The Experts below are selected from a list of 300 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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Single Domain Antibodies
Methods Mol Biol, 2012Co-Authors: Aurelien Olichon, Cécile Vincke, Ario De Marco, Serge MuyldermansAbstract:The preparation of antibody libraries starting from lymphocytes recovered from immunized members of the Camelidae enables to collect binders that underwent somatic maturation. However, the time and costs necessary to prepare a library for each new antigen may urge to look for alternatives such as those offered by large one-pot libraries. Here we describe how to obtain a suitable naïve library using material from nonimmunized llamas. Despite the lack of somatic maturation, the selection based on phage display allowed to isolate from such naïve libraries VHHs with af fi nity in the subnanomolar range and suitable for the standard immunoapplications.
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Antibody repertoire development in camelids
Developmental and Comparative Immunology, 2006Co-Authors: Erwin De Genst, Dirk Saerens, Serge Muyldermans, Katja ConrathAbstract:The humoral immune response of the Camelidae is unique as these animals are the only known mammals that seem to possess functional homodimeric heavy-chain antibodies besides the classical heteromeric antibodies composed of heavy (H) and light (L) chains. By definition, the heavy-chain antibodies lack the L-chain, and it was noticed that their H-chain is devoid of the typical first constant domain (CH1) and contains a dedicated variable domain, referred to as VHH. The VHH exon is assembled from separate V-D-J gene segments. The recombined VHH region is subjected to somatic hypermutations; however, the timing and actual mechanism of the class switch from μ to the dedicated γ-isotype remains elusive. Interestingly, antigen-specific VHHs are easily retrieved after panning of a phage-displayed rearranged V-gene pool cloned from an immunised camelid. These single-domain antigen binding entities possess a number of biophysical properties that offer particular advantages in various medical and biotechnological applications. © 2005 Elsevier Ltd. All rights reserved.
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emergence and evolution of functional heavy chain antibodies in Camelidae
Developmental and Comparative Immunology, 2003Co-Authors: Katja Conrath, Serge Muyldermans, Ulrich Wernery, Viet Khong NguyenAbstract:Antibodies of jawed-vertebrates are composed of paired heavy (H) and light (L) polypeptide chains. Surprisingly, the sera of camelids, nurse shark and wobbegong shark, and possibly ratfish contain antibodies that lack L-chains. In camelids, these Heavy-chain antibodies (HCAbs) are γ-isotypes, and are functional in antigen binding. In this review we focus on the dedicated immunoglobulin (Ig) genes that encode the HCAb in Camelidae (camels, dromedaries and llamas), about their origin, and how these camel immunoglobulins evolved and acquire a large and diverse repertoire of antigen binding sites in absence of the H–L combinatorial diversity.
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heavy chain antibodies in Camelidae a case of evolutionary innovation
Immunogenetics, 2002Co-Authors: Viet Khong Nguyen, Chen Su, Serge MuyldermansAbstract:The emergence in Camelidae species of functional antibodies devoid of light chains (referred to as heavy-chain antibodies or HCAbs) is an intriguing evolutionary event. Homodimeric HCAbs have also been documented in spotted ratfish (Cos5-Abs) and nurse shark (NAR). To reveal the evolutionary history of HCAbs, we evaluated the phylogenetic and phenotypic relationships among HCAbs and conventional antibodies across taxa and confirmed the current viewpoint that different groups of HCAbs have evolved independently in the three lineages. At least, in the camelids, HCAbs are not the result of resuscitation of dormant genes. They are derived from the conventional antibodies within the Camelidae lineage, and are apparently the outcome of more recent adaptive changes occurring in the compartment of heteromeric antibodies. The shared structural properties of HCAbs across taxa are therefore explained by convergent evolution due to similar constraints related to the absence of pairing to the light chain. It appears that innovative evolutionary changes in Camelidae have led to a new level of antigen binding repertoire diversification and have allowed acquisition of novel antigen-receptor properties.
