fc double mutant (DM, and parental IgG were administered into severe combined immunodeficiency (SCID mice bearing LAPC-9 xenografts and followed by whole-body PET imaging of mice at preselected time points. Regions of interest were manually drawn around tumor and nontarget tissues and evaluated for radioactivity accumulation. The 124I-hu1G8 IgG has its best time point for tumor high-contrast imaging at 168 hours postinjection. The 124I-hu1G8 minibody at 44 hours postinjection results in superior tumor high-contrast imaging compared to the other antibody formats. The 124I-hu1G8 minibody at 44 hours postinjection also has comparable percent tumor radioactivity compared to 124I-hu1G8 IgG at 168 hours postinjection. The 124I-hu1G8 minibody is the best engineered hu1G8 antibody format for imaging prostate cancer.

  15. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

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    Kui Zhu

    2014-04-01

    Full Text Available Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv, as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs, magnetic nanoparticles (MNPs, quantum dots (QDs and carbon nanomaterials (graphene and carbon nanotube, for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC, which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT, will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  16. Recent developments in antibody-based assays for the detection of bacterial toxins.

    Science.gov (United States)

    Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin

    2014-04-11

    Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  17. Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells

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    Banks Lawrence

    2011-01-01

    Full Text Available Abstract Background "High risk" Human Papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins. Methods In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™technology and Surface Plasmon Resonance. Results The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. Conclusions Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific sc

  18. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

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    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  19. Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-alpha toxin antibody fragment and alpha toxin.

    Science.gov (United States)

    Oganesyan, Vaheh; Barnes, Arnita; Tkaczyk, Christine; Ferguson, Andrew; Wu, Herren; Dall'Acqua, William F

    2013-03-01

    Staphylococcus aureus alpha toxin (AT) has been crystallized in complex with the Fab fragment of a human antibody (MEDI4893). This constitutes the first reported crystals of AT bound to an antibody. The monoclinic crystals belonged to space group P2₁, with unit-cell parameters a=85.52, b=148.50, c=93.82 Å, β=99.82°. The diffraction of the crystals extended to 2.56 Å resolution. The asymmetric unit contained two MEDI4893 Fab-AT complexes. This corresponds to a crystal volume per protein weight (VM) of 2.3 Å3 Da(-1) and a solvent content of 47%. The three-dimensional structure of this complex will contribute to an understanding of the molecular basis of the interaction of MEDI4893 with AT. It will also shed light on the mechanism of action of this antibody, the current evaluation of which in the field of S. aureus-mediated diseases makes it a particularly interesting case study. Finally, this study will provide the three-dimensional structure of AT in a monomeric state for the first time.

  20. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment.

    Science.gov (United States)

    McKinstry, William J; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T; Martin, Thomas J; Parker, Michael W

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

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

    Science.gov (United States)

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

    2015-01-01

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

  2. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage.

    Science.gov (United States)

    Cai, Binggang; Wang, Maorong; Zhu, Xuhui; Xu, Jing; Zheng, Wenkai; Zhang, Yiqing; Zheng, Feng; Feng, Zhenqing; Zhu, Jin

    2015-01-01

    Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  3. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4 Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage

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    Binggang Cai

    2015-10-01

    Full Text Available Lipopolysaccharides (LPS can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4 signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2 complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  4. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    Science.gov (United States)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  5. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

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    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

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

    Science.gov (United States)

    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.

  7. Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

    Institute of Scientific and Technical Information of China (English)

    WEI Jing-yan; ZHANG Han-qi; JIN Qin-han; LI Wei; LUO Gui-min; LI Shan-yu; MU Ying; ZHU Xue-jun; LIU Lei; GAO Li-zeng; SONG Da-qian; SUN Zhi-wei; YAN Gang-lin

    2005-01-01

    The single chain variable fragments of antibodies(scFvs) against eTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5,A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions (PCR)and cloned into expression vector pPELB and expressed as a soluble protein in E. coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant (KA)values range from 1.2×104 to 1.7×105 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.

  8. Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study

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    Grinstead, Jeffrey S.; Schuman, Jason T.; Campbell, Ann P.

    2003-11-13

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant ''reverse templates'' of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The 13CR T1 values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast

  9. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    Science.gov (United States)

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23.

  10. Construction of an anti-IL-1β scfv and TNFRI fusion protein and its therapeutic effect on RA mice model.

    Science.gov (United States)

    Kan, Fangming; Ren, Guiping; Guo, Mo; Qi, Jianying; Zhang, Yu; Han, Yang; Zhang, Yakun; Li, Deshan

