The Experts below are selected from a list of 228 Experts worldwide ranked by ideXlab platform
Serge Muyldermans - One of the best experts on this subject based on the ideXlab platform.
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Introduction to Heavy Chain Antibodies and Derived Nanobodies
Methods of Molecular Biology, 2012Co-Authors: Cécile Vincke, Serge MuyldermansAbstract:The immune response of infected or immunized dromedaries contains a diverse repertoire of conventional and Heavy Chain-only antibodies, both functional in antigen binding. By definition, a Heavy Chain antibody is devoid of a light Chain and in the case of the Heavy Chain antibodies in camelids the CH1 domain is also missing. Consequently a camelid Heavy Chain antibody associates with its cognate antigen via a single domain, the variable Heavy Chain domain of a Heavy Chain antibody or VHH. An antigen-specific VHH, also known as Nanobody, with excellent biochemical properties can be obtained in various ways. Their recombinant expression provides access to user-friendly tools for a wide variety of applications.
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disulfide bond introduction for general stabilization of immunoglobulin Heavy Chain variable domains
Journal of Molecular Biology, 2008Co-Authors: Dirk Saerens, Katja Conrath, Jochen Govaert, Serge MuyldermansAbstract:Several antibody fragment engineering techniques aim at intrinsic stability enhancement, but are not applied in a truly generic way. Here, a strategy is proposed whereby consistent gain in stability is accomplished by introducing a specific disulfide bond between two opposite β-strands in the hydrophobic core of the immunoglobulin Heavy-Chain variable domain of Heavy-Chain antibodies (Nanobody). Besides the rational design of a disulfide bond between residues 39 and 87, a Nanobody harboring an extra naturally occurring cystine between residues 54 and 78 was compared to an equivalent Nanobody without that cystine. Both novel disulfide cross-links were introduced in several Nanobodies in various combinations. Interestingly, only the extra naturally occurring cystine consistently increased the conformational and thermal stabilities of wild-type Nanobodies without affecting antigen binding.
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functional Heavy Chain antibodies fragments thereof library thereof and methods of production thereof
2002Co-Authors: Serge Muyldermans, Viet Khong NguyenAbstract:The present invention relates to functional Heavy Chain antibodies, functional single domain Heavy Chain antibodies, functional VH domains, or functional fragments thereof comprising an amino acid which is neither a charged amino acid nor a C at position 45, and comprising an amino acid at position 103 independently chosen from the group consisting of R, G, K, S, Q, L, and P, and optionally an amino acid at position 108 independently chosen from the group consisting of Q, L and R, said positions determined according to the Kabat numbering.
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unique single domain antigen binding fragments derived from naturally occurring camel Heavy Chain antibodies
Journal of Molecular Recognition, 1999Co-Authors: Serge Muyldermans, Marc LauwereysAbstract:The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two Heavy Chains and no light Chains. The amino acid sequence of the variable domain of the naturally occurring Heavy-Chain antibodies reveals the necessary adaptations to compensate for the absence of the light Chain. In contrast to the conventional antibodies, a large proportion of the Heavy-Chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these Heavy-Chain antibodies. The presence of the Heavy-Chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site. Copyright # 1999 John Wiley & Sons, Ltd.
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Potent enzyme inhibitors derived from dromedary Heavy-Chain antibodies
EMBO Journal, 1998Co-Authors: Marc Lauwereys, Mehdi Arbabi Ghahroudi, Wolfgang Hölzer, Erwin De Genst, Aline Desmyter, Lode Wyns, Jorg Kinne, Serge MuyldermansAbstract:Evidence is provided that dromedary Heavy-Chain antibodies, in vivo-matured in the absence of light Chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of Heavy-Chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector, By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.
Robert J Talmadge - One of the best experts on this subject based on the ideXlab platform.
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electrophoretic separation of rat skeletal muscle myosin Heavy Chain isoforms
Journal of Applied Physiology, 1993Co-Authors: Robert J TalmadgeAbstract:A new technique for the sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of rat skeletal muscle myosin Heavy-Chain (MHC) isoforms is presented. This technique allows for the sep...
Marc Lauwereys - One of the best experts on this subject based on the ideXlab platform.