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beta lactamase inhibitors derived from single domain antibody fragments elicited in the Camelidae
Antimicrobial Agents and Chemotherapy, 2001Co-Authors: Katja Conrath, Lode Wyns, Marc Lauwereys, Moreno Galleni, Andre Matagne, Jeanmarie Frere, Jorg Kinne, Serge MuyldermansAbstract:Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII β-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those β-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 β-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of β-lactamases.
Ario Marco - One of the best experts on this subject based on the ideXlab platform.
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Molecular dynamics simulations and docking enable to explore the biophysical factors controlling the yields of engineered nanobodies
Scientific Reports, 2016Co-Authors: Miguel A. Soler, Ario Marco, Sara FortunaAbstract:Nanobodies (VHHs) have proved to be valuable substitutes of conventional antibodies for molecular recognition. Their small size represents a precious advantage for rational mutagenesis based on modelling. Here we address the problem of predicting how Camelidae nanobody sequences can tolerate mutations by developing a simulation protocol based on all-atom molecular dynamics and whole-molecule docking. The method was tested on two sets of nanobodies characterized experimentally for their biophysical features. One set contained point mutations introduced to humanize a wild type sequence, in the second the CDRs were swapped between single-domain frameworks with Camelidae and human hallmarks. The method resulted in accurate scoring approaches to predict experimental yields and enabled to identify the structural modifications induced by mutations. This work is a promising tool for the in silico development of single-domain antibodies and opens the opportunity to customize single functional domains of larger macromolecules.
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bacterial cytoplasm as an effective cell compartment for producing functional vhh based affinity reagents and Camelidae igg like recombinant antibodies
Microbial Cell Factories, 2014Co-Authors: Selma Djender, Ronan Crepin, Aurelie Schneider, Anne Beugnet, Klervi E. Desrumeaux, Chiara Romani, Franck Perez, Sandrine Moutel, Ario MarcoAbstract: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.
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Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies
Microbial Cell Factories, 2014Co-Authors: Selma Djender, Ronan Crepin, Aurelie Schneider, Anne Beugnet, Klervi E. Desrumeaux, Chiara Romani, Franck Perez, Sandrine Moutel, Ario MarcoAbstract:© 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.
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Preparation of a naïve library of camelid single domain antibodies
Methods in Molecular Biology, 2012Co-Authors: Aurelien Olichon, Ario MarcoAbstract:The preparation of antibody libraries starting from lymphocytes recovered from immunized members of the Camelidae enables to collect binders that underwent somatic maturation. However, the time and costs necessary to prepare a library for each new antigen may urge to look for alternatives such as those offered by large one-pot libraries. Here we describe how to obtain a suitable naïve library using material from nonimmunized llamas. Despite the lack of somatic maturation, the selection based on phage display allowed to isolate from such naïve libraries VHHs with affinity in the subnanomolar range and suitable for the standard immunoapplications. © 2012 Springer Science+Business Media, LLC.
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User-friendly expression plasmids enable the fusion of VHHs to application-specific tags
Methods in Molecular Biology, 2012Co-Authors: Ario MarcoAbstract:The preparation of antibody libraries starting from lymphocytes recovered from immunized members of the Camelidae enables to collect binders that underwent somatic maturation. However, the time and costs necessary to prepare a library for each new antigen may urge to look for alternatives such as those offered by large one-pot libraries. Here we describe how to obtain a suitable naïve library using material from nonimmunized llamas. Despite the lack of somatic maturation, the selection based on phage display allowed to isolate from such naïve libraries VHHs with af fi nity in the subnanomolar range and suitable for the standard immunoapplications.
