The Experts below are selected from a list of 8499 Experts worldwide ranked by ideXlab platform

Thomas Schirrmann - One of the best experts on this subject based on the ideXlab platform.

  • Generation of Recombinant Antibodies against toxins and viruses by phage display for diagnostics and therapy
    Advances in Experimental Medicine and Biology, 2016
    Co-Authors: Tobias Unkauf, Sebastian Miethe, Anne C. Frenzel, Thomas Schirrmann, Viola Fühner, André Frenzel, Michael Hust
    Abstract:

    Antibodies are valuable molecules for the diagnostic and treatment of diseases caused bypathogens and toxins. Traditionally, these Antibodies are generated by hybridoma technology.An alternative to hybridoma technology is the use of antibody phage display to generate re-combinant Antibodies. This in vitro technology circumvents the limitations of the immunesystem and allows—in theory—the generation of Antibodies against all conceivable molecules.Phage display technology enables obtaining human Antibodies from na¨ıve antibody gene li-braries when either patients are not available or immunization is not ethically feasible. Onthe other hand, if patients or immunized/infected animals are available, it is common to con-struct immune phage display libraries to select in vivo affinity-matured Antibodies. Becausethe phage packaged DNA sequence encoding the Antibodies is directly available, the Antibodiescan be smoothly engineered according to the requirements of the final application. In thisreview, an overview of phage display derived Recombinant Antibodies against bacterial, viral,and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

  • Expression of Recombinant Antibodies
    Frontiers in Immunology, 2013
    Co-Authors: Anne C. Frenzel, André Frenzel, Michael Hust, Thomas Schirrmann
    Abstract:

    Recombinant Antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of Recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic Antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain Antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific Antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  • High level transient production of Recombinant Antibodies and antibody fusion proteins in HEK293 cells
    BMC Biotechnology, 2013
    Co-Authors: Volker Jager, Anne C. Frenzel, Konrad Büssow, André Frenzel, Michael Hust, Susanne Weber, Andreas Wagner, Thomas Schirrmann
    Abstract:

    BACKGROUND: The demand of monospecific high affinity binding reagents, particularly monoclonal Antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of Recombinant Antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.\n\nRESULTS: In this study transient expression of scFv-Fc Antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10--20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc Antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.\n\nCONCLUSION: Transient production of Recombinant scFv-Fc Antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput Recombinant antibody generation pipeline.

  • Generating Recombinant Antibodies for Research, Diagnostics and Therapy Using Phage Display
    Current Biotechnology, 2011
    Co-Authors: Anne C. Frenzel, Torsten Meyer, David Frode, Thomas Schirrmann, André Frenzel, Michael Hust
    Abstract:

    Since the development of antibody phage display, 20 years ago, this technique has been evolved to a valuable and robust tool for the development of Recombinant human Antibodies. Today, this technique is widely used for the generation of Antibodies for therapy and basic research. Automation processes and decrease of costs will enable the high- throughput design of monoclonal binders against a huge number of different antigens, presenting a valuable tool for proteome research. This review presents an overview on phage display technology and antibody libraries. The benefit of Recombinant Antibodies for basic research, diagnostic and therapy is shown.

  • Identification of a putative Crf splice variant and generation of Recombinant Antibodies for the specific detection of Aspergillus fumigatus
    PLoS ONE, 2009
    Co-Authors: Mark Schütte, Thibaut Pelat, Xenia Wezler, Philip Rosenstock, Dominik Hinz, Mike Hansenberg, Martina Inga Kirsch, Philippe Thullier, Ronald Frank, Thomas Schirrmann
    Abstract:

    Background: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of Recombinant Antibodies for the detection of A. fumigatus by ELISA. Results: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with Recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these Antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the Antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. Conclusion: Crf2 and the corresponding Recombinant Antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

Michael Hust - One of the best experts on this subject based on the ideXlab platform.

  • Recombinant Antibodies for diagnostics and therapy against pathogens and toxins generated by phage display.
    Proteomics Clinical Applications, 2016
    Co-Authors: Philipp Kuhn, Sebastian Miethe, Gustavo Marçal Schmidt Garcia Moreira, Viola Fühner, Tobias Unkauf, André Frenzel, Michael Hust
    Abstract:

    Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these Antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody phage display to generate Recombinant Antibodies. This in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of Antibodies against all conceivable molecules. Phage display technology enables obtaining human Antibodies from naive antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity-matured Antibodies. Because the phage packaged DNA sequence encoding the Antibodies is directly available, the Antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of phage display derived Recombinant Antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

