The Experts below are selected from a list of 144 Experts worldwide ranked by ideXlab platform
Julien Muzard - One of the best experts on this subject based on the ideXlab platform.
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creation of recombinant Antigen Binding molecules derived from hybridomas secreting specific antibodies
Nature Protocols, 2013Co-Authors: Conor Fields, David J Oconnell, Sujing Xiao, Philippe Billiald, Julien MuzardAbstract:Creation of recombinant Antigen-Binding molecules derived from hybridomas secreting specific antibodies
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Creation of recombinant Antigen-Binding molecules derived from hybridomas secreting specific antibodies
Nature Protocols, 2013Co-Authors: Conor Fields, Sujing Xiao, Philippe Billiald, David O'connell, Julien MuzardAbstract:This protocol describes the design and development of recombinant monovalent Antigen-Binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy ( VH ) and variable light ( VL ) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant Antigen-Binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.
Abner Louis Notkins - One of the best experts on this subject based on the ideXlab platform.
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polyreactive Antigen Binding b cells are the predominant cell type in the newborn b cell repertoire
European Journal of Immunology, 1998Co-Authors: Zhi Jian Chen, James C Wheeler, Wei Shi, Cheryl H Yarboro, Michael Gallagher, Abner Louis NotkinsAbstract:Polyreactive antibodies bind to a variety of different self and non-self Antigens. The B cells that make these antibodies express the polyreactive lg receptor on their surface. To determine the frequency of polyreactive Antigen-Binding B cells in peripheral blood, we incubated two different Antigens, one (insulin) labeled with fluorescein isothiocyanate and the other (beta-galactosidase) with phycoerythrin, with peripheral B cells. The percentage of cells that bound these Antigens was determined with the fluorescence-activated cells sorter. Approximately 21% of adult B cells bound insulin, 28% bound beta-galactosidase, and 11% bound both Antigens. In contrast to B cells in the adult repertoire, 49% of B cells in cord blood bound insulin, 54% bound beta-galactosidase, and 33% bound both Antigens. The properties of polyreactive Antigen-Binding B cells in adult and cord blood were similar, except for the fact that almost all the polyreactive Antigen-Binding B cells in cord blood were CD5 positive (93%), whereas only 40% of the polyreactive Antigen-Binding B cells in adult peripheral blood were CD5 positive, indicating that the CD5 marker is not directly linked to polyreactivity. The percentage of polyreactive Antigen-Binding B cells in patients with Sjogren's syndrome, systemic lupus erythematosus and rheumatoid arthritis was equal to or slightly below that found in the normal adult B cell repertoire. It is concluded that polyreactive Antigen-Binding B cells are a major constituent of the normal adult B cell repertoire and are the predominant cell type in the newborn B cell repertoire.
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polyreactive Antigen Binding b cells in the peripheral circulation are igd and b7
European Journal of Immunology, 1996Co-Authors: Zhi Jian Chen, Jim Wheeler, Fumio Shimizu, Abner Louis NotkinsAbstract:Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self Antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different Antigens. The present investigation was initiated to characterize these polyreactive Antigen-Binding B cells further. A panel of fluorescein isothiocyanate-labeled Antigens (insulin, IgG Fc fragment or beta-galactosidase) served as probes to select polyreactive Antigen-Binding B cells by cell sorting. Our experiment revealed that these polyreactive Antigen-Binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the Binding of Antigens to monoreactive receptors (usually high affinity), the Binding of Antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as Antigens that bound to monoreactive receptors. Despite the ability of these B7- cells to process Antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive Antigen-Binding B cells have properties that are consistent with the ability to induce immunological tolerance.
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Antigen‐Binding B cells and polyreactive antibodies
European journal of immunology, 1995Co-Authors: Zhi Jian Chen, Jim Wheeler, Abner Louis NotkinsAbstract:The present experiments were initiated to see if cells capable of Binding Antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self Antigens were incubated with B cells from normal individuals. Antigen-Binding cells were separated from non-Antigen-Binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound Antigens than in the B cell subset that did not bind Antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of Antigens (e.g., insulin, IgGFc and beta-galactosidase), whereas cell lines making monoreactive antibodies bound only a single Antigen. The Binding of Antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%-57%) by both homogeneous and heterogeneous Antigens. Both CD5+ and CD5- Antigen-Binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5+ Antigen-Binding (85%) as compared to the CD5- Antigen-Binding (50%) population. Comparison of CD5+ B cells that bound Antigens with CD5+ B cells that did not bind Antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of Binding a variety of different Antigens can make polyreactive antibodies and that Antigen Binding is a good marker for identifying polyreactive antibody-producing cells.
Conor Fields - One of the best experts on this subject based on the ideXlab platform.