    2014-01-01

    IL-1β and TNF-α play key roles in the inflammatory response. Their abnormal expression may cause the occurrence of various diseases, such as RA. Recently, medicines of target TNF-α and IL-1β have become popular in the clinical practice. Although these biological agents can get mostly good results, they are not effective in all patients. The reason for this result may be that these biological agents could not fully inhibit a variety of inflammatory cytokines in the inflammatory response. In the present study, a fusion protein gene which encoded human interleukin-1β scfv and soluble TNF receptor I (sTNFRI) was cloned. A number of in vitro assays demonstrated that anti-IL-1β scfv/TNFRI simultaneously bound to both targets. The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells, indicating that the fusion protein has the ability to neutralize both hTNF-α and hIL-1β. In this study, we established the chicken type II collagen-induced rheumatoid arthritis model in Kunming mice, and evaluated the pharmacological effect of the fusion protein in vivo. Model mice were randomly divided into 8 groups (n=8): CIA model control group, DEX treatment group (1 mg/kg), intraperitoneal treatment group (highdose: 5 mg/kg; medium-dose: 2 mg/kg; low-dose: 0.8 mg/kg), subcutaneous treatment group (high-dose: 5 mg/kg; medium- dose: 2 mg/kg; low-dose: 0.8 mg/kg), and healthy mice as control. The control group received the same volume of saline. The mice were administrated once every 2 days. Arthritis index, anti-CII antibody titers, cytokine levels, histopathological changes were examined. The results showed that anti-IL-1β scfv/TNFRI fusion protein could reduce the degree of joint swelling, inflammatory cell infiltration, synovial cell proliferation and the level of CII antibody in the sera. The Real-time PCR analysis showed that anti-IL-1β scfv/TNFRI had the ability to

  11. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

    Science.gov (United States)

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-01-01

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases. PMID:27786270

  12. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases.

    Science.gov (United States)

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-10-27

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

  13. Preparation and activity of conjugate of monoclonal antibody HAbl8 against hepatoma F(ab')2 fragment and staphylococcal enterotoxin A

    Institute of Scientific and Technical Information of China (English)

    Lian Jun Yang; Yan Fang Sui; Zhi Nan Chen

    2001-01-01

    AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION

  14. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    Science.gov (United States)

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

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

    Energy Technology Data Exchange (ETDEWEB)

    Surinder Batra, Ph D

    2006-02-27

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

  16. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); Li, Lin [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Shibo, E-mail: sjiang@nybloodcenter.org [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China)

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  17. Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance.

    Science.gov (United States)

    Thillaivinayagalingam, Pranavan; Newcombe, Anthony R; O'Donovan, Kieran; Francis, Richard; Keshavarz-Moore, Eli

    2007-12-01

    Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

  18. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    Directory of Open Access Journals (Sweden)

    Soraya S Pereira

    Full Text Available In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅ of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB, surface plasmon resonance (SPR device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with

  19. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    Science.gov (United States)

    Pereira, Soraya S; Moreira-Dill, Leandro S; Morais, Michelle S S; Prado, Nidiane D R; Barros, Marcos L; Koishi, Andrea C; Mazarrotto, Giovanny A C A; Gonçalves, Giselle M; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Pereira da Silva, Luiz H; Duarte dos Santos, Claudia N; Fernandes, Carla F C; Stabeli, Rodrigo G

    2014-01-01

    In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus

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

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

  1. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display

    Directory of Open Access Journals (Sweden)

    Meyer Torsten

    2012-06-01

    Full Text Available Abstract Background Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv against these five antigens were selected using antibody phage display and characterised. Conclusion In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

  2. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Sixiang [University of Wisconsin, Materials Science Program, Madison, WI (United States); Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Liu, Bai; Wong, Hing C. [Altor BioScience, Miramar, FL (United States); Cai, Weibo [University of Wisconsin, Materials Science Program, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States); University of Wisconsin, Departments of Radiology and Medical Physics, Madison, WI (United States)

    2015-07-15

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and {sup 64}Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of {sup 64}Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of {sup 64}Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. {sup 64}Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  3. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    Science.gov (United States)

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  4. Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

    Science.gov (United States)

    Tang, Dan-Yang; Yu, Yang; Zhao, Xuan-Jun; Schachner, Melitta; Zhao, Wei-Jiang

    2015-01-15

    L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases.

  5. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    Science.gov (United States)

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  6. Combining intracellular antibodies to restore function of mutated p53 in cancer.

    Science.gov (United States)

    Chan, Grace; Jordaan, Gwen; Nishimura, Robert N; Weisbart, Richard H

    2016-01-01

    TP53 is a tumor suppressor gene that is mutated in 50% of cancers, and its function is tightly regulated by the E3 ligase, Mdm2. Both p53 and Mdm2 are localized in the cell nucleus, a site that is impervious to therapeutic regulation by most antibodies. We identified a cell-penetrating lupus monoclonal anti-DNA antibody, mAb 3E10, that targets the nucleus, and we engineered mAb 3E10 to function as an intranuclear transport system to deliver therapeutic antibodies into the nucleus as bispecific single chain Fv (scFv) fragments. Bispecific scFvs composed of 3E10 include PAb421 (3E10-PAb421) that binds p53 and restores the function of mutated p53, and 3G5 (3E10-3G5) that binds Mdm2 and prevents destruction of p53 by Mdm2. We documented the therapeutic efficacy of these bispecific scFvs separately in previous studies. In this study, we show that combination therapy with 3E10-PAb421 and 3E10-3G5 augments growth inhibition of cells with p53 mutations compared to the effect of either antibody alone. By contrast, no enhanced response was observed in cells with wild-type p53 or in cells homozygous null for p53.

  7. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  8. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies.

    Science.gov (United States)

    Villani, Maria Elena; Morgun, Bogdan; Brunetti, Patrizia; Marusic, Carla; Lombardi, Raffaele; Pisoni, Ivan; Bacci, Camilla; Desiderio, Angiola; Benvenuto, Eugenio; Donini, Marcello

    2009-01-01

    The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

  9. 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).

    Science.gov (United States)

    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.

  10. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  11. Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment.