  • Recombinant Antibodies for diagnostics and therapy against pathogens and toxins generated by phage display
    Proteomics - Clinical Applications, 2016
    Co-Authors: Philipp Kuhn, Sebastian Miethe, Anne C. Frenzel, Gustavo Marçal Schmidt Garcia Moreira, Viola Fühner, Tobias Unkauf, André Frenzel, Michael Hust
    Abstract:

    © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these Antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody phage display to generate Recombinant Antibodies. This in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of Antibodies against all conceivable molecules. Phage display technology enables obtaining human Antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity-matured Antibodies. Because the phage packaged DNA sequence encoding the Antibodies is directly available, the Antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of phage display derived Recombinant Antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

  • Generation of Recombinant Antibodies against toxins and viruses by phage display for diagnostics and therapy
    Advances in Experimental Medicine and Biology, 2016
    Co-Authors: Tobias Unkauf, Sebastian Miethe, Anne C. Frenzel, Thomas Schirrmann, Viola Fühner, André Frenzel, Michael Hust
    Abstract:

    Antibodies are valuable molecules for the diagnostic and treatment of diseases caused bypathogens and toxins. Traditionally, these Antibodies are generated by hybridoma technology.An alternative to hybridoma technology is the use of antibody phage display to generate re-combinant Antibodies. This in vitro technology circumvents the limitations of the immunesystem and allows—in theory—the generation of Antibodies against all conceivable molecules.Phage display technology enables obtaining human Antibodies from na¨ıve antibody gene li-braries when either patients are not available or immunization is not ethically feasible. Onthe other hand, if patients or immunized/infected animals are available, it is common to con-struct immune phage display libraries to select in vivo affinity-matured Antibodies. Becausethe phage packaged DNA sequence encoding the Antibodies is directly available, the Antibodiescan be smoothly engineered according to the requirements of the final application. In thisreview, an overview of phage display derived Recombinant Antibodies against bacterial, viral,and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

  • Novel human Recombinant Antibodies against Mycobacterium tuberculosis antigen 85B
    BMC Biotechnology, 2014
    Co-Authors: Manon Fuchs, Ralf Spallek, Wiebke Prilop, Saskia Helmsing, Stefan Dübel, Susanne Kämpfer, Wulf Oehlmann, Mahavir Singh, Ronald Frank, Michael Hust
    Abstract:

    BACKGROUND: Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic.\n\nRESULTS: In this work, the first human single chain fragment variable (scFv) Antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these Antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of Recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection.\n\nCONCLUSIONS: Recombinant Antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These Antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.

  • Expression of Recombinant Antibodies
    Frontiers in Immunology, 2013
    Co-Authors: Anne C. Frenzel, André Frenzel, Michael Hust, Thomas Schirrmann
    Abstract:

    Recombinant Antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of Recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic Antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain Antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific Antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

Katrina Campbell - One of the best experts on this subject based on the ideXlab platform.

  • Progress in the development of immunoanalytical methods incorporating Recombinant Antibodies to small molecular weight biotoxins
    Analytical and Bioanalytical Chemistry, 2015
    Co-Authors: Owen Kavanagh, Christopher T Elliott, Katrina Campbell
    Abstract:

    Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) Antibodies have provided the binding element of the majority of these assays but Recombinant Antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain Antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins. Graphical AbstractSchematic representation of (A) affinity selection of phage-displayed Recombinant antibody (rAb) and (B) immunoglobulin antibody structures and the corresponding antibody fragments

  • Progress in the development of immunoanalytical methods incorporating Recombinant Antibodies to small molecular weight biotoxins
    Analytical and Bioanalytical Chemistry, 2015
    Co-Authors: Anal. Bioanal. Chem. 408 (2016) 915–932. Doi:10.1007/s00216-015-9183-3. [1] L. Tartaglione, A. Mazzeo, C. Dell’aversano, M, Anal. Bioanal. Chem. (2016) 5737–5743. Doi:10.1007/s00216-016-9675-9. [2] X.-q. Mei, X.-p. He, J.-t. Wang, Molecularly I, Anal. Bioanal. Chem. (2016) 3057–3058. Doi:10.1007/s00216-015-9292-z. [3] T. Younos, Luis M . Botana , M . Carmen Louzao, Anal. Bioanal. Chem. 407 (2015) 95–116. Doi:10.1007/s00216-014-8193-x. [4] K.l. Bruce, S.c. Leterme, A. V. Ellis, C.e. Le, Anal. Bioanal. Chem. 407 (2015) 6345–6356. Doi:10.1007/s00216-015-8637-y. [6] G. Orellana, L. Van Meulebroek, S. Van Vooren,, Anal. Bioanal. Chem. 407 (2015) 5487–5501. Doi:10.1007/s00216-015-8722-2. [7] A. Roy-lachapelle, M. Solliec, S. Sauvé, Deter, Anal. Bioanal. Chem. (2015) 5353–5363. Doi:10.1007/s00216-015-8695-1. [8] C. Hollingdale, K. Thomas, N. Lewis, K. B??kri, Anal. Bioanal. Chem. 407 (2015) 3743–3750. Doi:10.1007/s00216-015-8597-2. [9] R. Andrýs, J. Zurita, N. Zguna, K. Verschueren, Katrina Campbell, Owen Kavanagh, Christopher T Elliott, Anal. Bioanal. Chem. 407 (2014) 2985–2996. Doi:10.1007/s00216-014-8250-5. [10] P. Mccarron, S.d. Giddings, K.l. Reeves, P. H
    Abstract:

    Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) Antibodies have provided the binding element of the majority of these assays but Recombinant Antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain Antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

Michael J. Hornsby - One of the best experts on this subject based on the ideXlab platform.

  • Determination of equilibrium dissociation constants for Recombinant Antibodies by high-throughput affinity electrophoresis
    Scientific Reports, 2016
    Co-Authors: Eric K. Sackmann, Michael J. Hornsby, Karolina Wypisniak, Sammy S. Datwani, Amy E Herr
    Abstract:

    Determination of equilibrium dissociation constants for Recombinant Antibodies by high-throughput affinity electrophoresis

  • Determination of equilibrium dissociation constants for Recombinant Antibodies by high-throughput affinity electrophoresis
    Scientific Reports, 2016
    Co-Authors: Yuchen Pan, Michael J. Hornsby, Eric K. Sackmann, Karolina Wypisniak, Sammy S. Datwani, Amy E Herr
    Abstract:

    © The Author(s) 2016. High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality Recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (K D ) of each Recombinant antibody and the target antigen. To characterize the K D of Recombinant Antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The K D for each of a six-member fragment antigen-binding fragment library is reported using ∼25-fold less sample mass and ∼5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

  • a high through put platform for Recombinant Antibodies to folded proteins
    Molecular & Cellular Proteomics, 2015
    Co-Authors: Michael J. Hornsby, Allison Doak, Svitlana Usatyuk, Karolina Wypisniak, Tet Matsuguchi, Annika Sääf, Shane Miersch, Marcin Paduch, Daniel King, Kimberly Perry
    Abstract:

    Antibodies are key reagents in biology and medicine, but commercial sources are rarely Recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated Recombinant Antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple Antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site Recombinant-Antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

  • A High Through-put Platform for Recombinant Antibodies to Folded Proteins
    Molecular & Cellular Proteomics, 2015
    Co-Authors: Michael J. Hornsby, Allison Doak, Karolina Wypisniak, Tet Matsuguchi, Annika Sääf, Brian Lee, Shane Miersch, Marcin Paduch, Daniel King, Svitlana Usatyuk
    Abstract:

    © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Antibodies are key reagents in biology and medicine, but commercial sources are rarely Recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated Recombinant Antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC 50 < 20 nM), high stability (T m ∼ 80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they crossreacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple Antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site Recombinant-Antibodies. org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

Franck Perez - One of the best experts on this subject based on the ideXlab platform.

  • Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like Recombinant Antibodies
    Microbial Cell Factories, 2014
    Co-Authors: Selma Djender, Ronan Crepin, Aurelie Schneider, Anne Beugnet, Klervi E. Desrumeaux, Sandrine Moutel, Franck Perez, Chiara Romani, Ario De Marco
    Abstract:

    © 2014 Djender et al.; licensee BioMed Central Ltd. Background: The isolation of Recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. Results: We demonstrate that fusion constructs of Recombinant Antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). Conclusions: The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal Antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.

  • Recombinant Antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo
    Traffic, 2003
    Co-Authors: Clément Nizak, Silvia Martin-lluesma, Sandrine Moutel, Aurelien Roux, Bruno Goud, Thomas E Kreis, Franck Perez
    Abstract:

    Generation of specific Antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that Recombinant Antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant Antibodies. In addition, we show that intracellular expression of GFP-tagged Recombinant Antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile Antibodies that represent new tools to study protein function.

  • Recombinant Antibodies to the small GTPase Rab6 as conformation sensors
    Science, 2003
    Co-Authors: Clément Nizak, Elaine Del Nery, Sandrine Moutel, Solange Monier, Bruno Goud, Franck Perez
    Abstract:

    Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant Antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6·GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6·GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.

  • Recombinant Antibodies to the Small GTPase Rab6 as
    Science, 2003
    Co-Authors: Clément Nizak, Elaine Del Nery, Sandrine Moutel, Solange Monier, Bruno Goud, Franck Perez
    Abstract:

    Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant Antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.