Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.
Identifieur interne : 000A92 ( PubMed/Corpus ); précédent : 000A91; suivant : 000A93Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.
Auteurs : R. Kamatchi ; J. Charumathi ; R. Ravishankaran ; P. Kaliraj ; S. MeenakshisundaramSource :
- Journal of helminthology [ 1475-2697 ] ; 2016.
English descriptors
- KwdEn :
- Animals, Antigens, Helminth (blood), Antigens, Helminth (genetics), Antigens, Helminth (immunology), Elephantiasis, Filarial (blood), Elephantiasis, Filarial (diagnosis), Elephantiasis, Filarial (parasitology), Enzyme-Linked Immunosorbent Assay, Escherichia coli (genetics), Escherichia coli (metabolism), Gene Expression, Humans, Mice, Recombinant Proteins (genetics), Recombinant Proteins (immunology), Single-Chain Antibodies (analysis), Single-Chain Antibodies (genetics), Single-Chain Antibodies (immunology), Wuchereria bancrofti (genetics), Wuchereria bancrofti (immunology), Wuchereria bancrofti (isolation & purification).
- MESH :
- chemical , analysis : Single-Chain Antibodies.
- chemical , blood : Antigens, Helminth.
- chemical , genetics : Antigens, Helminth, Recombinant Proteins, Single-Chain Antibodies.
- chemical , immunology : Antigens, Helminth, Recombinant Proteins, Single-Chain Antibodies.
- blood : Elephantiasis, Filarial.
- diagnosis : Elephantiasis, Filarial.
- genetics : Escherichia coli, Wuchereria bancrofti.
- immunology : Wuchereria bancrofti.
- isolation & purification : Wuchereria bancrofti.
- metabolism : Escherichia coli.
- parasitology : Elephantiasis, Filarial.
- Animals, Enzyme-Linked Immunosorbent Assay, Gene Expression, Humans, Mice.
Abstract
Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.
DOI: 10.1017/S0022149X14000868
PubMed: 26693887
Links to Exploration step
pubmed:26693887Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.</title><author><name sortKey="Kamatchi, R" sort="Kamatchi, R" uniqKey="Kamatchi R" first="R" last="Kamatchi">R. Kamatchi</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Charumathi, J" sort="Charumathi, J" uniqKey="Charumathi J" first="J" last="Charumathi">J. Charumathi</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Ravishankaran, R" sort="Ravishankaran, R" uniqKey="Ravishankaran R" first="R" last="Ravishankaran">R. Ravishankaran</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Kaliraj, P" sort="Kaliraj, P" uniqKey="Kaliraj P" first="P" last="Kaliraj">P. Kaliraj</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Meenakshisundaram, S" sort="Meenakshisundaram, S" uniqKey="Meenakshisundaram S" first="S" last="Meenakshisundaram">S. Meenakshisundaram</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2016">2016</date><idno type="RBID">pubmed:26693887</idno><idno type="pmid">26693887</idno><idno type="doi">10.1017/S0022149X14000868</idno><idno type="wicri:Area/PubMed/Corpus">000A92</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000A92</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.</title><author><name sortKey="Kamatchi, R" sort="Kamatchi, R" uniqKey="Kamatchi R" first="R" last="Kamatchi">R. Kamatchi</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Charumathi, J" sort="Charumathi, J" uniqKey="Charumathi J" first="J" last="Charumathi">J. Charumathi</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Ravishankaran, R" sort="Ravishankaran, R" uniqKey="Ravishankaran R" first="R" last="Ravishankaran">R. Ravishankaran</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Kaliraj, P" sort="Kaliraj, P" uniqKey="Kaliraj P" first="P" last="Kaliraj">P. Kaliraj</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author><author><name sortKey="Meenakshisundaram, S" sort="Meenakshisundaram, S" uniqKey="Meenakshisundaram S" first="S" last="Meenakshisundaram">S. Meenakshisundaram</name><affiliation><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Journal of helminthology</title><idno type="eISSN">1475-2697</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antigens, Helminth (blood)</term><term>Antigens, Helminth (genetics)</term><term>Antigens, Helminth (immunology)</term><term>Elephantiasis, Filarial (blood)</term><term>Elephantiasis, Filarial (diagnosis)</term><term>Elephantiasis, Filarial (parasitology)</term><term>Enzyme-Linked Immunosorbent Assay</term><term>Escherichia