    Science.gov (United States)

    Isidro, Inês A; Portela, Rui M; Clemente, João J; Cunha, António E; Oliveira, Rui

    2016-09-01

    Despite the growing importance of the Pichia pastoris expression system as industrial workhorse, the literature is almost absent in systematic studies on how culture medium composition affects central carbon fluxes and heterologous protein expression. In this study we investigate how 26 variations of the BSM+PTM1 medium impact central carbon fluxes and protein expression in a P. pastoris X-33 strain expressing a single-chain antibody fragment. To achieve this goal, we adopted a hybrid metabolic flux analysis (MFA) methodology, which is a modification of standard MFA to predict the rate of synthesis of recombinant proteins. Hybrid MFA combines the traditional parametric estimation of central carbon fluxes with non-parametric statistical modeling of product-related quantitative or qualitative measurements as a function of central carbon fluxes. It was observed that protein yield variability was 53.6 % (relative standard deviation) among the different experiments. Protein yield is much more sensitive to medium composition than biomass growth, which is mainly determined by the carbon source availability and main salts. Hybrid MFA was able to describe accurately the protein yield with normalized RMSE of 6.3 % over 5 independent experiments. The metabolic state that promotes high protein yields is characterized by high overall metabolic rates through main central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy generating pathways.

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

    Institute of Scientific and Technical Information of China (English)

    肖静; 刘章锁; 王沛; 黄留玉; 宋宏彬; 赵明辉

    2010-01-01

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

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

    Science.gov (United States)

    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.

  14. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  15. Screening scFv Specific to Vcam-1 by Phage Display Library and Its Activity Evaluation%噬菌体展示库筛选构建血管细胞黏附分子-1单链抗体及其效价检测

    Institute of Scientific and Technical Information of China (English)

    刘纯宝; 宋夷龄; 张永学

    2015-01-01

    目的:从噬菌体重组抗体库中筛选获得靶向血管细胞黏附分子‐1(Vcam‐1)的单链抗体,纯化浓缩后进行亲和性鉴定,并与单克隆抗体效价进行比较。方法扩增Vcam‐1基因克隆质粒并转入真核细胞中表达获得Vcam‐1抗原蛋白,纯化后包被免疫管,通过4轮压力逐渐增大的“吸附‐洗脱‐扩增”过程筛选获得阳性克隆。对阳性克隆进行ELISA检测,选取效价高的克隆送予测序并转入大肠埃希菌进行表达,认定高表达的样品为最终的阳性克隆。将该阳性克隆转染入感受态细胞表达单链抗体,纯化后经ELISA检测并评价其抗原亲和性。结果真核细胞表达的Vcam‐1抗原蛋白的分子量为85~90 kD。以Vcam‐1抗原蛋白为免疫原筛选4轮所得的阳性克隆进行单噬菌体ELISA检测,从检测结果中选取9个效价高的克隆经基因测序共获得3个序列,其中1个序列对应的克隆高表达。表达的单链抗体分子量约30 kD ,ELISA检测其对Vcam‐1抗原蛋白有较高亲和性,效价较单克隆抗体低。结论利用噬菌体展示技术获得了靶向Vcam‐1的单链抗体,为随后的诊断和治疗应用奠定了基础。%Objective To screen out single chain variable fragment antibody (scFv)specific to vascular cell adhesion mole‐cule 1(Vcam‐1)from phage recombinant antibody library ,and to evaluate its activity and compare its activity with full‐length monoclonal antibody.Methods Amplification of Vcam‐1 was performed by PCR and Vcam‐1 gene plasmid was transferred into eukaryotic cells to express Vcam‐1 antigen protein.Immune cuvette was coated with purified Vcam‐1 antigen ,and the positive clones were screened out by 4 rounds of “adhesion‐elution‐proliferation” process with gradually increasing pressure.The posi‐tive clones were tested by ELISA method and high titer clones were chosen for gene sequencing.Then the high‐titer clones

  16. Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab' fragment of monoclonal antibody directed against type IV collagenase

    Institute of Scientific and Technical Information of China (English)

    WANG; Fengqiang; SHANG; Boyang; ZHEN; Yongsu

    2004-01-01

    Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab′-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab′ fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab′-LDM conjugate was 65 kD with a 1:1 molecular ratio of Fab′ and LDM. The Fab′-LDM conjugate maintained most part of the immunoreactivity of Fab′ fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab′-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab′-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab′-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab′-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab′-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.

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

    Science.gov (United States)

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

    2014-02-01

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

  18. Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    Fangfang Li; Fanping Meng; Quanxin Jin; Changyuan Sun; Yingxin Li; Honghua Li; Songzhu Jin

    2014-01-01

    Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.

  19. RA8, A human anti-CD25 antibody against human treg cells

    Energy Technology Data Exchange (ETDEWEB)

    Arias, Robyn; Flanagan, Meg; Miller, Keith D.; Nien, Yu-Chih; Hu, Peisheng; Gray, Dixon; Khawli, Leslie A.; Epstein, Alan L.

    2007-06-01

    Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 109 single chain variable fragments (scFv). Two unique clones with low KD values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25+ cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.