Jane C Wheeler - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial dna variation and systematics of the guanaco lama guanicoe artiodactyla Camelidae
Journal of Mammalogy, 2008Co-Authors: Juan Marin, Jane C Wheeler, Benito A Gonzalez, Eduardo R Palma, Angel E Spotorno, Cristian Bonacic, Ciara S Casey, Michael William Bruford, Elie PoulinAbstract:Guanacos (Lama guanicoe) are the most important native herbivorous species in the South American steppes and the dominant ungulate in a fauna rich in rodents but poor in large mammal species. Between 2 and 4 subspecies are usually recognized within Lama guanicoe, based on subtle morphological differences and geographic distribution. To evaluate whether molecular variation is consistent with the latter hypotheses, we analyzed the complete cytochrome-b and partial control region mitochondrial DNA sequences of L. guanicoe from 22 localities in Peru, Bolivia, Argentina, and Chile. Sequence analyses of both genes support the monophyly of the species but failed to distinguish the occurrence of subspecies along the geographic range. Despite that, the northernmost populations (Peru and northern Chile) showed some degree of genetic differentiation with respect to southern representatives from Argentina, Bolivia, and rest of Chile. Analysis of genetic diversity also showed a strong signal of past low population size and a recent population expansion.
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evolution and present situation of the south american Camelidae
Biological Journal of The Linnean Society, 1995Co-Authors: Jane C WheelerAbstract:Abstract This paper provides a review of South American camelid evolution, classification and present status. Particular attention is paid to the debate concerning origins of the domestic alpaca and Llama and the contribution of research on faunal remains from Andean archaeological sites towards resolving this issue. Changes in incisor morphology during the domestication process suggest that the alpaca may be descended from the vicuna, while a comparison of fibre production characteristics in preconquest and extant llama and alpaca breeds indicates that extensive hybridization between the two species is likely to have occurred since European contact. The potential role of hybridization in the formation of extant South American camelid populations has not been studied, and may be the root cause of taxonomic disputes.
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molecular evolution of the family Camelidae a mitochondrial dna study
Proceedings of The Royal Society B: Biological Sciences, 1994Co-Authors: Helen F Stanley, Miranda Kadwell, Jane C WheelerAbstract:We report the first molecular evolutionary analysis of the family Camelidae by analysing the full DNA sequence of the mitochondrial cytochrome b gene. Estimates for the time of divergence of the Old World (Camelini) and New World (Lamini) tribes obtained from sequence data are in agreement with those derived from the fossil record. The DNA sequence data were also used to test current hypotheses concerning the ancestors of the domesticated llama and alpaca. The results show that hybridization has occurred in the ancestry of both domesticated camelids, obscuring the origin of the domestic species.
Benito A Gonzalez - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial dna variation and systematics of the guanaco lama guanicoe artiodactyla Camelidae
Journal of Mammalogy, 2008Co-Authors: Juan Marin, Jane C Wheeler, Benito A Gonzalez, Eduardo R Palma, Angel E Spotorno, Cristian Bonacic, Ciara S Casey, Michael William Bruford, Elie PoulinAbstract:Guanacos (Lama guanicoe) are the most important native herbivorous species in the South American steppes and the dominant ungulate in a fauna rich in rodents but poor in large mammal species. Between 2 and 4 subspecies are usually recognized within Lama guanicoe, based on subtle morphological differences and geographic distribution. To evaluate whether molecular variation is consistent with the latter hypotheses, we analyzed the complete cytochrome-b and partial control region mitochondrial DNA sequences of L. guanicoe from 22 localities in Peru, Bolivia, Argentina, and Chile. Sequence analyses of both genes support the monophyly of the species but failed to distinguish the occurrence of subspecies along the geographic range. Despite that, the northernmost populations (Peru and northern Chile) showed some degree of genetic differentiation with respect to southern representatives from Argentina, Bolivia, and rest of Chile. Analysis of genetic diversity also showed a strong signal of past low population size and a recent population expansion.