coli (genetics)</term><term>Escherichia coli (metabolism)</term><term>Gene Expression</term><term>Humans</term><term>Mice</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (immunology)</term><term>Single-Chain Antibodies (analysis)</term><term>Single-Chain Antibodies (genetics)</term><term>Single-Chain Antibodies (immunology)</term><term>Wuchereria bancrofti (genetics)</term><term>Wuchereria bancrofti (immunology)</term><term>Wuchereria bancrofti (isolation & purification)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Single-Chain Antibodies</term></keywords><keywords scheme="MESH" type="chemical" qualifier="blood" xml:lang="en"><term>Antigens, Helminth</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antigens, Helminth</term><term>Recombinant Proteins</term><term>Single-Chain Antibodies</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antigens, Helminth</term><term>Recombinant Proteins</term><term>Single-Chain Antibodies</term></keywords><keywords scheme="MESH" qualifier="blood" xml:lang="en"><term>Elephantiasis, Filarial</term></keywords><keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Elephantiasis, Filarial</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term><term>Wuchereria bancrofti</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>Wuchereria bancrofti</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Wuchereria bancrofti</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="parasitology" xml:lang="en"><term>Elephantiasis, Filarial</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Enzyme-Linked Immunosorbent Assay</term><term>Gene Expression</term><term>Humans</term><term>Mice</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">26693887</PMID><DateCreated><Year>2015</Year><Month>12</Month><Day>23</Day></DateCreated><DateCompleted><Year>2016</Year><Month>10</Month><Day>24</Day></DateCompleted><DateRevised><Year>2016</Year><Month>12</Month><Day>30</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Electronic">1475-2697</ISSN><JournalIssue CitedMedium="Internet"><Volume>90</Volume><Issue>1</Issue><PubDate><Year>2016</Year><Month>Jan</Month></PubDate></JournalIssue><Title>Journal of helminthology</Title><ISOAbbreviation>J. Helminthol.</ISOAbbreviation></Journal><ArticleTitle>Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.</ArticleTitle><Pagination><MedlinePgn>74-80</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1017/S0022149X14000868</ELocationID><Abstract><AbstractText>Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Kamatchi</LastName><ForeName>R</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Charumathi</LastName><ForeName>J</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ravishankaran</LastName><ForeName>R</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kaliraj</LastName><ForeName>P</ForeName><Initials>P</Initials><AffiliationInfo><Affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Meenakshisundaram</LastName><ForeName>S</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>J Helminthol</MedlineTA><NlmUniqueID>2985115R</NlmUniqueID><ISSNLinking>0022-149X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000947">Antigens, Helminth</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D057127">Single-Chain Antibodies</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000947" MajorTopicYN="N">Antigens, Helminth</DescriptorName><QualifierName UI="Q000097" MajorTopicYN="Y">blood</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000276" MajorTopicYN="N">immunology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004605" MajorTopicYN="N">Elephantiasis, Filarial</DescriptorName><QualifierName UI="Q000097" MajorTopicYN="N">blood</QualifierName><QualifierName UI="Q000175" MajorTopicYN="Y">diagnosis</QualifierName><QualifierName UI="Q000469" MajorTopicYN="N">parasitology</QualifierName></MeshHeading><MeshHeading><DescriptorName 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PubStatus="medline"><Year>2016</Year><Month>10</Month><Day>25</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">26693887</ArticleId><ArticleId IdType="pii">S0022149X14000868</ArticleId><ArticleId IdType="doi">10.1017/S0022149X14000868</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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