  20. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Directory of Open Access Journals (Sweden)

    Nidiane D R Prado

    Full Text Available Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II, two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs and immunoglobulin frameworks (FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718 were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607 neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

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

    Science.gov (United States)

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

    2012-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiaocong Yu

    2016-01-01

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

  4. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  5. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  6. Preventive Activity against Influenza (H1N1 Virus by Intranasally Delivered RNA-Hydrolyzing Antibody in Respiratory Epithelial Cells of Mice

    Directory of Open Access Journals (Sweden)

    Seungchan Cho

    2015-09-01

    Full Text Available The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1 was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day for five days prior to infection demonstrated an antiviral activity (70% survival against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.

  7. Preparation and identification of the fragment of rabbit's polyclonal antibodies against human ouabain%抗哇巴因多克隆抗体F(ab)2 片段的研制及鉴定

    Institute of Scientific and Technical Information of China (English)

    张明娟; 吕卓人; 袁育康; 贺浪冲; 王颢

    2000-01-01

    Objective Anti-ouabain polyclonal antibody was digested with pepsin to prepare F fragment in order to find the base of reshaped antibody. Methods Anti-ouabain polyclonal antibody was prepared by immunizing rabbits. Then anti-ouabain polyclonal antibody was digested under different conditions. The reactants were analyzed and purified by High Performance Size Ex-clude Chromatography (HPSEC). The immune activities were detected and identified by Enzyme Linked Immunosorbent Assay (ELISA). Results The proper reactive conditions for preparation of anti-ouabain polyclonal antibody F(ab)2 fragment with pepsin were established. The eligible dose of pepsin, by which 100mg anti-onabain polyclonal antibody was digested better, was 2mg. The eligible digesuve time was 18h. The value of pH of reactive system was 3.0. Conclusion The active F fragment of anti-ouabain polyclonal antibody can be obtained with pepsin. The method is simple and feasible.%目的用胃蛋白酶酶解抗哇巴因多克隆抗体,制备出片段,为改型抗体的研究提 供依据。方法免疫家兔制备出抗哇巴因多克隆抗体,并用胃蛋白酶分别在不同的酶解条件下消化抗体,继之用高效液相排阻色谱法(HPSEC)对其进行分析、纯化,采用酶联免疫吸附法(ELISA)检测、鉴定其活性。结果用胃蛋白酶2mg/100mg抗哇巴因多克隆抗体,消化18h,反应体系的pH3.0时,可获得片段。结论用胃蛋白酶酶解抗哇巴因多克隆抗体是研制出有活性的片段的有效方法。

  8. Detection of the cyanobacterial toxin, microcystin-LR, using a novel recombinant antibody-based optical-planar waveguide platform.

    Science.gov (United States)

    Murphy, Caroline; Stack, Edwina; Krivelo, Svetlana; McPartlin, Daniel A; Byrne, Barry; Greef, Charles; Lochhead, Michael J; Husar, Greg; Devlin, Shauna; Elliott, Christopher T; O'Kennedy, Richard J

    2015-05-15

    Microcystins are a major group of cyanobacterial heptapeptide toxins found in freshwater and brackish environments. There is currently an urgent requirement for highly-sensitive, rapid and in-expensive detection methodologies for these toxins. A novel single chain fragment variable (scFv) fragment was generated and is the first known report of a recombinant anti-microcystin avian antibody. In a surface plasmon resonance-based immunoassay, the antibody fragment displayed cross-reactivity with seven microcystin congeners (microcystin-leucine-arginine (MC-LR) 100%, microcystin-tyrosine-arginine (MC-YR) 79.7%, microcystin-leucine-alanine (MC-LA) 74.8%, microcystin-leucine-phenylalanine (MC-LF) 67.5%, microcystin-leucine-tryptophan (MC-LW) 63.7%, microcystin-arginine-arginine (MC-RR) 60.1% and nodularin (Nod) 69.3%, % cross reactivity). Following directed molecular evolution of the parental clone the resultant affinity-enhanced antibody fragment was applied in an optimized fluorescence immunoassay on a planar waveguide detection system. This novel immuno-sensing format can detect free microcystin-LR with a functional limit of detection of 0.19 ng mL(-1)and a detection range of 0.21-5.9 ng mL(-1). The assay is highly reproducible (displaying percentage coefficients of variance below 8% for intra-day assays and below 11% for inter-day assays), utilizes an inexpensive cartridge system with low reagent volumes and can be completed in less than twenty minutes.

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

    Institute of Scientific and Technical Information of China (English)

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

    2000-01-01

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

  10. Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

    Science.gov (United States)

    Schwimmer, Lauren J; Huang, Betty; Giang, Hoa; Cotter, Robyn L; Chemla-Vogel, David S; Dy, Francis V; Tam, Eric M; Zhang, Fangjiu; Toy, Pamela; Bohmann, David J; Watson, Susan R; Beaber, John W; Reddy, Nithin; Kuan, Hua-Feng; Bedinger, Daniel H; Rondon, Isaac J

    2013-05-31

    Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

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

    Science.gov (United States)

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

    2014-12-10

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

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

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2011-11-01

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

  13. Biodistribution and planar gamma camera imaging of {sup 123}I- and {sup 131}I-labeled F(ab'){sub 2} and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor

    Energy Technology Data Exchange (ETDEWEB)