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taxonomic and biogeographical status of guanaco lama guanicoe artiodactyla Camelidae
Mammal Review, 2006Co-Authors: Benito A Gonzalez, Eduardo R Palma, Beatriz Zapata, Juan MarinAbstract:We review the status of the four currently recognized guanaco Lama guanicoe subspecies, and provide information about their taxonomy and distribution. The success of guanaco in inhabiting open habitats of South America is based mainly on the flexibility of their social behaviour and ecophysiological adaptations to harsh environments. 2. Lonnberg described the first subspecies, L. g. cacsilensis , at the beginning of the 20th century. Forty years later Krumbiegel described L. g. voglii , based on skull measurements and pelage colouration. The other two subspecies, L. g. huanacus and L. g. guanicoe , were classi- fied as subspecies by Krumbiegel based on pelage colouration and body size, while maintain- ing the original Latin names and descriptors. 3. Further guanaco populations have been incorporated into each of these subspecies, based on their proximity to the type locality but without attention to the homogeneity of phenotype or habitat and only limited consideration of Bergmann's rule based on scarce skulls. Two alternative geographical ranges were proposed in the middle and towards the end of the 20th century. Discrepancies occur in the geographical range of each subspecies. 4. Molecular studies based on mitochondrial DNA cytochrome b sequences recognized only two subspecies: the Peruvian L. guanicoe cacsilensis and the rest of the populations grouped in the clade recognized as L. g. guanicoe . We conclude that the evolutionary biology of L. guanicoe requires a significant revision with respect to biogeography. Phylogeographical data hold particular value in developing conservation strategies, particularly for some of the reduced and marginal populations and/or subspecies and will support IUCN (The World Conservation Union) Red List classification.
Katja Conrath - One of the best experts on this subject based on the ideXlab platform.
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Antibody repertoire development in camelids
Developmental and Comparative Immunology, 2006Co-Authors: Erwin De Genst, Dirk Saerens, Serge Muyldermans, Katja ConrathAbstract:The humoral immune response of the Camelidae is unique as these animals are the only known mammals that seem to possess functional homodimeric heavy-chain antibodies besides the classical heteromeric antibodies composed of heavy (H) and light (L) chains. By definition, the heavy-chain antibodies lack the L-chain, and it was noticed that their H-chain is devoid of the typical first constant domain (CH1) and contains a dedicated variable domain, referred to as VHH. The VHH exon is assembled from separate V-D-J gene segments. The recombined VHH region is subjected to somatic hypermutations; however, the timing and actual mechanism of the class switch from μ to the dedicated γ-isotype remains elusive. Interestingly, antigen-specific VHHs are easily retrieved after panning of a phage-displayed rearranged V-gene pool cloned from an immunised camelid. These single-domain antigen binding entities possess a number of biophysical properties that offer particular advantages in various medical and biotechnological applications. © 2005 Elsevier Ltd. All rights reserved.
-
emergence and evolution of functional heavy chain antibodies in Camelidae
Developmental and Comparative Immunology, 2003Co-Authors: Katja Conrath, Serge Muyldermans, Ulrich Wernery, Viet Khong NguyenAbstract:Antibodies of jawed-vertebrates are composed of paired heavy (H) and light (L) polypeptide chains. Surprisingly, the sera of camelids, nurse shark and wobbegong shark, and possibly ratfish contain antibodies that lack L-chains. In camelids, these Heavy-chain antibodies (HCAbs) are γ-isotypes, and are functional in antigen binding. In this review we focus on the dedicated immunoglobulin (Ig) genes that encode the HCAb in Camelidae (camels, dromedaries and llamas), about their origin, and how these camel immunoglobulins evolved and acquire a large and diverse repertoire of antigen binding sites in absence of the H–L combinatorial diversity.
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beta lactamase inhibitors derived from single domain antibody fragments elicited in the Camelidae
Antimicrobial Agents and Chemotherapy, 2001Co-Authors: Katja Conrath, Lode Wyns, Marc Lauwereys, Moreno Galleni, Andre Matagne, Jeanmarie Frere, Jorg Kinne, Serge MuyldermansAbstract:Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII β-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those β-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 β-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of β-lactamases.
