The Experts below are selected from a list of 168 Experts worldwide ranked by ideXlab platform
Patrick Chames - One of the best experts on this subject based on the ideXlab platform.
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Single-domain antibodies: A versatile and rich source of binders for breast cancer diagnostic approaches
Molecular BioSystems, 2012Co-Authors: Klervi Even-desrumeaux, Véronique Secq, Daniel Baty, Patrick Fourquet, Patrick ChamesAbstract:Noninvasive early detection of breast cancer through the use of biomarkers is urgently needed since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. A crucial issue in this approach is the availability of relevant markers and corresponding monoclonal antibodies suitable for the development of effective immunodiagnostic modalities. The identification of such markers from human pathological lesions and the isolation of specific antibodies using conventional approaches remain major challenges. Camelids produce functional antibodies devoid of light chains in which the single N-terminal domain of the heavy chain is fully capable of antigen binding. When produced as an independent domain, these so-called single-domain antibody fragments (sdAbs) or nanobodies have several advantages for biotechnological applications owing to their unique properties of size (13 kDa), stability, solubility, and Expression Yield. In this work, we have generated phage display libraries from animals immunized with breast cancer biopsies. These libraries were used to isolate sdAbs against known and relevant antigens such as HER2, or several cancer-specific sdAbs against unknown targets. We describe the identification of one these targets, cytokeratin 19, using affinity purification in combination with mass spectrometry. Some of these sdAbs were used in several straightforward diagnostic applications such as immunohistochemical analysis of tumor samples, multiplexed cytometric bead array analysis of crude samples, or an immune enrichment procedure of rare cells. Here, we demonstrate that phage display-based selection of single-domain antibodies is an efficient and high-throughput compatible approach to generate binders with excellent characteristics for the fast development of diagnostic and prognostic modalities.
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strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high throughput compatible cost effective antibody array generation
Molecular BioSystems, 2010Co-Authors: Klervi Evendesrumeaux, Daniel Baty, Patrick ChamesAbstract:Antibody microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnoses and proteomic analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and Expression Yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecules for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs, produced in crude bacterial lysates, to generate a proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and a direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers.
Arne Skerra - One of the best experts on this subject based on the ideXlab platform.
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proba complementation of an auxotrophic e coli strain improves plasmid stability and Expression Yield during fermenter production of a recombinant antibody fragment
Gene, 2001Co-Authors: Markus Fiedler, Arne SkerraAbstract:Abstract The proline-auxotrophic Escherichia coli K12 strain JM83 harbouring an Expression vector providing the proBA gene in trans was utilized for the fermenter production of the partially humanized IN-1 antibody Fab fragment. Thus, plasmid-mediated complementation of the chromosomal proBA deletion was employed as a second selection mechanism, together with a chloramphenicol resistance, in order to (i) abolish plasmid loss and (ii) benefit from E. coli JM83 as an Expression strain with approved periplasmic protein secretion characteristics in the presence of a minimal medium. Starting from the generic vector pASK75, which makes use of the tightly regulated and chemically inducible tet promoter for foreign gene Expression, a set of new vectors carrying the entire or part of the proBA operon was constructed and compared concerning their capability of functional ΔproBA complementation as well as recombinant protein Yield. As a result, the vector pMF1 was developed, where transcription of the proBA operon is controlled by its own constitutive promoter and terminator sequences, permitting the transformed JM83 strain to grow under glucose/ammonia minimal culture conditions. When pMF1 was used for the fermenter production of the IN-1 Fab fragment, no plasmid loss was observed during the growth and induction phases, and the Yield of functionally purified recombinant protein was found to be considerably improved.
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proBA complementation of an auxotrophic E. coli strain improves plasmid stability and Expression Yield during fermenter production of a recombinant antibody fragment.
Gene, 2001Co-Authors: Markus Fiedler, Arne SkerraAbstract:The proline-auxotrophic Escherichia coli K12 strain JM83 harbouring an Expression vector providing the proBA gene in trans was utilized for the fermenter production of the partially humanized IN-1 antibody F(ab) fragment. Thus, plasmid-mediated complementation of the chromosomal proBA deletion was employed as a second selection mechanism, together with a chloramphenicol resistance, in order to (i) abolish plasmid loss and (ii) benefit from E. coli JM83 as an Expression strain with approved periplasmic protein secretion characteristics in the presence of a minimal medium. Starting from the generic vector pASK75, which makes use of the tightly regulated and chemically inducible tet promoter for foreign gene Expression, a set of new vectors carrying the entire or part of the proBA operon was constructed and compared concerning their capability of functional Delta proBA complementation as well as recombinant protein Yield. As a result, the vector pMF1 was developed, where transcription of the proBA operon is controlled by its own constitutive promoter and terminator sequences, permitting the transformed JM83 strain to grow under glucose/ammonia minimal culture conditions. When pMF1 was used for the fermenter production of the IN-1 F(ab) fragment, no plasmid loss was observed during the growth and induction phases, and the Yield of functionally purified recombinant protein was found to be considerably improved.
Daniel Baty - One of the best experts on this subject based on the ideXlab platform.