    Burvenich, Ingrid J.G. [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium)]. E-mail: ingrid.burvenich@ugent.be; Schoonooghe, Steve [Department of Biomedical Research, Flanders Institute of Biotechnology (VIB), University of Ghent, B-9000 Ghent (Belgium); Blanckaert, Peter [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Bacher, Klaus [Department of Medical Physics and Radiation Protection, Ghent University, B-9000 Ghent (Belgium); Vervoort, Liesbet [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Coene, Elisabeth [N. Goormaghtigh Institute of Pathology, Ghent University, B-9000 Ghent (Belgium); Mertens, Nico [Department of Biomedical Research, Flanders Institute of Biotechnology (VIB), University of Ghent, B-9000 Ghent (Belgium); Vos, Filip de [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Slegers, Guido [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium)

    2007-04-15

    Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab'){sub 2} and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice. Methods: F(ab'){sub 2} and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of {sup 125}I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of {sup 131}I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these {sup 123}I-labeled 14C5 fragments for radioimmunodetection (RID). Results: Saturation binding experiments showed highest affinity for {sup 125}I-labeled F(ab'){sub 2} fragments (K {sub d}=0.37{+-}0.10 nmol/L) and lowest affinity for {sup 125}I-labeled Fab fragments (K {sub d}=2.25{+-}0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t1/2{alpha}) value for Fab, F(ab'){sub 2} and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t1/2{beta} value for Fab, F(ab'){sub 2} and mAb 14C5 was 439, 627 and 4067 min, respectively. {sup 131}I-Fab fragments showed highest tumor uptake 3 h after injection (2.4{+-}0.8 %ID/g), {sup 131}I-labeled F(ab'){sub 2} showed highest tumor uptake 6 h after injection (4.7{+-}0.7 %ID/g) and for {sup 131}I-labeled mAb highest tumor uptake was observed at 24 h (10.7{+-}2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than {sup 123}I-mAb 14C5. Conclusion: Fab and F(ab'){sub 2} fragments derived from intact mAb 14C5 have

  14. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known...... an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...

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

    Directory of Open Access Journals (Sweden)

    Foroogh Nejatollahi

    2013-01-01

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

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

    Science.gov (United States)

    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.

  17. Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

    Institute of Scientific and Technical Information of China (English)

    史耕先; 靳玉兰; 王壮志; 崔巍; 刘音; 王讯; 朱立平

    2001-01-01

    The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa's staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells.

  18. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  19. Expression and optimization of anti-AFB1 scFv in Escherichia coli%抗黄曲霉毒素B1单链抗体在大肠杆菌中的表达及优化

    Institute of Scientific and Technical Information of China (English)

    杨炼; 张艳红; 丁虎生; 王丽云; 陈卫; 张灏

    2009-01-01

    [Objective] A drawback of the expression of single chain antibody fragment (scFv) in prokaryotic system is the protein accumulation in the cytoplasm as inclusion body. We aimed at high-level production of an anti-aflatoxin Bl scFv in functional form. [Methods] The gene of scFv-H4 was cloned into pET22b vector and transformed into E. coli BL21(DE3) and Origami (DE3), respectively. The amount of functional scFv-H4 was optimized in terms of IPTG concentration and induction temperature. [Results] scFv-H4 could be expressed in both BL21(DE3) and Origami (DE3). Compared with BL21(DE3), Origami(DE3) could express multifunctional scFv-H4 (35 mg/mL) and less in inclusion body (11% of the total expression) . The expression of scFv-H4 was significantly affected by induction temperature rather than IPTG concentration. [Conclusion] The p£T22b could be used for high-level expression of the functional scFv-H4 in Origami (DE3) , which has an oxidative cytoplasm. In addition, the induction at low temperature avoided the formation of inclusion body.%[目的]继杂交瘤技术后,重组抗体技术是新一代的抗体制备技术.然而如何用原核系统中较多地表达具有生物活性的单链抗体,避免包涵体形成仍是一个需要探讨的问题.[方法]将目的基因scFv-H4克隆到载体pET22b上,分别转入大肠杆菌BL21(DE3)和Origami(DE3)中,通过改变诱导温度和IPTG浓度,比较具有生物活性的蛋白量以及包涵体的量.[结果]在BL21(DE3)中,pET22b能产生大量表达scFv-H4,而BL21(DE3)的含有trxA和gor双突变的衍生菌Origami(DE3)表达的scFv-H4的总量较少,但是具有生物活性的蛋白量较多(35 mg/L培养物),具有生物活性的蛋白比例也较BL2l(DE3)高.另外IFrG的浓度对scFv-H4表达没有显著影响,而较高的诱导温度会促使表达的蛋白形成包涵体.[结论]在较低的温度下,pET22b能在Origami(DE3)能较好地表达具有生物活性的scFv-H4,减少包涵体的比例,为后续的抗

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

    Institute of Scientific and Technical Information of China (English)

    张志超; 胡学军; 包永明; 杨青; 张红梅; 安利佳

    2002-01-01

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

  1. [Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25-35)].

    Science.gov (United States)

    Kolobov, V V; Davydova, T V; Fomina, V G

    2014-01-01

    Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp 1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ25-35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp 1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.

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

    Institute of Scientific and Technical Information of China (English)

    左伟勇; 王永娟; 陆辉; 洪伟鸣; 刘莉

    2013-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    陈秀芹; 阎锡蕴

    2003-01-01

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

  4. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S;

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known....... Serum samples from autoimmune (NZBxNZW)F1 mice, healthy BALB/c mice, patients with SLE, RA and normal healthy individuals were analyzed for presence and amount of circulating anti-dsDNA antibodies and nucleosomal DNA. Here we use a quantitative PCR to measure circulating DNA in sera. We demonstrate...

