Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library
Identifieur interne : 000507 ( Main/Exploration ); précédent : 000506; suivant : 000508Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library
Auteurs : Mehdi Houimel [Tunisie] ; Koussay Dellagi [Tunisie]Source :
- Journal of virological methods [ 0166-0934 ] ; 2009.
Descripteurs français
- Pascal (Inist)
- Wicri :
- topic : Homme.
English descriptors
- KwdEn :
- Antibodies, Viral (genetics), Antibodies, Viral (immunology), Antibodies, Viral (isolation & purification), Epitope Mapping, Fab-Fragment, Female, Glycoproteins (immunology), Human, Humans, Immunoglobulin Fab Fragments (genetics), Immunoglobulin Fab Fragments (immunology), Immunoglobulin Fab Fragments (isolation & purification), Immunoglobulin Variable Region (genetics), Immunoglobulin Variable Region (immunology), Immunoglobulin Variable Region (isolation & purification), Isolation, Lymphocytes (immunology), Male, Method, Microbiology, Neutralization, Neutralization Tests, Neutralizing antibody, Peptide Library, Phage display, RNA (genetics), RNA (isolation & purification), Rabies (genetics), Rabies (immunology), Rabies Vaccines (immunology), Rabies virus, Rabies virus (immunology), Recombinant virus, Viral Proteins (immunology).
- MESH :
- chemical , genetics : Antibodies, Viral, Immunoglobulin Fab Fragments, Immunoglobulin Variable Region, RNA.
- chemical , immunology : Antibodies, Viral, Glycoproteins, Immunoglobulin Fab Fragments, Immunoglobulin Variable Region, Rabies Vaccines, Viral Proteins.
- chemical , isolation & purification : Antibodies, Viral, Immunoglobulin Fab Fragments, Immunoglobulin Variable Region, RNA.
- genetics : Rabies.
- immunology : Lymphocytes, Rabies, Rabies virus.
- Epitope Mapping, Female, Humans, Male, Neutralization Tests, Peptide Library.
Abstract
A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 107 Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the KD range 7 x 10-9 to 5 × 10-8 M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11).
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Affiliations:
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Le document en format XML
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sortKey="Dellagi, Koussay" sort="Dellagi, Koussay" uniqKey="Dellagi K" first="Koussay" last="Dellagi">Koussay Dellagi</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire d'Immunopathologie Vaccinologie et Génétique Moléculaire, Institut Pasteur de Tunis</s1><s3>TUN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ></inist:fA14><country>Tunisie</country><wicri:noRegion>Laboratoire d'Immunopathologie Vaccinologie et Génétique Moléculaire, Institut Pasteur de Tunis</wicri:noRegion></affiliation></author></analytic><series><title level="j" type="main">Journal of virological methods</title><title level="j" type="abbreviated">J. virol. methods</title><idno type="ISSN">0166-0934</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of virological methods</title><title level="j" type="abbreviated">J. virol. methods</title><idno type="ISSN">0166-0934</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Antibodies, Viral (genetics)</term><term>Antibodies, Viral (immunology)</term><term>Antibodies, Viral (isolation & purification)</term><term>Epitope Mapping</term><term>Fab-Fragment</term><term>Female</term><term>Glycoproteins (immunology)</term><term>Human</term><term>Humans</term><term>Immunoglobulin Fab Fragments (genetics)</term><term>Immunoglobulin Fab Fragments (immunology)</term><term>Immunoglobulin Fab Fragments (isolation & purification)</term><term>Immunoglobulin Variable Region (genetics)</term><term>Immunoglobulin Variable Region (immunology)</term><term>Immunoglobulin Variable Region (isolation & purification)</term><term>Isolation</term><term>Lymphocytes (immunology)</term><term>Male</term><term>Method</term><term>Microbiology</term><term>Neutralization</term><term>Neutralization Tests</term><term>Neutralizing antibody</term><term>Peptide Library</term><term>Phage display</term><term>RNA (genetics)</term><term>RNA (isolation & purification)</term><term>Rabies (genetics)</term><term>Rabies (immunology)</term><term>Rabies Vaccines (immunology)</term><term>Rabies virus</term><term>Rabies virus (immunology)</term><term>Recombinant virus</term><term>Viral Proteins (immunology)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antibodies, Viral</term><term>Immunoglobulin Fab Fragments</term><term>Immunoglobulin Variable Region</term><term>RNA</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antibodies, Viral</term><term>Glycoproteins</term><term>Immunoglobulin Fab Fragments</term><term>Immunoglobulin Variable Region</term><term>Rabies Vaccines</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Antibodies, Viral</term><term>Immunoglobulin Fab Fragments</term><term>Immunoglobulin Variable Region</term><term>RNA</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Rabies</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>Lymphocytes</term><term>Rabies</term><term>Rabies virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Epitope Mapping</term><term>Female</term><term>Humans</term><term>Male</term><term>Neutralization Tests</term><term>Peptide Library</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Homme</term><term>Virus rage</term><term>Isolement</term><term>Anticorps neutralisant</term><term>Virus recombinant</term><term>Technique phage display</term><term>Fragment peptidique Fab</term><term>Neutralisation</term><term>Microbiologie</term><term>Méthode</term></keywords><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Homme</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 10<sup>7</sup> Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the K<sub>D</sub> range 7 x 10<sup>-9</sup> to 5 × 10<sup>-8</sup> M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11).</div></front></TEI><affiliations><list><country><li>Tunisie</li></country></list><tree><country name="Tunisie"><noRegion><name sortKey="Houimel, Mehdi" sort="Houimel, Mehdi" uniqKey="Houimel M" first="Mehdi" last="Houimel">Mehdi Houimel</name></noRegion><name sortKey="Dellagi, Koussay" sort="Dellagi, Koussay" uniqKey="Dellagi K" first="Koussay" last="Dellagi">Koussay Dellagi</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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