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creation of recombinant Antigen Binding molecules derived from hybridomas secreting specific antibodies
Nature Protocols, 2013Co-Authors: Conor Fields, David J Oconnell, Sujing Xiao, Philippe Billiald, Julien MuzardAbstract:Creation of recombinant Antigen-Binding molecules derived from hybridomas secreting specific antibodies
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Creation of recombinant Antigen-Binding molecules derived from hybridomas secreting specific antibodies
Nature Protocols, 2013Co-Authors: Conor Fields, Sujing Xiao, Philippe Billiald, David O'connell, Julien MuzardAbstract:This protocol describes the design and development of recombinant monovalent Antigen-Binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy ( VH ) and variable light ( VL ) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant Antigen-Binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.
Zhi Jian Chen - One of the best experts on this subject based on the ideXlab platform.
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polyreactive Antigen Binding b cells are the predominant cell type in the newborn b cell repertoire
European Journal of Immunology, 1998Co-Authors: Zhi Jian Chen, James C Wheeler, Wei Shi, Cheryl H Yarboro, Michael Gallagher, Abner Louis NotkinsAbstract:Polyreactive antibodies bind to a variety of different self and non-self Antigens. The B cells that make these antibodies express the polyreactive lg receptor on their surface. To determine the frequency of polyreactive Antigen-Binding B cells in peripheral blood, we incubated two different Antigens, one (insulin) labeled with fluorescein isothiocyanate and the other (beta-galactosidase) with phycoerythrin, with peripheral B cells. The percentage of cells that bound these Antigens was determined with the fluorescence-activated cells sorter. Approximately 21% of adult B cells bound insulin, 28% bound beta-galactosidase, and 11% bound both Antigens. In contrast to B cells in the adult repertoire, 49% of B cells in cord blood bound insulin, 54% bound beta-galactosidase, and 33% bound both Antigens. The properties of polyreactive Antigen-Binding B cells in adult and cord blood were similar, except for the fact that almost all the polyreactive Antigen-Binding B cells in cord blood were CD5 positive (93%), whereas only 40% of the polyreactive Antigen-Binding B cells in adult peripheral blood were CD5 positive, indicating that the CD5 marker is not directly linked to polyreactivity. The percentage of polyreactive Antigen-Binding B cells in patients with Sjogren's syndrome, systemic lupus erythematosus and rheumatoid arthritis was equal to or slightly below that found in the normal adult B cell repertoire. It is concluded that polyreactive Antigen-Binding B cells are a major constituent of the normal adult B cell repertoire and are the predominant cell type in the newborn B cell repertoire.
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polyreactive Antigen Binding b cells in the peripheral circulation are igd and b7
European Journal of Immunology, 1996Co-Authors: Zhi Jian Chen, Jim Wheeler, Fumio Shimizu, Abner Louis NotkinsAbstract:Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self Antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different Antigens. The present investigation was initiated to characterize these polyreactive Antigen-Binding B cells further. A panel of fluorescein isothiocyanate-labeled Antigens (insulin, IgG Fc fragment or beta-galactosidase) served as probes to select polyreactive Antigen-Binding B cells by cell sorting. Our experiment revealed that these polyreactive Antigen-Binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the Binding of Antigens to monoreactive receptors (usually high affinity), the Binding of Antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as Antigens that bound to monoreactive receptors. Despite the ability of these B7- cells to process Antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive Antigen-Binding B cells have properties that are consistent with the ability to induce immunological tolerance.
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Antigen‐Binding B cells and polyreactive antibodies
European journal of immunology, 1995Co-Authors: Zhi Jian Chen, Jim Wheeler, Abner Louis NotkinsAbstract:The present experiments were initiated to see if cells capable of Binding Antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self Antigens were incubated with B cells from normal individuals. Antigen-Binding cells were separated from non-Antigen-Binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound Antigens than in the B cell subset that did not bind Antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of Antigens (e.g., insulin, IgGFc and beta-galactosidase), whereas cell lines making monoreactive antibodies bound only a single Antigen. The Binding of Antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%-57%) by both homogeneous and heterogeneous Antigens. Both CD5+ and CD5- Antigen-Binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5+ Antigen-Binding (85%) as compared to the CD5- Antigen-Binding (50%) population. Comparison of CD5+ B cells that bound Antigens with CD5+ B cells that did not bind Antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of Binding a variety of different Antigens can make polyreactive antibodies and that Antigen Binding is a good marker for identifying polyreactive antibody-producing cells.
Sujing Xiao - One of the best experts on this subject based on the ideXlab platform.
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creation of recombinant Antigen Binding molecules derived from hybridomas secreting specific antibodies
Nature Protocols, 2013Co-Authors: Conor Fields, David J Oconnell, Sujing Xiao, Philippe Billiald, Julien MuzardAbstract:Creation of recombinant Antigen-Binding molecules derived from hybridomas secreting specific antibodies
-
Creation of recombinant Antigen-Binding molecules derived from hybridomas secreting specific antibodies
Nature Protocols, 2013Co-Authors: Conor Fields, Sujing Xiao, Philippe Billiald, David O'connell, Julien MuzardAbstract:This protocol describes the design and development of recombinant monovalent Antigen-Binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy ( VH ) and variable light ( VL ) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant Antigen-Binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.