  5. Characterization of the interaction between human complement protein C4 and a single-chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

    NARCIS (Netherlands)

    Seifar, R.M.; Cool, Robbert; Quax, Wim; Bischoff, Rainer

    2004-01-01

    Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surf

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

    Science.gov (United States)

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

    2016-10-01

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

  7. Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

    NARCIS (Netherlands)

    Heskamp, S.; Laarhoven, H.W.M. van; Molkenboer-Kuenen, J.D.M.; Bouwman, W.H.; Graaf, W.T. van der; Oyen, W.J.G.; Boerman, O.C.

    2012-01-01

    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal

  8. Randomized, placebo-controlled trial of the anti-tumor necrosis factor antibody fragment afelimomab in hyperinflammatory response during severe sepsis : The RAMSES Study

    NARCIS (Netherlands)

    Reinhart, K; Menges, T; Gardlund, B; Zwaveling, JH; Smithes, M; Vincent, JL; Tellado, JM; Salgado-Remigio, A; Zimlichman, R; Withington, S; Tschaikowsky, K; Brase, R; Damas, P; Kupper, H; Kempeni, J; Eiselstein, J; Kaul, M

    2001-01-01

    Objective: This study investigated whether treatment with the anti-tumor necrosis factor-or monoclonal antibody afelimomab would improve survival in septic patients with serum interleukin (IL)-6 concentrations of >1000 pg/ml, Design: Multicenter, double-blind, randomized, placebo-controlled study. S

  9. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C.

    Science.gov (United States)

    Strokappe, Nika; Szynol, Agnieszka; Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

    2012-01-01

    Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.

  10. Lactobacillus helveticus MIMLh5-specific antibodies for detection of S-layer protein in Grana Padano protected-designation-of-origin cheese.

    Science.gov (United States)

    Stuknyte, Milda; Brockmann, Eeva-Christine; Huovinen, Tuomas; Guglielmetti, Simone; Mora, Diego; Taverniti, Valentina; Arioli, Stefania; De Noni, Ivano; Lamminmäki, Urpo

    2014-01-01

    Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  12. 抗胃癌单链抗体免疫毒素重组质粒的构建及表达%Construction of Anti-carcinoma ScFv Immunotoxin Recombina nt Plasmid and its Expression

    Institute of Scientific and Technical Information of China (English)

    韩梅; 戴寿芝; 靳更林; 寿成超

    2001-01-01

    Objectives To construct the recombinant plasm id that expresses anti-carcinoma ScF v immunotoxin.Methods By recombinant technique,the cDNA coding the anti-carcinoma ScFv antibody was recombined with the cDNA coding Pseudomonas areuginosa exotoxin to form the recombinanr plasmid.The E Coli.BL-21,after bein g transformed with the recombinant plasmid,was induced by IPTG to express the an ti-carcinoma ScFv immumotoxin.Results The recombinantion of pla smid was identifide to be correct by endonuclease restriction analysis and the B L-21 that contain the recombinant plasmid abundantly expressed the immunotoxin b y IPTG inducement.The size of the expressed protein is similar to that anticipat ed in MW. Conclusions The recombinant plasmid that can express the anti -carcinoma ScFv immunotoxin is successfully obtained.%目的 构建能表达抗胃癌单链抗体免疫毒素的重组质粒并进行表达。方法 应用基因工程方法,将抗胃癌的单链抗体基因与绿脓杆菌外毒素基因进行重组,得到的重组质粒转化大肠杆菌BL-21 ,鉴定后的阳性克隆经IPTG诱导表达。结果 重组质粒经酶切鉴定正确,IPTG诱导后可看到明显的表达带,分子大小与预计值相符。结论 得到了能表达抗胃癌单链抗体免疫毒素的重组质粒。

  13. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    2002-01-01

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of H

  14. Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site.

    Science.gov (United States)

    Talavera, Ariel; Mackenzie, Jenny; Garrido, Greta; Friemann, Rosmarie; López-Requena, Alejandro; Moreno, Ernesto; Krengel, Ute

    2011-07-01

    The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.

  15. A single-dose cytomegalovirus-based vaccine encoding tetanus toxin fragment C induces sustained levels of protective tetanus toxin antibodies in mice.

    Science.gov (United States)

    Tierney, Rob; Nakai, Toru; Parkins, Christopher J; Caposio, Patrizia; Fairweather, Neil F; Sesardic, Dorothea; Jarvis, Michael A

    2012-04-26

    The current commercially available vaccine used to prevent tetanus disease following infection with the anaerobic bacterium Clostridium tetani is safe and effective. However, tetanus remains a major source of mortality in developing countries. In 2008, neonatal tetanus was estimated to have caused >59,000 deaths, accounting for 1% of worldwide infant mortality, primarily in poorer nations. The cost of multiple vaccine doses administered by injection necessary to achieve protective levels of anti-tetanus toxoid antibodies is the primary reason for low vaccine coverage. Herein, we show that a novel vaccine strategy using a cytomegalovirus (CMV)-based vaccine platform induces protective levels of anti-tetanus antibodies that are durable (lasting >13 months) in mice following only a single dose. This study demonstrates the ability of a 'single-dose' CMV-based vaccine strategy to induce durable protection, and supports the potential for a tetanus vaccine based on CMV to impact the incidence of tetanus in developing countries.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  17. A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells.