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unique single domain antigen binding fragments derived from naturally occurring camel Heavy Chain antibodies
Journal of Molecular Recognition, 1999Co-Authors: Serge Muyldermans, Marc LauwereysAbstract:The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two Heavy Chains and no light Chains. The amino acid sequence of the variable domain of the naturally occurring Heavy-Chain antibodies reveals the necessary adaptations to compensate for the absence of the light Chain. In contrast to the conventional antibodies, a large proportion of the Heavy-Chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these Heavy-Chain antibodies. The presence of the Heavy-Chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site. Copyright # 1999 John Wiley & Sons, Ltd.
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Potent enzyme inhibitors derived from dromedary Heavy-Chain antibodies
EMBO Journal, 1998Co-Authors: Marc Lauwereys, Mehdi Arbabi Ghahroudi, Wolfgang Hölzer, Erwin De Genst, Aline Desmyter, Lode Wyns, Jorg Kinne, Serge MuyldermansAbstract:Evidence is provided that dromedary Heavy-Chain antibodies, in vivo-matured in the absence of light Chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of Heavy-Chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector, By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.
Lode Wyns - One of the best experts on this subject based on the ideXlab platform.
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Potent enzyme inhibitors derived from dromedary Heavy-Chain antibodies
EMBO Journal, 1998Co-Authors: Marc Lauwereys, Mehdi Arbabi Ghahroudi, Wolfgang Hölzer, Erwin De Genst, Aline Desmyter, Lode Wyns, Jorg Kinne, Serge MuyldermansAbstract:Evidence is provided that dromedary Heavy-Chain antibodies, in vivo-matured in the absence of light Chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of Heavy-Chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector, By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.
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comparison of llama vh sequences from conventional and Heavy Chain antibodies
Molecular Immunology, 1997Co-Authors: Khoa Bang Vu, Mehdi Arbabi Ghahroudi, Lode Wyns, Serge MuyldermansAbstract:Forty different PCR clones encoding a llama variable Heavy Chain domain were analysed. The majority of these clones are derived from Heavy-Chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric Heavy-Chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.
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selection and identification of single domain antibody fragments from camel Heavy Chain antibodies
FEBS Letters, 1997Co-Authors: Arbabi M Ghahroudi, Raymond Hamers, Aline Desmyter, Lode Wyns, Serge MuyldermansAbstract:Functional Heavy-Chain γ-immunoglobulins lacking light Chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its Heavy-Chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.
Ritsu Kamiya - One of the best experts on this subject based on the ideXlab platform.
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A Chlamydomonas outer arm dynein mutant with a truncated beta Heavy Chain.
Journal of Cell Biology, 1993Co-Authors: H Sakakibara, Saeko Takada, Stephen M. King, George B. Witman, Ritsu KamiyaAbstract:A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein Heavy Chains, the oda4-s7 axoneme contains the alpha and gamma Heavy Chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta Heavy Chain. These observations indicate that this mutant has a truncated beta Heavy Chain, and that the NH2-terminal part of the beta Heavy Chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha Heavy Chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm Heavy Chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta Heavy Chain impairs outer-arm function more seriously than does the lack of the alpha Heavy Chain, suggesting that the alpha and beta Chains play different roles in outer arm function.
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A Chlamydomonas outer arm dynein mutant missing the alpha Heavy Chain.
Journal of Cell Biology, 1991Co-Authors: H Sakakibara, D R Mitchell, Ritsu KamiyaAbstract:A novel Chlamydomonas flagellar mutant (oda-11) missing the alpha Heavy Chain of outer arm dynein but retaining the beta and gamma Heavy Chains was isolated. Restriction fragment length polymorphism analysis with an alpha Heavy Chain locus genomic probe indicated that the oda-11 mutation was genetically linked with the structural gene of the alpha Heavy Chain. In cross-section electron micrographs, the oda-11 axoneme lacked the outermost appendage of the outer arm, indicating that the alpha Heavy Chain should be located in this region in the wild-type outer arm. This mutant swam at 119 microns/s at 25 degrees C, i.e., at an intermediate speed between those of wild type (194 microns/s) and of oda-1 (62 microns/s), a mutant missing the entire outer dynein arm. The flagellar beat frequency (approximately 50 Hz) was also between those of wild type (approximately 60 Hz) and oda-1 (approximately 26 Hz). These results indicate that the outer dynein arm of Chlamydomonas can be assembled without the alpha Heavy Chain, and that the outer arm missing the alpha Heavy Chain retains partial function.