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Single-domain antibodies: A versatile and rich source of binders for breast cancer diagnostic approaches
Molecular BioSystems, 2012Co-Authors: Klervi Even-desrumeaux, Véronique Secq, Daniel Baty, Patrick Fourquet, Patrick ChamesAbstract:Noninvasive early detection of breast cancer through the use of biomarkers is urgently needed since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. A crucial issue in this approach is the availability of relevant markers and corresponding monoclonal antibodies suitable for the development of effective immunodiagnostic modalities. The identification of such markers from human pathological lesions and the isolation of specific antibodies using conventional approaches remain major challenges. Camelids produce functional antibodies devoid of light chains in which the single N-terminal domain of the heavy chain is fully capable of antigen binding. When produced as an independent domain, these so-called single-domain antibody fragments (sdAbs) or nanobodies have several advantages for biotechnological applications owing to their unique properties of size (13 kDa), stability, solubility, and Expression Yield. In this work, we have generated phage display libraries from animals immunized with breast cancer biopsies. These libraries were used to isolate sdAbs against known and relevant antigens such as HER2, or several cancer-specific sdAbs against unknown targets. We describe the identification of one these targets, cytokeratin 19, using affinity purification in combination with mass spectrometry. Some of these sdAbs were used in several straightforward diagnostic applications such as immunohistochemical analysis of tumor samples, multiplexed cytometric bead array analysis of crude samples, or an immune enrichment procedure of rare cells. Here, we demonstrate that phage display-based selection of single-domain antibodies is an efficient and high-throughput compatible approach to generate binders with excellent characteristics for the fast development of diagnostic and prognostic modalities.
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strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high throughput compatible cost effective antibody array generation
Molecular BioSystems, 2010Co-Authors: Klervi Evendesrumeaux, Daniel Baty, Patrick ChamesAbstract:Antibody microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnoses and proteomic analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and Expression Yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecules for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs, produced in crude bacterial lysates, to generate a proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and a direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers.
Yufei Wang - One of the best experts on this subject based on the ideXlab platform.
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a novel nanobody targeting middle east respiratory syndrome coronavirus mers cov receptor binding domain has potent cross neutralizing activity and protective efficacy against mers cov
Journal of Virology, 2018Co-Authors: Guangyu Zhao, Lei He, Shihui Sun, Hongjie Qiu, Wanbo Tai, Jiawei Chen, Jiangfan Li, Yuehong Chen, Yan Guo, Yufei WangAbstract:The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great Expression Yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high Yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics. IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high Expression Yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.
Markus Fiedler - One of the best experts on this subject based on the ideXlab platform.
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proba complementation of an auxotrophic e coli strain improves plasmid stability and Expression Yield during fermenter production of a recombinant antibody fragment
Gene, 2001Co-Authors: Markus Fiedler, Arne SkerraAbstract:Abstract The proline-auxotrophic Escherichia coli K12 strain JM83 harbouring an Expression vector providing the proBA gene in trans was utilized for the fermenter production of the partially humanized IN-1 antibody Fab fragment. Thus, plasmid-mediated complementation of the chromosomal proBA deletion was employed as a second selection mechanism, together with a chloramphenicol resistance, in order to (i) abolish plasmid loss and (ii) benefit from E. coli JM83 as an Expression strain with approved periplasmic protein secretion characteristics in the presence of a minimal medium. Starting from the generic vector pASK75, which makes use of the tightly regulated and chemically inducible tet promoter for foreign gene Expression, a set of new vectors carrying the entire or part of the proBA operon was constructed and compared concerning their capability of functional ΔproBA complementation as well as recombinant protein Yield. As a result, the vector pMF1 was developed, where transcription of the proBA operon is controlled by its own constitutive promoter and terminator sequences, permitting the transformed JM83 strain to grow under glucose/ammonia minimal culture conditions. When pMF1 was used for the fermenter production of the IN-1 Fab fragment, no plasmid loss was observed during the growth and induction phases, and the Yield of functionally purified recombinant protein was found to be considerably improved.
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proBA complementation of an auxotrophic E. coli strain improves plasmid stability and Expression Yield during fermenter production of a recombinant antibody fragment.
Gene, 2001Co-Authors: Markus Fiedler, Arne SkerraAbstract:The proline-auxotrophic Escherichia coli K12 strain JM83 harbouring an Expression vector providing the proBA gene in trans was utilized for the fermenter production of the partially humanized IN-1 antibody F(ab) fragment. Thus, plasmid-mediated complementation of the chromosomal proBA deletion was employed as a second selection mechanism, together with a chloramphenicol resistance, in order to (i) abolish plasmid loss and (ii) benefit from E. coli JM83 as an Expression strain with approved periplasmic protein secretion characteristics in the presence of a minimal medium. Starting from the generic vector pASK75, which makes use of the tightly regulated and chemically inducible tet promoter for foreign gene Expression, a set of new vectors carrying the entire or part of the proBA operon was constructed and compared concerning their capability of functional Delta proBA complementation as well as recombinant protein Yield. As a result, the vector pMF1 was developed, where transcription of the proBA operon is controlled by its own constitutive promoter and terminator sequences, permitting the transformed JM83 strain to grow under glucose/ammonia minimal culture conditions. When pMF1 was used for the fermenter production of the IN-1 F(ab) fragment, no plasmid loss was observed during the growth and induction phases, and the Yield of functionally purified recombinant protein was found to be considerably improved.