    Science.gov (United States)

    Tur, Mehmet Kemal; Rothe, Achim; Huhn, Michael; Goerres, Ute; Klimka, Alexander; Stöcker, Michael; Engert, Andreas; Fischer, Rainerr; Finner, Ricarda; Barth, Stefan

    2003-04-01

    Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.

  18. Autoreactive antibodies are present in sheep with Johne's disease and cross-react with Mycobacterium avium subsp. paratuberculosis antigens.

    Science.gov (United States)

    Berger, Sven; Bannantine, John P; Griffin, J Frank T

    2007-07-01

    Mycobacterial infection has been linked to the generation of autoantibodies, including anti-neutrophil cytoplasmic autoantibodies (ANCA), in clinical studies and small animal models. In an attempt to identify antibodies that react with both self and mycobacterial antigens in naturally infected ruminants, we generated a phage display library comprising single chain antibody fragments (scFv) from sheep with Johne's disease (JD). The library was screened simultaneously against ovine small intestinal tissue and Mycobacterium avium subsp. paratuberculosis (MAP). A clone (termed AutoH1) reacted strongly with host tissue and MAP, recognizing a proteinase-susceptible 32 kDa determinant in ovine gut tissues and lymphatics, and in blood granulocytes but not mononuclear cells. In granulocytes, binding was to cytoplasmic granules and cell membranes; in MAP, AutoH1 bound bacterial cell wall determinants. We further identified a synthetic peptide sequence recognized by AutoH1, using this to generate a carrier:peptide fusion protein (paH1). Sera of normal and JD sheep were screened for AutoH1-like autoantibody activity; 7/11 JD animals showed autoreactivity that could be blocked by paH1, while 0/21 normal animals showed no such serological reactivity. It is possible that the severe pathology observed in ruminant JD may have an autoimmune component, possibly due to ANCA-type binding; this remains to be further investigated.

  19. Conformation Selective Antibody Enables Genome Profiling and Leads to Discovery of Parallel G-Quadruplex in Human Telomeres.

    Science.gov (United States)

    Liu, Hui-Yun; Zhao, Qi; Zhang, Tian-Peng; Wu, Yue; Xiong, Yun-Xia; Wang, Shi-Ke; Ge, Yuan-Long; He, Jin-Hui; Lv, Peng; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Ren, Jian; Zhao, Yong; Huang, Zhi-Shu

    2016-10-20

    G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo.

  20. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

    Directory of Open Access Journals (Sweden)

    Shuli Zhao

    2012-03-01

    Full Text Available Doxorubicin (DOX was conjugated to a single-chain variable fragment (scFv against human midkine (MK, and the conjugate (scFv-DOX was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex as a linker. The molecular formula of this immunoconjugate was scFv(Dex1.3(DOX20. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8% for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%. Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

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

    Science.gov (United States)

    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.

  2. Dulaglutide, a long-acting GLP-1 analog fused with an Fc antibody fragment for the potential treatment of type 2 diabetes

    DEFF Research Database (Denmark)

    Jimenez-Solem, Espen; Rasmussen, Mette H; Christensen, Mikkel;

    2010-01-01

    that dulaglutide reduces plasma glucose, and has an insulinotropic effect increasing insulin and C-peptide levels. Two phase II clinical trials demonstrated a dose-dependent reduction in glycated hemoglobin (HbA1c) of up to 1.52% compared with placebo. Side effects associated with dulaglutide administration were...... mainly gastrointestinal. To date, there have been no reports on the formation of antibodies against dulaglutide, but, clearly, long-term data will be needed to asses this and other possible side effects. The results of several phase III clinical trials are awaited for clarification of the expected...

  3. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J. (SVIMR-A); (HeidelbergU); (WEHI); (Melbourne)

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  4. Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

    Science.gov (United States)

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dhagat, Urmi; Owczarek, Catherine M; Hardy, Matthew P; Fabri, Louis J; Scotney, Pierre D; Nash, Andrew D; Wilson, Nicholas J; Lopez, Angel F; Parker, Michael W

    2014-03-01

    Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.

  5. Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

    Science.gov (United States)

    Hahn, M; Winkler, D; Welfle, K; Misselwitz, R; Welfle, H; Wessner, H; Zahn, G; Scholz, C; Seifert, M; Harkins, R; Schneider-Mergener, J; Höhne, W

    2001-11-23

    The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.

  6. Half molecular exchange of IgGs in the blood of healthy humans: chimeric lambda-kappa-immunoglobulins containing HL fragments of antibodies of different subclasses (IgG1-IgG4).

    Science.gov (United States)

    Sedykh, Sergey E; Lekchnov, Evgenii A; Prince, Viktor V; Buneva, Valentina N; Nevinsky, Georgy A

    2016-10-20

    In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

  7. Development of a recombinant antibody towards PAPP-A for immunohistochemical use in multiple animal species

    DEFF Research Database (Denmark)

    Mikkelsen, Jakob H; Steffensen, Lasse B; Oxvig, Claus

    2014-01-01

    normal physiology and under different pathological conditions. However, antibodies for the detection of PAPP-A in non-human tissues have been lacking, although needed for use with several animal models which are currently being developed. To develop a monoclonal antibody suitable...... for the immunohistochemical detection of PAPP-A, we therefore selected a phage-derived scFv antibody, PAC1, specifically recognizing an epitope of PAPP-A, which is highly conserved between multiple animal species. We first converted this antibody into bivalent IgG, and verified its ability to recognize PAPP-A in sections...

  8. Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies.

    Directory of Open Access Journals (Sweden)

    Dongmei Hu

    Full Text Available Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv gene libraries with 4 x 10(6 DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.

  9. Quantum fragmentation

    CERN Document Server

    Peschanski, R

    1993-01-01

    Phenomenological and theoretical aspects of fragmentation for elementary particles (resp. nuclei) are discussed. It is shown that some concepts of classical fragmentation remain relevant in a microscopic framework, exhibiting non-trivial properties of quantum relativistic field theory (resp. lattice percolation). Email contact: pesch@amoco.saclay.cea.fr

  10. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  11. 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.

    Science.gov (United States)

    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.

  12. Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support.

    Science.gov (United States)

    Sushma, Krishnan; Bilgimol, Chuvappumkal Joseph; Vijayalakshmi, Mookambeswaran A; Satheeshkumar, Padikara Kutty

    2012-04-01

    Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC₅₀ value of 15 μg. To the best of our knowledge, this is the first report showing the application of methacrylate based chromatographic supports for matrix-assisted refolding and purification of Escherichia coli inclusion bodies. The results are promising to elaborate the methodology further to exploit the potential positive features of monoliths in protein refolding science.

  13. Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

    Science.gov (United States)

    Vega, Celina G; Bok, Marina; Vlasova, Anastasia N; Chattha, Kuldeep S; Gómez-Sebastián, Silvia; Nuñez, Carmen; Alvarado, Carmen; Lasa, Rodrigo; Escribano, José M; Garaicoechea, Lorena L; Fernandez, Fernando; Bok, Karin; Wigdorovitz, Andrés; Saif, Linda J; Parreño, Viviana

    2013-01-01

    Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

  14. Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to Rotavirus VP6 Protect Neonatal Gnotobiotic Piglets against Human Rotavirus-Induced Diarrhea

    Science.gov (United States)

    Vlasova, Anastasia N.; Chattha, Kuldeep S.; Gómez-Sebastián, Silvia; Nuñez, Carmen; Alvarado, Carmen; Lasa, Rodrigo; Escribano, José M.; Garaicoechea, Lorena L.; Fernandez, Fernando; Bok, Karin; Wigdorovitz, Andrés; Saif, Linda J.; Parreño, Viviana

    2013-01-01

    Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea. PMID:23658521

  15. Recombinant monovalent llama-derived antibody fragments (VHH to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

    Directory of Open Access Journals (Sweden)

    Celina G Vega

    Full Text Available Group A Rotavirus (RVA is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH to protect against human rotavirus in gnotobiotic (Gn piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256 for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

  16. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.

    Science.gov (United States)

    Graille, M; Stura, E A; Corper, A L; Sutton, B J; Taussig, M J; Charbonnier, J B; Silverman, G J

    2000-05-09

    Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

  17. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR/sub 2/), also identifies a 72Kd secreted fragment of CR/sub 2/ that contains the C3d-binding site

    Energy Technology Data Exchange (ETDEWEB)

    Myones, B.L.; Ross, G.D.

    1986-03-05

    CR/sub 2/ is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR/sub 2/, blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR/sub 2/, does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR/sub 2/ because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of /sup 125/I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from /sup 125/I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence /sup 3/H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of /sup 125/I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR/sub 2/ molecule, also identifies a 72Kd shed fragment of CR/sub 2/ isolated from Raji cell media, which contains the C3d-binding site.

  18. Characterization of botulinum neurotoxin type A neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity.

    Directory of Open Access Journals (Sweden)

    Christelle Mazuet

    Full Text Available Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent, intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab'(2, Fab', scFv, chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs. F(ab'(2 fragments, native or treated with polyethyleneglycol (PEG, were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17, recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A

  19. Characterization of a single-chain variable fragment recognizing a linear epitope of aβ: a biotechnical tool for studies on Alzheimer's disease?

    Directory of Open Access Journals (Sweden)

    Silke Dornieden

    Full Text Available 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.

  20. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

    Science.gov (United States)

    Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

    2015-04-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

  1. Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands.

    Science.gov (United States)

    Sumitani, M; Kasashima, K; Yamamoto, D S; Yagi, K; Yuda, M; Matsuoka, H; Yoshida, S

    2013-02-01

    We have previously developed a robust salivary gland-specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti-P. falciparum circumsporozoite protein (PfCSP) single-chain antibody (scFv) fused to DsRed in a secretory form (mDsRed-2A10 scFv). Fluorescence microscopy showed that the mDsRed-2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission-blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed-2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland-specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans.

  2. Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.

    Science.gov (United States)

    Alanen, Heli I; Walker, Kelly L; Lourdes Velez Suberbie, M; Matos, Cristina F R O; Bönisch, Sarah; Freedman, Robert B; Keshavarz-Moore, Eli; Ruddock, Lloyd W; Robinson, Colin

    2015-03-01

    Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

  3. Magma Fragmentation

    Science.gov (United States)

    Gonnermann, Helge M.

    2015-05-01

    Magma fragmentation is the breakup of a continuous volume of molten rock into discrete pieces, called pyroclasts. Because magma contains bubbles of compressible magmat